Regular Plasmid Gene Expression Vector

Overview

Delivering plasmid vectors into mammalian cells by conventional transfection is one of the most widely used procedures in biomedical research. While a number of more sophisticated gene delivery vector systems have been developed over the years such as lentiviral vectors, adenovirus vectors, AAV vectors and piggyBac, conventional plasmid transfection remains the workhorse of gene delivery in many labs. This is largely due to its technical simplicity as well as good efficiency in a wide range of cell types. A key feature of transfection with regular plasmid vectors is that it is transient, with only a very low fraction of cells stably integrating the plasmid in the genome (typically less than 1%).

For further information about this vector system, please refer to the papers below.

ReferencesTopic
Mol Biotechnol. 16:151 (2000)Overview of vector design for mammalian gene expression
EMBO J. 12:2539 (1993)Transcription blocker prevent transcriptional interference

Highlights

Our vector is optimized for high copy number replication in E. coli and high-efficiency transfection. Cells transfected with the vector can be selected and/or visualized based on marker gene expression as chosen by the user.

Advantages

Technical simplicity: Delivering plasmid vectors into cells by conventional transfection is technically straightforward, and far easier than virus-based vectors which require the packaging of live virus.

Very large cargo space: Our vector can accommodate ~30 kb of total DNA. The plasmid backbone only occupies about 3 kb, leaving plenty of room to accommodate the user's sequence of interest.

High-level expression: Conventional transfection of plasmids can often result in very high copy numbers in cells (up to several thousand copies per cell). This can lead to very high expression levels of the genes carried on the vector.

Disadvantages

Non-integration of vector DNA: Conventional transfection of plasmid vectors is also referred to as transient transfection because the vector stays mostly as episomal DNA in cells without integration. However, plasmid DNA can integrate permanently into the host genome at a very low frequency (one per 102 to 106 cells depending on cell type). If a drug resistance or fluorescence marker is incorporated into the plasmid, cells stably integrating the plasmid can be derived by drug selection or cell sorting after extended culture.

Limited cell type range: The efficiency of plasmid transfection can vary greatly from cell type to cell type. Non-dividing cells are often more difficult to transfect than dividing cells, and primary cells are often harder to transfect than immortalized cell lines. Some important cell types, such as neurons and pancreatic β cells, are notoriously difficult to transfect. Additionally, plasmid transfection is largely limited to in vitro applications and rarely used in vivo.

Non-uniformity of gene delivery: Although a successful transfection can result in very high average copy number of the transfected plasmid vector per cell, this can be highly non-uniform. Some cells can carry many copies while others carry very few or none. This is unlike transduction by virus-based vectors which tends to result in relatively uniform gene delivery into cells.

Key components

Promoter: The promoter that drives your gene of interest is placed here.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

CMV promoter: Human cytomegalovirus immediate early promoter. It drives the ubiquitous expression of the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

BGH pA: Bovine growth hormone polyadenylation. It facilitates transcriptional termination of the upstream ORF.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

Lentivirus Gene Expression Vector

Overview

The lentiviral vector system is a highly efficient vehicle for introducing genes permanently into mammalian cells. Presently, it is one of the two most commonly used methods for gene delivery into mammalian cells (the other being conventional plasmid transfection). Features that make this system so popular include the ability to choose which promoter will drive the gene of interest and the ability to infect a very wide range of cell types.

Lentiviral vectors are derived from HIV, which is a member of the retrovirus family. Wildtype lentivirus has a plus-strand linear RNA genome.

A lentiviral vector is first constructed as a plasmid in E. coli. It is then transfected into packaging cells along with several helper plasmids. Inside the packaging cells, vector DNA located between the two long terminal repeats (LTRs) is transcribed into RNA, and viral proteins expressed by the helper plasmids further package the RNA into virus. Live virus is then released into the supernatant, which can be used to infect target cells directly or after concentration.

When the virus is added to target cells, the RNA cargo is shuttled into cells where it is reverse transcribed into DNA and randomly integrated into the host genome. Any gene(s) that were placed in-between the two LTRs during vector construction are permanently inserted into host DNA alongside the rest of viral genome.

By design, lentiviral vectors lack the genes required for viral packaging and transduction (these genes are instead carried by helper plasmids used during virus packaging). As a result, virus produced from lentiviral vectors has the important safety feature of being replication incompetent (meaning that they can transduce target cells but cannot replicate in them).

For further information about this vector system, please refer to the papers below.

ReferencesTopic
J Virol. 72:8463 (1998)The 3rd generation lentivirus vectors
J Virol. 72:9873 (1998)Self-inactivating lentivirus vectors
Science. 272:263 (1996)Transduction of non-dividing cells by lentivirus vectors
Curr Gene Ther. 5:387 (2005)Tropism of lentiviral vectors
J Virol. 77:4685 (2003)Impact of cPPT to lentivirus vector transduction
J Virol. 73:2886 (1999)WPRE enhances the expression of transgenes
Nat Protoc. 1:241 (2006)Production and purification of lentiviral vectors

Highlights

Our vector is derived from the third-generation lentiviral vector system. It is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient viral transduction of a wide range of cells, efficient vector integration into the host genome, and high-level transgene expression.

Advantages

Permanent integration of vector DNA: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, lentiviral transduction can deliver genes permanently into host cells due to the integration of the viral vector into the host genome.

High viral titer: Our lentiviral vector can be packaged into high titer virus. When lentivirus is obtained through our virus packaging service, titer can reach >108 transducing unit per ml (TU/ml). At this titer, transduction efficiency for cultured mammalian cells can approach 100% when an adequate amount of viral is used.

Very broad tropism: Our packaging system adds the VSV-G envelop protein to the viral surface. This protein has broad tropism. As a result, cells from all commonly used mammalian species (and even some non-mammalian species) can be transduced. Furthermore, almost any mammalian cell type can be transduced (e.g. dividing cells and non-dividing cells, primary cells and established cell lines, stem cells and differentiated cells, adherent cells and non-adherent cells). Neurons, which are often impervious to conventional transfection, can be readily transduced by our lentiviral vector. Lentiviral vectors packaged with our system have broader tropism than adenoviral vectors (which have low transduction efficiency for some cell types) or MMLV retroviral vectors (which have difficulty transducing non-dividing cells).

Customizable internal promoter: Our vector is designed to self-inactivate the promoter activity in its 5' LTR upon integration into the genome. As a result, users can put in their own promoter to drive their gene of interest within the vector. This is a distinct advantage over our MMLV retrovirus vectors, which rely on the promoter function of 5' LTR to drive the ubiquitous expression of the gene of interest.

Relative uniformity of gene delivery: Generally, viral transduction can deliver vectors into cells in a relatively uniform manner. In contrast, conventional transfection of plasmid vectors can be highly non-uniform, with some cells receiving a lot of copies while other cells receiving few copies or none.

Effectiveness in vitro and in vivo: While our vector is mostly used for in vitro transduction of cultured cells, it can also be used to transduce cells in live animals.

Safety: The safety of our vector is ensured by two features. One is the partition of genes required for viral packaging and transduction into several helper plasmids; the other is self-inactivation of the promoter activity in the 5' LTR upon vector integration. As a result, it is essentially impossible for replication competent virus to emerge during packaging and transduction. The health risk of working with our vector is therefore minimal.

Disadvantages

Limited cargo space: The wildtype lentiviral genome is ~9.2 kb. In our vector, the components necessary for viral packaging and transduction occupy ~2.8 kb, which leaves ~6.4 kb to accommodate the user's DNA of interest. When the vector goes beyond this size limit, viral titer can be severely reduced. Our vector is routinely used for inserting several functional elements besides the ORF of the gene of interest, such as promoter and drug resistance. A large ORF plus these additional elements could exceed 6.4 kb, and the result could be compromised viral production.

Technical complexity: The use of lentiviral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technical demanding and time consuming relative to conventional plasmid transfection.

Key components

RSV promoter: Rous sarcoma virus promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into live virus.

Δ5' LTR: A deleted version of the HIV-1 5' long terminal repeat. In wildtype lentivirus, 5' LTR and 3' LTR are essentially identical in sequence. They reside on two ends of the viral genome and point in the same direction. Upon viral integration, the 3' LTR sequence is copied onto the 5' LTR. The LTRs carry both promoter and polyadenylation function, such that in wildtype virus, the 5' LTR acts as a promoter to drive the transcription of the viral genome, while the 3' LTR acts as a polyadenylation signal to terminate the upstream transcript. On our vector, Δ5' LTR is deleted for a region that is required for the LTR's promoter activity normally facilitated by the viral transcription factor Tat. This does not affect the production of viral RNA during packaging because the promoter function is supplemented by the RSV promoter engineered upstream of Δ5' LTR.

Ψ: HIV-1 packaging signal required for the packaging of viral RNA into virus.

RRE: HIV-1 Rev response element. It allows the nuclear export of viral RNA by the viral Rev protein during viral packaging.

cPPT: HIV-1 Central polypurine tract. It creates a "DNA flap" that increases nuclear importation of the viral genome during target cell infection. This improves vector integration into the host genome, resulting in higher transduction efficiency.

Promoter: The promoter driving your gene of interest is placed here.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here.

WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances viral RNA stability in packaging cells, leading to higher titer of packaged virus.

mPGK promoter: Mouse phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

ΔU3/3' LTR: A truncated version of the HIV-1 3' long terminal repeat that deletes the U3 region. This leads to the self-inactivation of the promoter activity of the 5' LTR upon viral vector integration into the host genome (due to the fact that 3' LTR is copied onto 5' LTR during viral integration). The polyadenylation signal contained in ΔU3/3' LTR serves to terminates all upstream transcripts produced both during viral packaging and after viral integration into the host genome.

SV40 early pA: Simian virus 40 early polyadenylation signal. It further facilitates transcriptional termination after the 3' LTR during viral RNA transcription during packaging. This elevates the level of functional viral RNA in packaging cells, thus improving viral titer.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

MMLV Retrovirus Gene Expression Vector

Overview

The MMLV retroviral vector system is an efficient vehicle for introducing genes permanently into mammalian cells. It became particularly popular as a gene delivery method for making iPS cells.

MMLV retroviral vectors are derived from Moloney murine leukemia virus, which is a member of the retrovirus family. Wildtype MMLV virus has a plus-strand linear RNA genome.

An MMLV retroviral vector is first constructed as a plasmid in E. coli. It is then transfected into packaging cells along with several helper plasmids. Inside the packaging cells, vector DNA located between the two long terminal repeats (LTRs) is transcribed into RNA, and viral proteins expressed by the helper plasmids further package the RNA into virus. Live virus is then released into the supernatant, which can be used to infect target cells directly or after concentration.

When the virus is added to target cells, the RNA cargo is shuttled into cells where it is reverse transcribed into DNA and randomly integrated in the host genome. Any gene(s) that were placed in-between the two LTRs during vector construction are permanently inserted into host DNA alongside the rest of viral genome.

By design, MMLV retroviral vectors lack the genes required for viral packaging and transduction (these genes are carried by helper plasmids or integrated into packaging cells instead). As a result, viruses produced from the vectors have the important safety feature of being replication incompetent (meaning that they can transduce target cells but cannot replicate in them).

For further information about this vector system, please refer to the papers below.

ReferencesTopic
Exp Hematol. 31:1007 (2003)Review
J Virol. 61:1639 (1987)Extended packaging signal increases the titer of MMLV vectors
Gene Ther. 7:1063 (2000)Tropism of MMLV vectors depends on packaging cell lines
Nat Protoc. 6:346 (2011)Tropism of MMLV vectors depends on packaging plasmids

Highlights

Our vector is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient viral transduction of a wide range of cells, efficient vector integration into the host genome, and high-level transgene expression.

Advantages

Permanent integration of vector DNA: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, retroviral transduction can deliver genes permanently into host cells due to integration of the viral vector into the host genome.

Broad tropism: Our packaging system adds the VSV-G envelop protein to the viral surface. This protein has broad tropism. As a result, from commonly used mammalian species such as human, mouse and rat can be transduced. Furthermore, many cell types can be transduced, though our vector has difficulty transducing non-dividing cells (see disadvantages below).

Large cargo space: The wildtype MMLV retroviral genome is ~8 kb. In our vector, the components necessary for viral packaging and transduction occupy ~2.5 kb, which leaves ~5.5 kb to accommodate the user's DNA of interest. Because our vector is designed for the insertion of only an ORF, this cargo space is sufficient for most applications.

High-level expression: The 5' LTR contains a strong ubiquitous promoter that drives high-level expression of the user's gene of interest.

Relative uniformity of gene delivery: Generally, viral transduction can deliver vectors into cells in a relatively uniform manner. In contrast, conventional transfection of plasmid vectors can be highly non-uniform, with some cells receiving a lot of copies while other cells receiving few copies or none.

Effectiveness in vitro and in vivo: While our vector is mostly used for in vitro transduction of cultured cells, it can also be used to transduce cells in live animals.

Safety: The safety of our vector is ensured by partitioning genes required for viral packaging and transduction into several helper plasmids or integrating them into packaging cells. As a result, live virus produced from our vector is replication incompetent.

Disadvantages

Dependence on 5' LTR promoter: Expression of the gene of interest in our vector is driven by the ubiquitous promoter function in the 5' LTR. This is a distinct disadvantage as compared to our lentiviral vectors which allow the user to put in their own promoter to drive their gene of interest.

Moderate viral titer: Viral titer from our vector reach ~107/ml in the supernatant of packaging cells without further concentration. This is about an order of magnitude lower than our lentiviral vectors.

Difficulty transducing non-dividing cells: Our vector has difficulty transducing non-dividing cells.

Technical complexity: The use of MMLV retroviral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technical demanding and time consuming relative to conventional plasmid transfection.

Key components

5' MoMuLV LTR: MMLV retrovirus 5' long terminal repeat. In wildtype MMLV retrovirus, 5' LTR and 3' LTR are essentially identical in sequence. They reside on two ends of the viral genome and point in the same direction. Upon viral integration, the 3' LTR sequence is copied onto the 5' LTR. The LTRs carry both promoter and polyadenylation function, such that the 5' LTR acts as a promoter to drive the transcription of the viral genome, while the 3' LTR acts as a polyadenylation signal to terminate the upstream transcript.

ψ plus pack2: MMLV retrovirus packaging signal required for the packaging of viral RNA into virus.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here. Its expression is driven by the ubiquitous promoter function in the 5' LTR.

3' MoMuLV LTR: MMLV retrovirus 3' long terminal repeat. The polyadenylation signal contained in 3' LTR serves to terminates the transcript from the upstream ORF.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

Adenovirus Gene Expression Vector

Overview

The adenoviral vector system is effective in transducing many (but not all) mammalian cell types, where the vector remains as episomal DNA without integration into the host genome. It is the preferred gene delivery system in vivo, often used in gene therapy and vaccination.

Adenoviral vectors are derived from adenovirus, which causes the common cold. Wildtype adenovirus has a double-stranded linear DNA genome.

An adenoviral vector is first constructed as a plasmid in E. coli. It is then transfected into packaging cells, where the region of the vector between the two inverted terminal repeats (ITRs) is packaged into live virus.

When the virus is added to target cells, the DNA cargo is delivered into cells where it enters the nucleus and remains as episomal DNA without integration into the host genome. Any gene(s) that were placed in-between the two ITRs during vector construction are introduced into target cells along with the rest of viral genome.

By design, adenoviral vectors lack the E1A, E1B and E3 genes (delta E1 + delta E3). The first two are required for the production of live virus (these two genes are engineered into the genome of packaging cells). As a result, virus produced from the vectors have the important safety feature of being replication incompetent (meaning that they can transduce target cells but cannot replicate in them).

For further information about this vector system, please refer to the papers below.

ReferencesTopic
Proc Natl Acad Sci U S A. 91:8802 (1994)The 2nd generation adenovirus vectors
J Gen Virol. 36:59 (1977)A packaging cell line for adenovirus vectors
J Virol. 79:5437 (2005)Replication-competent adenovirus (RCA) formation in 293 Cells
Gene Ther. 3:75 (1996)A cell line for testing RCA

Highlights

Our vector is derived from the adenovirus serotype 5 (Ad5). It is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient transduction of host cells, and high-level transgene expression.

Advantages

Low risk of host genome disruption: Upon transduction into host cells, adenoviral vectors remain as episomal DNA in the nucleus. The lack of integration into the host genome can be a desirable feature for in vivo human applications, as it reduces the risk of host genome disruption that might lead to cancer.

Very high viral titer: After our adenoviral vector is transfected into packaging cells to produce live virus, the virus can be further amplified to very high titer by re-infecting packaging cells. This is unlike lentivirus, MMLV retrovirus, or AAV, which cannot be amplified by re-infection. When adenovirus is obtained through our virus packaging service, titer can reach >109 plaque-forming unit per ml (PFU/ml).

Broad tropism: Cells from commonly used mammalian species such as human, mouse and rat can be transduced with our vector. But some cell types have proven difficult to transduce (see disadvantages below).

Large cargo space: Our vector has about ~8.3 kb of cargo space to accommodate the user's DNA of interest (such as promoter, ORF, and fluorescence marker). This is bigger than the ~6.4 kb cargo space in our lentiviral expression vector, and is sufficient for most applications.

Effectiveness in vitro and in vivo: Our vector is often used to transduce cells in live animals, but it can also be used effectively in vitro.

Safety: The safety of our vector is ensured by the fact that it lacks genes essential for virus production (these genes are engineered into the genome of packaging cells). Virus made from our vector is therefore replication incompetent except when it is used to transduce packaging cells.

Disadvantages

Non-integration of vector DNA: The adenoviral genome does not integrate into the genome of transduced cells. Rather, it exists as episomal DNA, which can be lost over time, especially in dividing cells. 

Difficulty transducing certain cell types: While our adenoviral vectors can transduce many different cell types including non-dividing cells, it is inefficient against certain cell types such as endothelia, smooth muscle, differentiated airway epithelia, peripheral blood cells, neurons, and hematopoietic cells.

Strong immunogenicity: Live virus from adenoviral vectors can elicit strong immune response in animals, thus limiting certain in vivo applications.

Technical complexity: The use of adenoviral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technical demanding and time consuming.

Key components

5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.

Ψ: Adenovirus packaging signal required for the packaging of viral DNA into virus.

Promoter: The promoter that drives your gene of interest is placed here.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here.

TK pA: Herpes simplex virus thymidine kinase polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

ΔAd5: Portion of Ad5 genome between the two ITRs minus the E1A, E1B and E3 regions.

3' ITR: 3' inverted terminal repeat.

pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin exist in medium copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

PacI: PacI restriction site (PacI is a rare cutter that cuts at TTAATTAA). The two PacI restriction sites on the vector can be used to linearize the vector and remove the vector backbone from the viral sequence, which is necessary for efficient packaging.

Adeno-associated Virus Gene Expression Vector

Overview

The adeno-associated virus (AAV) vector system is a popular and versatile tool for in vitro and in vivo gene delivery. AAV is effective in transducing many mammalian cell types, and, unlike adenovirus, has very low immunogenicity, being almost entirely nonpathogenic in vivo. This makes AAV the ideal viral vector system for many animal studies.

An AAV vector is first constructed as a plasmid in E. coli. It is then transfected into packaging cells along with helper plasmids, where the region of the vector between the two inverted terminal repeats (ITRs) is packaged into live virus.

When the virus is added to target cells, the double-stranded linear DNA genome is delivered into cells where it enters the nucleus and remains as episomal DNA without integration into the host genome. Any gene(s) placed in-between the two ITRs are introduced into target cells along with the rest of viral genome.

A major practical advantage of AAV is that in most cases AAV can be handled in biosafety level 1 (BSL1) facilities. This is due to AAV being inherently replication-deficient, producing little or no inflammation, and causing no known human disease.

Many strains of AAV have been identified in nature. They are divided into different serotypes based on different antigenicity of the capsid protein on the viral surface. Different serotypes can render the virus with different tissue tropism (i.e. tissue specificity of infection). When our AAV vectors are packaged into virus, different serotypes can be conferred to the virus by using different capsid proteins for the packaging. The table below lists different AAV serotypes and their tissue tropism.

SerotypeTissue tropism
AAV1SM, CNS, lung, retina, pancreas, heart, liver
AAV2SM, CNS, liver, kidney, retina
AAV3SM
AAV4CNS, retina, lung, kidney
AAV5SM, CNS, lung, retina
AAV6SM, heart, lung, adipose, liver
AAV7SM, retina, CNS, liver
AAV8SM, CNS, retina, liver, pancreas, heart, kidney, adipose
AAV9SM, lung, liver, heart, pancreas, CNS, retina, testes, kidney
AAVrh10SM, lung, liver, heart, pancreas, CNS, retina, kidney

For further information about this vector system, please refer to the papers below.

ReferencesTopic
Methods in Enzy. 507:229-54 (2012)Review of AAV virology and uses
Curr Opin Pharmacol. 24:59-67 (2015)AAV use in gene therapy, and serotype differences

Highlights

Our AAV vector system is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient transduction of host cells, and high-level transgene expression. This viral vector can be packaged into virus using all known capsid serotypes, is capable of very high transduction efficiency, and presents low safety risk.

Advantages

Safety: AAV is the safest viral vector system available. AAV is inherently replication-deficient, and is not known to cause any human diseases.

Low risk of host genome disruption: Upon transduction into host cells, AAV vectors remain as episomal DNA in the nucleus. The lack of integration into the host genome can be a desirable feature for in vivo human applications, as it reduces the risk of host genome disruption that might lead to cancer.

High viral titer: Our AAV vector can be packaged into high titer virus. When AAV virus is obtained through our virus packaging service, titer can reach >1011 genome copy per ml (GC/ml).

Broad tropism: A wide range of cell and tissue types from commonly used mammalian species such as human, mouse and rat can be readily transduced with our AAV vector when it is packaged into the appropriate serotype. But some cell types may be difficult to transduce, depending on the serotype used (see disadvantages below).

Effectiveness in vitro and in vivo: Our vector is often used to transduce cells in live animals, but it can also be used effectively in vitro.

Disadvantages

Small cargo space: AAV has the smallest cargo capacity of any of our viral vector systems. AAV can accommodate a maximum of 4.7kb of sequence between the ITRs.

Difficulty transducing certain cell types: Our AAV vector system can transduce many different cell types including non-dividing cells when packaged into the appropriate serotype. However, different AAV serotypes have tropism for different cell types, and certain cell types may be hard to transduce by any serotype.

Technical complexity: The use of viral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technical demanding and time consuming relative to conventional plasmid transfection. These demands can be alleviated by choosing our virus packaging services when ordering your vector.

Key components

5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.

Promoter: The promoter that drives your gene of interest is placed here.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

3' ITR: 3' inverted terminal repeat. See description for 5’ ITR.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

PiggyBac Gene Expression Vector

Overview

Our piggyBac vector system is highly effective for inserting foreign DNA into the host genome of mammalian cells. This system is technically simple, utilizing plasmid transfection (rather than viral transduction) to permanently integrate your gene(s) of interest into the host genome.

The system is derived from the piggyBac transposon, which is originally isolated from the cabbage looper (Trichoplusia ni; a moth species). Based on sequence homology, the piggyBac transposon was found to belong to a class of transposons common to many animals.

The piggyBac system contains two vectors, both engineered as E. coli plasmids. One vector, referred to as the helper plasmid, encodes the transposase. The other vector, referred to as the transposon plasmid, contains two terminal repeats (TRs) bracketing the region to be transposed. The gene to be delivered into host cells is cloned into this region.

When the helper and transposon plasmids are co-transfected into target cells, the transposase produced from the helper would recognize the two TRs on the transposon, and insert the flanked region including the two TRs into the host genome. Insertion typically occurs at host chromosomal sites that contain the TTAA sequence, which is duplicated on the two flanks of the integrated fragment.

PiggyBac is a class II transposon, meaning that it moves in a cut-and-paste manner, hopping from place to place without leaving copies behind. (In contrast, class I transposons move in a copy-and-paste manner.) Because the helper plasmid is only transiently transfected into host cells, it will get lost over time. With the loss of the helper plasmid, the integration of the transposon in the genome of host cells becomes permanent. If these cells are transfected with the helper plasmid again, the transposon could get excised from the genome of some cells, footprint free.

For further information about this vector system, please refer to the papers below.

ReferencesTopic
Mol Cell Biochem. 354:301 (2011)Review
Cell. 122:473 (2005)Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice

Highlights of our vector

Our piggyBac transposon plasmid along with the helper plasmid are optimized for high copy number replication in E. coli, efficient transfection into a wide range of target cells, and high-level expression of the transgene carried on the vector.

Advantages

Permanent integration of vector DNA: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, transfection of mammalian cells with the piggyBac transposon plasmid along with the helper plasmid can deliver genes carried on the transposon permanently into host cells due to the integration of the transposon into the host genome.

Technical simplicity: Delivering plasmid vectors into cells by conventional transfection is technically straightforward, and far easier than virus-based vectors which require the packaging of live virus.

Very large cargo space: Our transposon vector can accommodate ~30 kb of total DNA. The plasmid backbone and transposon-related sequences only occupies about 3 kb, leaving plenty of room to accommodate the user's sequence of interest.

Disadvantages

Limited cell type range: The delivery of piggyBac vectors into cells relies on transfection. The efficiency of transfection can vary greatly from cell type to cell type. Non-dividing cells are often more difficult to transfect than dividing cells, and primary cells are often harder to transfect than immortalized cell lines. Some important cell types, such as neurons and pancreatic β cells, are notoriously difficult to transfect. Additionally, plasmid transfection is largely limited to in vitro applications and rarely used in vivo. These issues limit the use of the piggyBac system.

Key components

5' ITR: 5' inverted terminal repeat. When a DNA sequence is flanked by two ITRs, the piggyBac transpose can recognize them, and insert the flanked region including the two ITRs into the host genome.

Promoter: The promoter driving your gene of interest is placed here.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here.

rBG pA: Rabbit β-globin polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

CMV promoter: Human cytomegalovirus immediate early promoter. It drives the ubiquitous expression of the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

BGH pA: Bovine growth hormone polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

3' ITR: 3' inverted terminal repeat.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Tol2 Gene Expression Vector

Overview

Our Tol2 vector system is highly effective for inserting foreign DNA into the genome of host cells. This system is technically simple, utilizing plasmid transfection (rather than viral transduction) to permanently integrate your gene(s) of interest into the host genome.

The system is derived from the Tol2 transposon, which is originally isolated from the teleost fish, medaka (Oryzias latipes). Based on sequence homology, the Tol2 transposon was found to be closely related to the hAT family of non-autonomous elements found throughout vertebrate genomes.

The Tol2 system contains two vectors, both engineered as E. coli plasmids. One vector, referred to as the helper plasmid, encodes the transposase. The other vector, referred to as the transposon plasmid, contains two inverted terminal repeats (ITRs) bracketing the region to be transposed. The gene to be delivered into host cells is cloned into this region of the transposon plasmid.

When the transposon and helper plasmids are co-transfected into target cells, the transposase produced from the helper plasmid would recognize the two ITRs on the transposon, and inserts the flanked region including the two ITRs into the host genome. Insertion occurs without any significant bias with respect to insertion site sequence. This is unlike transposon systems which have specific target consensus sites. For example, piggyBac transposons typically inserts at sites containing the sequence TTAA.

Tol2 is a class II transposon, meaning that it moves in a cut-and-paste manner, hopping from place to place without leaving copies behind. (In contrast, class I transposons move in a copy-and-paste manner.) Tol2 integrates as a single copy through a cut-and-paste mechanism. At each insertion site, the Tol2 transposase creates an 8 bp duplication, resulting in identical 8 bp direct repeats flanking each transposon integration site in the genome.

There are two alternative methods for introducing the transposase into target cells. The helper plasmid can be transiently transfected into cells, where it will temporarily drive expression of the transposase. Alternatively, target cells can be injected with Tol2 mRNA generated by in vitro transcription from the helper plasmid. In either case, the transposase will only be expressed for a short time. With the loss of the helper plasmid or degradation of transposase mRNA, the integration of the transposon in the host genome becomes permanent. If Tol2 transposase is reintroduced into the cells, the transposon could get excised from the genome of some cells.

For further information about this vector system, please refer to the papers below.

References Topic
Genome Biol. 8(Suppl 1): S7 (2007) Review of Tol2 vectors
Genetics 174: 639–649 (2006) Identification of minimal sequences for Tol2 transposable elements
PLoS Genetics 2: e169 (2006) Large cargo-capacity transposition with a minimal Tol2 transposon

Highlights

Our Tol2 transposon vector system enables efficient insertion of sequences up to 11 kb into the genome of target cells. The Tol2 transposon plasmid along with the helper plasmid are optimized for high copy number replication in E. coli, efficient transfection into a wide range of target cells, and high-level expression of the transgene carried on the vector.

Advantages

Permanent integration of vector DNA: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, transfection of mammalian cells with the Tol2 transposon plasmid along with the helper plasmid (or introduction of Tol2 mRNA) can deliver genes carried on the transposon permanently into host cells due to the integration of the transposon into the host genome.

Technical simplicity: Delivering plasmid vectors into cells by conventional transfection is technically straightforward, and far easier than virus-based vectors which require the packaging of live virus.

Very large cargo space: Our Tol2 transposon vector can accommodate ~11 kb of total DNA. The plasmid backbone and transposon-related sequences only occupies about 3 kb, leaving plenty of room to accommodate the user's sequence of interest.

Disadvantages

Limited cell type range: The delivery of Tol2 vectors into cells relies on transfection. The efficiency of transfection can vary greatly from cell type to cell type. Non-dividing cells are often more difficult to transfect than dividing cells, and primary cells are often harder to transfect than immortalized cell lines. Some important cell types, such as neurons and pancreatic β cells, are notoriously difficult to transfect. These issues limit the use of the Tol2 system.

Key components

5' ITR: Tol2 5' terminal repeat. When a DNA sequence is flanked by two ITRs, the Tol2 transpose can recognize them, and insert the flanked region including the two ITRs into the host genome.

Promoter: The promoter driving your gene of interest is placed here.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

3' ITR: Tol2 3' terminal repeat.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Regular Plasmid Inducible Gene Expression Vector (Tet-On)

Overview

The Tet-On inducible gene expression system is a powerful tool to control the timing of expression of the gene(s) of interest in mammalian cells. Our Tet-On inducible gene expression vectors are designed to achieve nearly complete silencing of a gene of interest in the absence of tetracycline and its analogs (e.g. doxycycline), and strong, rapid expression in response to the addition of tetracycline or one of its analogs (e.g. doxycycline). This is achieved through a multicomponent system which incorporates active silencing by the tTS protein in the absence of tetracycline and strong activation by the rtTA protein in the presence of tetracycline.

Delivering plasmid vectors into mammalian cells by conventional transfection is one of the most widely used procedures in biomedical research. While a number of more sophisticated gene delivery vector systems have been developed over the years such as lentiviral vectors, adenovirus vectors, AAV vectors and piggyBac, conventional plasmid transfection remains the workhorse of gene delivery in many labs. This is largely due to its technical simplicity as well as good efficiency in a wide range of cell types. A key feature of transfection with regular plasmid vectors is that it is transient, with only a very low fraction of cells stably integrating the plasmid in the genome (typically less than 1%).

For further information about this vector system, please refer to the papers listed below.

References Topic
Science 268:1766-1769 (1995) Development of rtTA.
J Gene Med. 1:4-12 (1999) Development of tTS.

Highlights

Our Tet-On inducible gene expression vectors are designed to achieve nearly complete silencing of the gene(s) of interest in the absence of tetracycline, and strong, rapid expression in response to the addition of tetracycline. Our vector is optimized for high copy number replication in E. coli and high-efficiency transfection in many mammalian cell lines.

Advantages

Switch-like gene activation: Unlike rtTA only Tet-On systems that usually have significant leaky expression in the absence of induction, our Tet-On gene expression vectors act as true tetracycline-regulated on-and-off switch for controlling gene expression, which can minimize the background expression without induction and result in high sensitivity and high dynamic range of the tetracycline induction.

Technical simplicity: Delivering plasmid vectors into cells by conventional transfection is technically straightforward, and far easier than virus-based vectors which require the packaging of viral vector plasmids into live virus.

Very large cargo space: Our regular plasmid vectors can accommodate ~30 kb of total DNA. The plasmid backbone only occupies about 4.6 kb, including Tet-On components, leaving plenty of room to accommodate the user's gene(s) of interest and promoter for driving Tet-regulatory proteins.

High-level expression: The TRE promoter can drive very high levels of expression of the gene(s) of interest in its induced state. Additionally, conventional transfection of plasmids often results in very high copy numbers in cells (up to several thousand copies per cell). This can lead to very high expression levels of the gene(s) carried on the vector.

Disadvantages

Non-integration of vector DNA: Conventional transfection of plasmid vectors is also referred to as transient transfection because the vector stays mostly as episomal DNA in cells without integration. However, plasmid DNA can integrate permanently into the host genome at a very low frequency (one per 102 to 106 cells depending on cell type). If a drug resistance or fluorescence marker is incorporated into the plasmid, cells stably integrating the plasmid can be derived by drug selection or cell sorting after extended culture.

Limited cell type range: The efficiency of plasmid transfection can vary greatly from cell type to cell type. Non-dividing cells are often more difficult to transfect than dividing cells, and primary cells are often harder to transfect than immortalized cell lines. Some important cell types, such as neurons and pancreatic β cells, are notoriously difficult to transfect. Additionally, plasmid transfection is largely limited to in vitro applications and rarely used in vivo.

Non-uniformity of gene delivery: Although a successful transfection can result in very high average copy number of the transfected plasmid vector per cell, this can be highly non-uniform. Some cells can carry many copies while others may carry very few or none. This is unlike transduction by virus which tends to result in relatively uniform gene delivery into cells.

Key components

TRE: Tetracycline-responsive element promoter (2nd generation). This element can be regulated by a class of transcription factors (e.g. tTA, rtTA and tTS) whose activities are dependent on tetracycline or its analogs (e.g. doxycycline).

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

Promoter: The promoter chosen to drive expression of the tTS/rtTA cassette.

tTS: Tetracycline-controlled transcriptional silencer. This protein binds to TRE promoter to actively suppress gene transcription only in the absence of tetracycline and its analogs (e.g. doxycycline).

T2A: Self-cleaving 2A peptide from thosea asigna virus that allows multiple proteins to be made from a polycistronic transcript containing multiple ORFs separated by T2A. The cleavage occurs through a putative “ribosomal skipping” mechanism.

rtTA: Reverse tetracycline responsive transcriptional activator M2 (2nd generation). This protein binds to TRE promoter to activate gene transcription only in the presence of tetracycline or its analogs (e.g. doxycycline). It has higher sensitivity to the inducing drug and lower leaky activity in the absence of the drug compared to its predecessor.

BGH pA: Bovine growth hormone polyadenylation. It facilitates transcriptional termination of the tTS/rtTA cassette.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Regular Plasmid Conditional Gene Expression Vector (LoxP-Stop-LoxP)

Overview

This regular plasmid gene expression vector system utilizes the LoxP-Stop-LoxP (LSL) cassette to achieve Cre-mediated conditional activation of gene expression in mammalian cells and animals. The LSL cassette comprises a LoxP-flanked (aka floxed) triple repeat of the SV40 polyadenylation sequence. The user-selected promoter is placed upstream of the cassette while the user’s gene of interest is placed downstream of it. In the absence of Cre recombinase, the cassette completely blocks transcription of the gene of interest. When Cre is introduced into cells carrying this vector, the cassette is excised, allowing the user-selected promoter to drive the transcription of the gene of interest.

While this vector system can be used in tissue culture cells, it is particular suitable for the generation of transgenic animals. When a transgenic animal carrying such a vector is crossed to an animal carrying a tissue-specific Cre transgene, the progeny animals carrying both types of transgenes would turn on the gene of interest specifically in cells where the tissue-specific Cre is expressed.

For using this vector system in cell culture, antibiotic or fluorescence based markers can be added to the vector to allow selection or visualization of transfected cells, including the isolation of cells that have permanently integrated the vector in the genome.

For further information about this vector system and Cre-mediated recombination, please refer to the papers below.

ReferencesTopic
Mol Biotechnol. 16:151 (2000)Overview of vector design for mammalian gene expression
EMBO J. 12:2539 (1993)Transcription blocker prevent transcriptional interference
J Biol Chem. 259:1509-14 (1984)Purification and properties of the Cre recombinase protein
Genesis. 26:99-109. (2000)Review of the Cre/LoxP recombination system

Highlights

This vector is designed for Cre-mediated conditional gene expression in mammalian cells and animals. Expression of the gene of interest is initially silent, but can be permanently activated by coexpression of Cre recombinase, which will excise a 3x SV40 polyadenylation sequence upstream of the gene of interest. After treatment with Cre, expression of the gene of interest is under the control of the user-selected promoter.

Advantages

Stable gene activation: Treatment with Cre recombinase will permanently remove the 3x SV40 polyadenylation sequence which blocks downstream transcription. This will allow transcription of the gene of interest, driven by the promoter chosen by the user.

Technical simplicity: Delivering plasmid vectors into cells by conventional transfection is technically straightforward, and far easier than virus-based vectors which require the packaging of live virus.

Very large cargo space: Our vector can accommodate ~30 kb of total DNA. The plasmid backbone only occupies about 3 kb, leaving plenty of room to accommodate the user's sequence of interest.

High-level expression: Conventional transfection of plasmids can often result in very high copy numbers in cells (up to several thousand copies per cell). This can lead to very high expression levels of the genes carried on the vector.

Suitability for in vivo applications: While this vector system can be used in tissue culture cells, it is particularly suitable for the generation of transgenic animals for the purpose of Cre-mediated conditional gene expression.

Disadvantages

Non-integration of vector DNA: When used in cell culture, plasmid DNA generally integrates into the host genome at only a very low frequency (one per 102 to 106 cells depending on cell type). Drug resistance or fluorescence markers incorporated into the plasmid can be used to isolate cells stably integrating the plasmid by drug selection or cell sorting after extended culture.

Limited cell type range: The efficiency of plasmid delivery in cell culture can vary greatly from cell type to cell type, and often requires optimization. Primary cells are often harder to transfect than immortalized cell lines, and some cell types are notoriously difficult to transfect.

Non-uniformity of gene delivery: Although a successful transfection can result in very high average copy number of the transfected plasmid vector per cell, this can be highly non-uniform. Some cells can carry many copies while others carry very few or none. This is unlike transduction by virus-based vectors which tends to result in relatively uniform gene delivery into cells.

Key components

Promoter: The promoter that will drive expression of your gene of interest after treatment with Cre recombinase.

LoxP: Recombination site for Cre recombinase. When Cre is present the region flanked by the two LoxP sites will be excised.

3x SV40 pA: Three repeats of the simian virus 40 late polyadenylation signal. This terminates transcription from the upstream promoter, preventing expression of the downstream gene of interest.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

CMV promoter: Human cytomegalovirus immediate early promoter. It drives the ubiquitous expression of the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

BGH pA: Bovine growth hormone polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

PiggyBac Conditional Gene Expression Vector (LoxP-Stop-LoxP)

Overview

This piggyBac gene expression vector system utilizes the LoxP-Stop-LoxP (LSL) cassette to achieve Cre-mediated conditional activation of gene expression in mammalian cells and animals. The LSL cassette comprises a LoxP-flanked (aka floxed) triple repeat of the SV40 polyadenylation sequence. The user-selected promoter is placed upstream of the cassette while the user’s gene of interest is placed downstream of it. In the absence of Cre recombinase, the cassette completely blocks transcription of the gene of interest. When Cre is introduced into cells carrying this vector, the cassette is excised, allowing the user-selected promoter to drive the transcription of the gene of interest.

While this vector system can be used in tissue culture cells, it is particular suitable for the generation of transgenic animals. When a transgenic animal carrying such a vector is crossed to an animal carrying a tissue-specific Cre transgene, the progeny animals carrying both types of transgenes would turn on the gene of interest specifically in cells where the tissue-specific Cre is expressed.

For using this vector system in cell culture, antibiotic or fluorescence based markers can be added to the vector to allow selection or visualization of transfected cells, including the isolation of cells that have permanently integrated the vector in the genome.

This transposon-based, piggyBac vector system contains two vectors, both engineered as E. coli plasmids. One vector, referred to as the helper plasmid, encodes the transposase. The other vector, referred to as the transposon plasmid, contains two terminal repeats (TRs) bracketing the region to be transposed. The user-selected promoter, LSL casette, and the gene of interest are cloned into this region. When the helper and transposon plasmids are both present in target cells, the transposase produced from the helper plasmid recognizes the two TRs on the transposon, and inserts the flanked region including the two TRs into the host genome. Insertion typically occurs at host chromosomal sites that contain the TTAA sequence, which is duplicated on the two flanks of the integrated fragment.

PiggyBac is a class II transposon, meaning that it moves in a cut-and-paste manner, hopping from place to place without leaving copies behind. (In contrast, class I transposons move in a copy-and-paste manner.) Because the helper plasmid is only transiently transfected into host cells, it will get lost over time. With the loss of the helper plasmid, the integration of the transposon in the genome of host cells becomes permanent. If these cells are transfected with the helper plasmid again, the transposon could get excised from the genome of some cells, footprint free.

For further information about this vector system and Cre-mediated recombination, please refer to the papers below.

ReferencesTopic
Mol Cell Biochem. 354:301 (2011)Review of piggyBac
Cell. 122:473 (2005)Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice
EMBO J. 12:2539 (1993)Transcription blocker prevent transcriptional interference
J Biol Chem. 259:1509-14 (1984)Purification and properties of the Cre recombinase protein
Genesis. 26:99-109. (2000)Review of the Cre/LoxP recombination system

Highlights

This piggyBac transposon-based vector is designed for Cre-mediated conditional gene expression in mammalian cells and animals. Expression of the gene of interest is initially silent, but can be permanently activated by coexpression of Cre recombinase, which will excise a 3x SV40 polyadenylation sequence upstream of the gene of interest. After treatment with Cre, expression of the gene of interest is under the control of the user-selected promoter.

Advantages

Stable gene activation: Treatment with Cre recombinase will permanently remove the 3x SV40 poly sequence which inhibits downstream transcription. This will allow transcription of the gene of interest, driven by the promoter chosen by the user.

Permanent integration of vector DNA: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, transfection of mammalian cells with the piggyBac transposon plasmid along with the helper plasmid can deliver genes carried on the transposon permanently into host cells due to the integration of the transposon into the host genome.

Technical simplicity: Delivering plasmid vectors into cells by conventional transfection is technically straightforward, and far easier than virus-based vectors which require the packaging of live virus.

Very large cargo space: Our transposon vectors can accommodate ~30 kb of total DNA. The plasmid backbone and transposon-related sequences only occupies about 3 kb, leaving plenty of room to accommodate the user's sequence of interest.

Disadvantages

Limited cell type range: The delivery of piggyBac vectors into cells relies on transfection. The efficiency of plasmid delivery can vary greatly from cell type to cell type, and often requires optimization. Primary cells are often harder to transfect than immortalized cell lines, and some cell types are notoriously difficult to transfect.

Key components

5' ITR: 5' inverted terminal repeat. When a DNA sequence is flanked by two ITRs, the piggyBac transpose can recognize them, and insert the flanked region including the two ITRs into the host genome.

Promoter: The promoter that will drive expression of your gene of interest after treatment with Cre recombinase.

LoxP: Recombination site for Cre recombinase. When Cre is present the region flanked by the two LoxP sites will be excised.

3x SV40 late pA: Repeats of the simian virus 40 late polyadenylation signal. This terminates transcription, preventing expression of the downstream gene of interest prior to excision with LoxP-flanked region with Cre.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here.

rBG pA: Rabbit β-globin polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

CMV promoter: Human cytomegalovirus immediate early promoter. It drives the ubiquitous expression of the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

BGH pA: Bovine growth hormone polyadenylation. It facilitates transcriptional termination of the upstream ORF.

3' ITR: 3' inverted terminal repeat.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Lentivirus shRNA Knockdown Vector

Overview

The Lentivirus shRNA Knockdown vector system is a highly efficient method for stably knocking down expression of a target gene in a wide variety of mammalian cells. Once the viral genome is reverse transcribed and permanently integrated into the host cell genome, the shRNA is expressed from the human U6 promoter, leading to degradation of target gene mRNA. The permanent nature of knockdown by lentivirus has several major advantages over transient knockdown by synthetic siRNA (see Advantages section below).

By design, our lentiviral vectors lack the genes required for viral packaging and transduction (these genes are instead carried by helper plasmids used during virus packaging). As a result, virus produced from lentiviral vectors has the important safety feature of being replication incompetent (meaning that they can transduce target cells but cannot replicate in them).

For general information about lentiviral vectors, see our Guide to Vector Systems section on Lentiviral Expression Vectors, and for further information about Lentiviral shRNA Knockdown vectors, please refer to the paper below.

ReferencesTopic
RNA. 9:493-501 (2003)Development of lentiviral shRNA vectors

Highlights

Our Lentivirus shRNA Knockdown vectors are derived from the third-generation lentiviral vector system. This system is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient viral transduction of a wide range of cells, and efficient vector integration into the host genome. The human U6 promoter drives high-level, constitutive transcription of the shRNA in mammalian cells, while our optimized shRNA stem-loop sequences mediate efficient shRNA processing and target gene knockdown.

Advantages

Permanent knockdown: Lentiviral integration into the host cell genome is an irreversible process, and the U6 promoter directs constitutive expression of the shRNA. For these reasons, the knockdown of the target gene is typically stable and permanent. This can be an important advantage for several experimental goals. It allows long-term analysis of the knockdown phenotype in cell culture or in vivo. It facilitates the isolation of clones having different levels of knockdown and/or different phenotype. When the knockdown vector carries a fluorescence marker such as EGFP, it also allows cells with different amounts of lentiviral integration (and hence potentially different levels of knockdown) to be isolated by flow sorting cells with different fluorescence intensity.

High viral titer: Our vector can be packaged into high-titer virus (>108 TU/ml when virus is obtained through our virus packaging service). At this viral titer, transduction efficiency for cultured mammalian cells can approach 100% when an adequate amount of viral supernatant is used.

Very broad tropism: Our packaging system adds the VSV-G envelop protein to the viral surface. This protein has broad tropism. As a result, cells from all commonly used mammalian species (and even some non-mammalian species) can be transduced. Furthermore, almost any mammalian cell type can be transduced (e.g. dividing cells and non-dividing cells, primary cells and established cell lines, stem cells and differentiated cells, adherent cells and non-adherent cells). Neurons, which are often impervious to conventional transfection, can be readily transduced by our lentiviral vector. Lentiviral vectors packaged with our system have broader tropism than adenoviral vectors (which have low transduction efficiency for some cell types) or MMLV retroviral vectors (which have difficulty transducing non-dividing cells).

Relative uniformity of vector delivery: Generally, viral transduction can deliver vectors into cells in a relatively uniform manner. In contrast, conventional transfection of plasmid vectors can be highly non-uniform, with some cells receiving a lot of copies while other cells receiving few copies or none.

Effectiveness in vitro and in vivo: Lentiviral vector systems can be used effectively in cultured cells and in live animals.

Safety: The safety of our vector is ensured by two features. One is the partition of genes required for viral packaging and transduction into several helper plasmids; the other is self-inactivation of the promoter activity in the 5' LTR upon vector integration. As a result, it is essentially impossible for replication competent virus to emerge during packaging and transduction. The health risk of working with our vector is therefore minimal.

Disadvantages

Technical complexity: The use of lentiviral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technical demanding and time consuming relative to conventional plasmid transfection.

Permanent knockdown: Lentiviral integration into the host cell genome is an irreversible process, and the U6 promoter directs constitutive expression of the shRNA. For these reasons, the target gene cannot easily be reactivated once it is knocked down by the Lentivirus shRNA Knockdown vector. This can be an advantage or a disadvantage, depending on experimental goals.

Key components

RSV promoter: Rous sarcoma virus promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into live virus.

Δ5' LTR: A deleted version of the HIV-1 5' long terminal repeat. In wildtype lentivirus, 5' LTR and 3' LTR are essentially identical in sequence. They reside on two ends of the viral genome and point in the same direction. Upon viral integration, the 3' LTR sequence is copied onto the 5' LTR. The LTRs carry both promoter and polyadenylation function, such that in wildtype virus, the 5' LTR acts as a promoter to drive the transcription of the viral genome, while the 3' LTR acts as a polyadenylation signal to terminate the upstream transcript. On our vector, Δ5' LTR is deleted for a region that is required for the LTR's promoter activity normally facilitated by the viral transcription factor Tat. This does not affect the production of viral RNA during packaging because the promoter function is supplemented by the RSV promoter engineered upstream of Δ5' LTR.

Ψ: HIV-1 packaging signal required for the packaging of viral RNA into virus.

RRE: HIV-1 Rev response element. It allows the nuclear export of viral RNA by the viral Rev protein during viral packaging.

cPPT: HIV-1 Central polypurine tract. It creates a "DNA flap" that increases nuclear importation of the viral genome during target cell infection. This improves vector integration into the host genome, resulting in higher transduction efficiency.

U6 Promoter: Drives expression of the shRNA. This is the promoter of the human U6 snRNA gene, an RNA polymerase III promoter which efficiently expresses short RNAs.

Sense, Antisense: These sequences are derived from your target sequences, and are transcribed to form the stem portion of the “hairpin” structure of the shRNA.

Loop: This optimized sequence is transcribed to form the loop portion of the shRNA “hairpin” structure.

Terminator: Terminates transcription of the shRNA.

hPGK promoter: Human phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression of the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances viral RNA stability in packaging cells, leading to higher titer of packaged virus.

ΔU3/3' LTR: A truncated version of the HIV-1 3' long terminal repeat that deletes the U3 region. This leads to the self-inactivation of the promoter activity of the 5' LTR upon viral vector integration into the host genome (due to the fact that 3' LTR is copied onto 5' LTR during viral integration). The polyadenylation signal contained in ΔU3/3' LTR serves to terminates all upstream transcripts produced both during viral packaging and after viral integration into the host genome.

SV40 early pA: Simian virus 40 early polyadenylation signal. It further facilitates transcriptional termination after the 3' LTR during viral RNA transcription during packaging. This elevates the level of functional viral RNA in packaging cells, thus improving viral titer.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Adenovirus shRNA Knockdown Vector

Overview

The Adenovirus shRNA Knockdown vector system is an efficient method for stably knocking down expression of a target gene in many (but not all) mammalian cell types. The vector remains as episomal DNA in cells, without integrating into the host genome, and is often the preferred vector system for in vivo use.

Adenoviral vectors are derived from adenovirus, a double-stranded linear DNA virus which causes the common cold.

An adenoviral vector is first constructed as a plasmid in E. coli, and is then transfected into packaging cells, where the region of the vector between the two inverted terminal repeats (ITRs) is packaged into live virus.

After the viral genome is delivered into target cells, it enters the nucleus and remains as episomal DNA. The shRNA is stably expressed from a human U6 promoter, leading to degradation of target gene mRNA within infected cells.

By design, our adenoviral vectors lack the E1A, E1B and E3 genes (delta E1 + delta E3). The first two are required for the production of live virus (these two genes are engineered into the genome of packaging cells). As a result, virus produced from the vectors have the important safety feature of being replication incompetent (meaning that they can transduce target cells but cannot replicate in them).

For further information about this vector system, please refer to the papers below.

ReferencesTopic
Proc Natl Acad Sci U S A. 91:8802 (1994)The 2nd generation adenovirus vectors
J Gen Virol. 36:59 (1977)A packaging cell line for adenovirus vectors
J Virol. 79:5437 (2005)Replication-competent adenovirus (RCA) formation in 293 Cells
Gene Ther. 3:75 (1996)A cell line for testing RCA

Highlights

Our vector is derived from the adenovirus serotype 5 (Ad5). It is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient transduction of host cells, and effective gene knockdown. The human U6 promoter drives high-level, constitutive transcription of the shRNA in mammalian cells, while our optimized shRNA stem-loop sequences mediate efficient shRNA processing and target gene knockdown.

Advantages

Low risk of host genome disruption: Upon transduction into host cells, adenoviral vectors remain as episomal DNA in the nucleus. The lack of integration into the host genome can be a desirable feature for in vivo human applications, as it reduces the risk of host genome disruption that might lead to cancer.

Very high viral titer: After our adenoviral vector is transfected into packaging cells to produce live virus, the virus can be further amplified to very high titer by re-infecting packaging cells. This is unlike lentivirus, MMLV retrovirus, or AAV, which cannot be amplified by re-infection. When adenovirus is obtained through our virus packaging service, titer can reach >1010 plaque-forming unit per ml (PFU/ml).

Broad tropism: Cells from commonly used mammalian species such as human, mouse and rat can be transduced with our adenoviral vectors. But some cell types have proven difficult to transduce (see disadvantages below).

Effectiveness in vitro and in vivo: Our vector is often used to transduce cells in live animals, but it can also be used effectively in vitro.

Safety: The safety of our vector is ensured by the fact that it lacks genes essential for virus production (these genes are engineered into the genome of packaging cells). Virus made from our vector is therefore replication incompetent except when it is used to transduce packaging cells.

Disadvantages

Non-integration of vector DNA: The adenoviral genome does not integrate into the genome of transduced cells. Rather, it exists as episomal DNA, which can be lost over time, especially in dividing cells.

Difficulty transducing certain cell types: While our adenoviral vectors can transduce many different cell types including non-dividing cells, it is inefficient against certain cell types such as endothelia, smooth muscle, differentiated airway epithelia, peripheral blood cells, neurons, and hematopoietic cells.

Strong immunogenicity: Live virus from adenoviral vectors can elicit strong immune response in animals, thus limiting certain in vivo applications.

Technical complexity: The use of viral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technical demanding and time consuming relative to conventional plasmid transfection. These demands can be alleviated by choosing our virus packaging services when ordering your vector.

Key components

5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.

Ψ: Adenovirus packaging signal required for the packaging of viral DNA into virus.

U6 Promoter: Drives expression of the shRNA. This is the promoter of the human U6 snRNA gene, an RNA polymerase III promoter which efficiently expresses short RNAs.

Sense, Antisense: These sequences are derived from your target sequences, and are transcribed to form the stem portion of the “hairpin” structure of the shRNA.

Loop: This optimized sequence is transcribed to form the loop portion of the shRNA “hairpin” structure.

Terminator: Terminates transcription of the shRNA.

hPGK promoter: Human phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression of the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

ΔAd5: Portion of Ad5 genome between the two ITRs minus the E1A, E1B and E3 regions.

3' ITR: 3' inverted terminal repeat. See description for 5’ ITR.

pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin exist in medium copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

PacI: PacI restriction site (PacI is a rare cutter that cuts at TTAATTAA). The two PacI restriction sites on the vector can be used to linearize the vector and remove the vector backbone from the viral sequence, which is necessary for efficient packaging.

Adeno-associated virus shRNA Knockdown Vector

Overview

Our Adeno-associated virus (AAV) shRNA Knockdown vector system is an efficient method for stably knocking down expression of a target gene in a wide variety of mammalian cell types, in vitro or in vivo. Due to the low immunogenicity and cytotoxicity of AAV, this is the ideal shRNA vector for many animal studies.

An AAV vector is first constructed as a plasmid in E. coli. It is then transfected into packaging cells along with helper plasmids, where the region of the vector between the two inverted terminal repeats (ITRs) is packaged into live virus. After the viral genome is delivered into the target cells, it enters the nucleus and remains as episomal DNA. The shRNA is stably expressed from a human U6 promoter, leading to degradation of target gene mRNA within infected cells.

A major practical advantage of AAV is that in most cases AAV can be handled in biosafety level 1 (BSL1) facilities. This is due to AAV being inherently replication-deficient, producing little or no inflammation, and causing no known human disease.

Many strains of AAV have been identified in nature. They are divided into different serotypes based on different antigenicity of the capsid protein on the viral surface. Different serotypes can render the virus with different tissue tropism (i.e. tissue specificity of infection). When our AAV vectors are packaged into virus, different serotypes can be conferred to the virus by using different capsid proteins for the packaging. The table below lists different AAV serotypes and their tissue tropism.

SerotypeTissue tropism
AAV1SM, CNS, lung, retina, pancreas, heart, liver
AAV2SM, CNS, liver, kidney, retina
AAV3SM
AAV4CNS, retina, lung, kidney
AAV5SM, CNS, lung, retina
AAV6SM, heart, lung, adipose, liver
AAV7SM, retina, CNS, liver
AAV8SM, CNS, retina, liver, pancreas, heart, kidney, adipose
AAV9SM, lung, liver, heart, pancreas, CNS, retina, testes, kidney
AAVrh10SM, lung, liver, heart, pancreas, CNS, retina, kidney

For further information about this vector system, please refer to the papers below.

ReferencesTopic
Methods in Enzy. 507:229-54 (2012)Review of AAV virology and uses
Curr Opin Pharmacol. 24:59-67 (2015)AAV use in gene therapy, and serotype differences

Highlights

Our AAV vector system is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient transduction of host cells, and high-level transgene expression. This viral vector can be packaged into virus using all known capsid serotypes, is capable of very high transduction efficiency, and presents low safety risk.

Advantages

Safety: AAV is the safest viral vector system available. AAV is inherently replication-deficient, and is not known to cause any human diseases.

Low risk of host genome disruption: Upon transduction into host cells, AAV vectors remain as episomal DNA in the nucleus. The lack of integration into the host genome can be a desirable feature for in vivo human applications, as it reduces the risk of host genome disruption that might lead to cancer.

Stable knockdown: AAV generally remains in a stable episomal state within the nucleus, and the U6 promoter directs constitutive expression of the shRNA. For these reasons, the knockdown of the target gene is typically stable and long-lasting.

High viral titer: Our AAV vector can be packaged into high titer virus. When AAV virus is obtained through our virus packaging service, titer can reach >1011 genome copy per ml (GC/ml).

Broad tropism: A wide range of cell and tissue types from commonly used mammalian species such as human, mouse and rat can be readily transduced with our AAV vector when it is packaged into the appropriate serotype. But some cell types may be difficult to transduce, depending on the serotype used (see disadvantages below).

Effectiveness in vitro and in vivo: Our vector is often used to transduce cells in live animals, but it can also be used effectively in vitro.

Disadvantages

Difficulty transducing certain cell types: Our AAV vector system can transduce many different cell types including non-dividing cells when packaged into the appropriate serotype. However, different AAV serotypes have tropism for different cell types, and certain cell types may be hard to transduce by any serotype.

Stable knockdown: AAV generally remains in a stable episomal state within the nucleus, and the U6 promoter directs constitutive expression of the shRNA. For these reasons, the target gene cannot easily be reactivated once it is knocked down by the AAV shRNA Knockdown vector. This can be an advantage or a disadvantage, depending on experimental goals.

Technical complexity: The use of viral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technical demanding and time consuming relative to conventional plasmid transfection. These demands can be alleviated by choosing our virus packaging services when ordering your vector.

Key components

5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.

U6 Promoter: Drives expression of the shRNA. This is the promoter of the human U6 snRNA gene, an RNA polymerase III promoter which efficiently expresses short RNAs.

Sense, Antisense: These sequences are derived from your target sequences, and are transcribed to form the stem portion of the “hairpin” structure of the shRNA.

Loop: This optimized sequence is transcribed to form the loop portion of the shRNA “hairpin” structure.

Terminator: Terminates transcription of the shRNA.

hPGK promoter: Human phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression of the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

3' ITR: 3' inverted terminal repeat. See description for 5’ ITR.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

PiggyBac shRNA Knockdown Vector

Overview

Our piggyBac shRNA Knockdown vector system is a simple and efficient method for stably knocking down expression of a target gene in a wide variety of cell types. This transposon-based system utilizes plasmid transfection (rather than viral transduction) to permanently integrate an shRNA expression cassette into the host cell genome. The shRNA is expressed from the human U6 promoter, leading to degradation of target gene mRNA. The permanent nature of knockdown by the piggyBac system has several major advantages over transient knockdown by synthetic siRNA (see Advantages section below).

The piggyBac shRNA knockdown system contains two vectors, both engineered as E. coli plasmids. One vector, referred to as the helper plasmid, encodes the transposase. The other vector, referred to as the transposon plasmid, contains two terminal repeats (TRs) bracketing the region to be transposed, which includes the shRNA-expression cassette.

When the helper and transposon plasmids are co-transfected into target cells, the transposase produced from the helper plasmid recognizes the two TRs on the transposon, and inserts the flanked region including the two TRs into the host genome. PiggyBac is a class II transposon, meaning that it moves in a cut-and-paste manner, hopping from place to place without leaving copies behind. (In contrast, class I transposons move in a copy-and-paste manner.) Because the helper plasmid is only transiently transfected into host cells, it will get lost over time. With the loss of the helper plasmid, the integration of the shRNA-expressing transposon in the genome of host cells becomes permanent. If these cells are transfected with the helper plasmid again, the transposon could get excised from the genome of some cells, footprint free.

For further information about this vector system, please refer to the papers below.

ReferencesTopic
Mol Cell Biochem. 354:301 (2011)Review
Cell. 122:473 (2005)Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice

Highlights

Our piggyBac transposon vector along with the helper plasmid are optimized for high copy number replication in E. coli, and efficient transfection into a wide range of target cells. The human U6 promoter drives high-level, constitutive transcription of the shRNA in mammalian cells, while our optimized shRNA stem-loop sequences mediate efficient shRNA processing and target gene knockdown.

Advantages

Permanent integration and knockdown: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, transfection of mammalian cells with the piggyBac transposon plasmid along with the helper plasmid can deliver DNA sequences carried on the transposon permanently into host cells due to the integration of the transposon into the host genome. Additionally, the U6 promoter directs constitutive expression of the shRNA. For these reasons, the knockdown of the target gene is stable and permanent. This can be an important advantage for many experimental goals. It allows long-term analysis of the knockdown phenotype. It facilitates the isolation of clones having different levels of knockdown and/or different phenotypes. When the knockdown vector carries a fluorescence marker such as EGFP, it also allows cells with different amounts of transposon integration (and hence potentially different levels of knockdown) to be isolated by flow sorting cells with different fluorescence intensity.

Reversibility: If cells carrying a piggyBac shRNA transposon are transfected with the helper plasmid again, the transposon may be excised from the genome of some cells, footprint free, eliminating expression of the shRNA from those cells. However, this will occur in only a subset of cells.

Technical simplicity Delivering plasmid vectors into cells by conventional transfection is technically straightforward, and far easier than virus-based vectors which require the packaging of live virus.

Safety: Conventional transfection does not have the safety concerns which are often associated with viral vectors.

Disadvantages

Limited cell type range: The delivery of piggyBac vectors into cells relies on transfection. The efficiency of transfection can vary greatly from cell type to cell type. Non-dividing cells are often more difficult to transfect than dividing cells, and primary cells are often harder to transfect than immortalized cell lines. Some important cell types, such as neurons and pancreatic β cells, are notoriously difficult to transfect. Additionally, plasmid transfection is largely limited to in vitro applications and rarely used in vivo. These issues limit the use of the piggyBac system.

Key components

5' ITR: 5' inverted terminal repeat. When a DNA sequence is flanked by two ITRs, the piggyBac transpose can recognize them, and insert the flanked region including the two ITRs into the host genome.

U6 Promoter: Drives expression of the shRNA. This is the promoter of the human U6 snRNA gene, an RNA polymerase III promoter which efficiently expresses short RNAs.

Sense, Antisense: These sequences are derived from your target sequences, and are transcribed to form the stem portion of the “hairpin” structure of the shRNA.

Loop: This optimized sequence is transcribed to form the loop portion of the shRNA “hairpin” structure.

Terminator: Terminates transcription of the shRNA.

hPGK promoter: Human phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression of the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

rBG pA: Rabbit β-globin polyadenylation signal. It facilitates transcriptional termination of the upstream marker gene.

3' ITR: 3' inverted terminal repeat. See description for 5’ ITR.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Regular Plasmid CRISPR Vector (Single gRNA)

Overview

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) nuclease expression vectors are among several types of emerging genome editing tools that can quickly and efficiently create mutations at target sites of a genome (the other two popular ones being ZFN and TALEN). These plasmid vectors encode a sequence-specific RNA-guided DNA nuclease (or nickase) enzyme, which can be used to edit the DNA sequence of specific user-defined sites in the genome.

Cas9 is a member of a class of RNA-guided DNA nucleases which are part of a natural prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and bacteriophage. Within the cell, the Cas9 enzyme forms a complex with a guide RNA (gRNA), which provides targeting specificity through direct interaction with homologous 18-22nt target sequences in the genome. Hybridization of the gRNA to the target site localizes Cas9, which then cuts the target site in the genome. For simplicity, our CRISPR/Cas9 Vectors are designed to efficiently express both the Cas9 nuclease (or nickase) enzyme and the guide RNA (gRNA) from a single vector.

Two variants of Cas9 enzyme are available in our CRISPR/Cas9 Expression Vectors. The standard humanized Cas9 (hCas9) variant efficiently generates double-strand breaks (DSBs) at target sites, while the “nickase” mutant form (hCas9-D10A) generates only single-stranded cuts in DNA. If hCas9-D10A nickase is used in conjunction with two gRNAs targeting the two opposite strands of a single target site, then the nickase enzyme will generate single strand cuts on both strands, resulting in DSBs at the target site. This approach generally reduces off-target effects of CRISPR/Cas9 expression because targeting by both gRNAs is necessary for DSBs to be generated.

Cellular repair of DSBs by the nonhomologous end-joining pathway (NHEJ) usually results in small deletions, or more rarely insertions and base substitutions. When these mutations disrupt a protein-coding region (e.g. a deletion causing a frameshift), the result is a functional gene knockout. Alternatively, and less efficiently, DSBs can be repaired by homology-directed repair (HDR), using exogenous donor DNA template, which is co-introduced with the CRISPR/Cas9 vector. This can result in replacement of the target genomic DNA sequence with template sequence, generating small targeted base changes, such as point mutations. Nicked genomic DNA also frequently undergoes homology-directed repair (HDR), and if exogenous template DNA is introduced into the cell along with a targeted hCas9-D10A nickase, then small base changes can be generated.

Most DNA sequence can be effectively targeted using the CRISPR/Cas9 system. However, there is a strict requirement for an NGG (sometimes NAG) sequence, known as protospacer adjacent motif (PAM), which is located on the immediate 3’ end of the gRNA recognition sequence within the target DNA.

For further information about this vector system, please refer to the papers below.

References Topic
Science 339:819-23 (2013) Description of genome editing using the CRISPR/Cas9 system
Cell. 154:1380–9 (2013) Use of Cas9 D10A double nicking for increased specificity
Nat. Biotech. 31:827–832 (2013) Specificity of RNA-guided Cas9 nucleases

Highlights

Our Simple CRISPR/Cas9 Expression Vectors are designed for quickly and efficiently creating small deletions at target sites in a cellular genome. To introduce mutations at a specific target site, a gRNA is chosen which matches the target DNA sequence. Nuclease variants can be chosen to introduce DSBs (with hCas9) or single-strand cuts (with hCas9-D10A nickase).

Advantages

Transient expression: Transfection of the CRISPR/Cas9 plasmid vector results in strong transient expression of the Cas9 protein and gRNA within the target cells. Without drug selection, the plasmid will be lost over time eliminating the Cas9 and gRNA from the target cells after genome editing has taken place.

Simplicity: The simple homology relationship between the gRNA and the target makes the CRISPR/Cas9 system conceptually simple and easy to design.

Disadvantages

Lower specificity: Some off-target activity has been reported for the CRISPR/Cas9 system, and in general the TALEN system has lower off-target activity than CRISPR/Cas9. However, off-target effects can be significantly mitigated by using hCas9-D10A nickase in conjunction with two gRNAs (as described above).

PAM requirement: CRISPR/Cas9 target sites must contain an NGG sequence, known as PAM, located on the immediate 3’ end of the gRNA recognition sequence.

Key components

U6 Promoter: This drives high level expression of the gRNA.

Guide sequence: Specifies the target sequence of the Cas9 nuclease.

gRNA scaffold: Structural portion of the gRNA to allow complexing with Cas9.

Terminator: Terminates transcription of the gRNA.

CBh promoter: Chicken beta-actin promoter. Drives expression of Cas9 nuclease.

Nuclease: Cas9 nuclease variant chosen by user.

BGH pA: Bovine growth hormone polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Lentivirus gRNA Expression Vector (Single gRNA)

Overview

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) nuclease expression vectors are among several types of emerging genome editing tools that can quickly and efficiently create mutations at target sites of a genome (the other two popular ones being ZFN and TALEN).

The lentivirus single gRNA expression vector system expresses a single guide RNA (gRNA) within transduced cells, which when coexpressed with the Cas9 nuclease, can lead to cleavage of the DNA sequence at a specific user-defined site in the genome. This vector is designed to be used together with another vector encoding the Cas9 nuclease.

Cas9 is a member of a class of RNA-guided DNA nucleases which are part of a natural prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and bacteriophage. Within the cell, the Cas9 enzyme forms a complex with a guide RNA (gRNA), which provides targeting specificity through direct interaction with homologous 18-22nt target sequences in the genome. Hybridization of the gRNA to the target site localizes Cas9, which then cuts the target site in the genome.

There are two commonly used variants of the Cas9 enzyme. The standard humanized Cas9 (hCas9) variant efficiently generates double-strand breaks (DSBs) at target sites. “Nickase” mutant form (Cas9_D10A) generate only single-stranded cuts in DNA, and if Cas9_D10A nickase is used in conjunction with two gRNAs targeting the two opposite strands of a single target site, then the nickase enzyme will generate single strand cuts on both strands, resulting in DSBs at the target site. This approach generally reduces off-target effects of CRISPR/Cas9 expression because targeting by both gRNAs is necessary for DSBs to be generated.

Design your Cas9 (or Cas9 variant) expression lentiviral vectors >>

Cellular repair of DSBs by the nonhomologous end-joining pathway (NHEJ) usually results in small deletions, or more rarely insertions and base substitutions. When these mutations disrupt a protein-coding region (e.g. a deletion causing a frameshift), the result is a functional gene knockout. Alternatively, and less efficiently, DSBs can be repaired by homology-directed repair (HDR), using exogenous donor DNA template, which is co-introduced with the CRISPR/Cas9 vector. This can result in replacement of the target genomic DNA sequence with template sequence, generating small targeted base changes, such as point mutations. Nicked genomic DNA also frequently undergoes homology-directed repair (HDR), and if exogenous template DNA is introduced into the cell along with a targeted Cas9_D10A nickase, then small base changes can be generated.

Design your homologous recombination donor vectors >>

Most DNA sequence can be effectively targeted using the CRISPR/Cas9 system. However, there is a strict requirement for an NGG (sometimes NAG) sequence, known as protospacer adjacent motif (PAM), which is located on the immediate 3’ end of the gRNA recognition sequence within the target DNA.

For further information on this vector system, please refer to the papers listed below.

References Topic
Science 339:819-23 (2013) Description of genome editing using the CRISPR/Cas9 system
Cell. 154:1380–9 (2013) Use of Cas9 D10A double nicking for increased specificity
Nat. Biotech. 31:827–832 (2013) Specificity of RNA-guided Cas9 nucleases
J Virol. 72:8463 (1998) The 3rd generation lentivirus vectors
J Virol. 72:9873 (1998) Self-inactivating lentivirus vectors
Science. 272:263 (1996) Transduction of non-dividing cells by lentivirus vectors
Curr Gene Ther. 5:387 (2005) Tropism of lentiviral vectors
J Virol. 77:4685 (2003) Impact of cPPT on lentivirus vector transduction
Nat Protoc. 1:241 (2006) Production and purification of lentiviral vectors

Highlights

Our Lentiviral CRISPR/Cas9 Expression Vectors are designed for quickly and efficiently expressing gRNAs for directing Cas9 nucleases to create small deletions at target sites in a cellular genome. To introduce mutations at a specific target site, a gRNA is chosen which matches the target DNA sequence. The nuclease must be expressed from another vector.

Advantages

Permanent integration of vector DNA: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, lentiviral transduction permanently inserts the user-designed DNA segments into host cells due to the integration of the viral vector into the host genome.

Simplicity: The simple homology relationship between the gRNA and the target makes the CRISPR/Cas9 system conceptually simple and easy to design.

High viral titer: Our lentiviral vector can be packaged into high titer virus. When lentivirus is obtained through our virus packaging service, the titer can reach >108 transducing unit per ml (TU/ml). At this titer, transduction efficiency for cultured mammalian cells can approach 100% when an adequate amount of viral is used.

Very broad tropism: Our packaging system adds the VSV-G envelop protein to the viral surface. This protein has broad tropism. As a result, cells from all commonly used mammalian species (and even some non-mammalian species) can be transduced. Furthermore, almost any mammalian cell type can be transduced (e.g. dividing cells and non-dividing cells, primary cells and established cell lines, stem cells and differentiated cells, adherent cells and non-adherent cells). Neurons, which are often impervious to conventional transfection, can be readily transduced by our lentiviral vector. Lentiviral vectors packaged with our system have broader tropism than adenoviral vectors (which have low transduction efficiency for some cell types) or MMLV retroviral vectors (which have difficulty transducing non-dividing cells).

Ability to concentrate viral particles: Our packaging system adds the VSV-G envelop protein to the viral surface. The structural robustness of this protein allows viral particles to be concentrated by high-speed centrifugation.

Relative uniformity of gene delivery: Generally, viral transduction can deliver vectors into cells in a relatively uniform manner. In contrast, conventional transfection of plasmid vectors can be highly non-uniform, with some cells receiving a lot of copies while other cells receiving few copies or none. Effectiveness in vitro and in vivo: While our vector is mostly used for in vitro transduction of cultured cells, it can also be used to transduce cells in live animals.

Effectiveness in vitro and in vivo: While our vector is mostly used for in vitro transduction of cultured cells, it can also be used to transduce cells in live animals.

Safety: The safety of our vector is ensured by two features. One is the partition of genes required for viral packaging and transduction into several helper plasmids; the other is self-inactivation of the promoter activity in the 5' LTR upon vector integration. As a result, it is essentially impossible for replication competent virus to emerge during packaging and transduction. The health risk of working with our vector is therefore minimal.

Disadvantages

Technical complexity: The use of lentiviral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technical demanding and time consuming relative to conventional plasmid transfection.

Requires separate nuclease expression vector: Our lentiviral single gRNA vectors drive expression of a gRNA but not a Cas9 nuclease. A separate vector must be used along with this vector system in order to perform genome editing.

Lower specificity: Some off-target activity has been reported for the CRISPR/Cas9 system, and in general the TALEN system has lower off-target activity than CRISPR/Cas9. Off-target effects can be mitigated by using Cas9_D10A nickase in conjunction with two gRNAs (as described above), however this vector system includes only a single gRNA.

PAM requirement: CRISPR/Cas9 target sites must contain an NGG sequence, known as PAM, located on the immediate 3’ end of the gRNA recognition sequence.

Key components

RSV promoter: Rous sarcoma virus promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into live virus.

Δ5' LTR: A deleted version of the HIV-1 5' long terminal repeat. In wildtype lentivirus, 5' LTR and 3' LTR are essentially identical in sequence. They reside on two ends of the viral genome and point in the same direction. Upon viral integration, the 3' LTR sequence is copied onto the 5' LTR. The LTRs carry both promoter and polyadenylation function, such that in wildtype virus, the 5' LTR acts as a promoter to drive the transcription of the viral genome, while the 3' LTR acts as a polyadenylation signal to terminate the upstream transcript. On our vector, Δ5' LTR is deleted for a region that is required for the LTR's promoter activity normally facilitated by the viral transcription factor Tat. This does not affect the production of viral RNA during packaging because the promoter function is supplemented by the RSV promoter engineered upstream of Δ5' LTR.

Ψ: HIV-1 packaging signal required for the packaging of viral RNA into virus.

RRE: HIV-1 Rev response element. It allows the nuclear export of viral RNA by the viral Rev protein during viral packaging.

cPPT:  HIV-1 Central polypurine tract. It creates a "DNA flap" that increases nuclear importation of the viral genome during target cell infection. This improves vector integration into the host genome, resulting in higher transduction efficiency.

U6 Promoter: This drives high level expression of the gRNA.

gRNA: Allow in vitro transcription for RNA preparation. Scaffold gRNA sequence is included.

Terminator: Terminates transcription of the gRNA.

hPGK promoter: Human phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances transcriptional termination in the 3' LTR during viral RNA transcription, which leads to higher levels of functional viral RNA in packaging cells and hence greater viral titer. It also enhances transcriptional termination during the transcription of the user's gene of interest on the vector, leading to their higher expression levels.

ΔU3/3' LTR: A truncated version of the HIV-1 3' long terminal repeat that deletes the U3 region. This leads to the self-inactivation of the promoter activity of the 5' LTR upon viral vector integration into the host genome (due to the fact that 3' LTR is copied onto 5' LTR during viral integration). The polyadenylation signal contained in ΔU3/3' LTR serves to terminates all upstream transcripts produced both during viral packaging and after viral integration into the host genome.

SV40 early pA: Simian virus 40 early polyadenylation signal. It further facilitates transcriptional termination after the 3' LTR during viral RNA transcription during packaging. This elevates the level of functional viral RNA in packaging cells, thus improving viral titer.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Regular Plasmid LacZ Reporter Vector (for in vivo enhancer testing)

Overview

This vector system is designed for efficient analysis of mammalian enhancers in mouse models. Typically, a putative enhancer of interest is cloned into this vector, and the resulting construct is used to make transgenic mice. Expression of the LacZ reporter in transgenic embryos or adult mice can then be used as a readout of enhancer activity.

This vector system is useful for identifying enhancer elements, determining tissue-specificity of enhancers, comparing enhancer variants, lineage-tracing, and many other applications.

For further information about this vector system, please refer to the papers below.

ReferencesTopic
Nature. 444:499-502 (2006)Use of the vector systemtocarry out genome-wide testing of putative enhancers in the mouse
Development. 105:707-714 (1989)Cloning of the Hsp68 minimal promoter

Highlights

Our vector is based on a regular plasmid system. The putative enhancer to be tested is placed immediately upstream of the Hsp68 minimal promoter (Hsp68_mini),which controls the expression of the downstream LacZ reporter. An active enhancer would stimulate the minimal promoter, driving LacZ expression. In the absence of enhancer activity, Hsp68_mini has very weak basal activity, and therefore produces little or no LacZ expression. LacZ is used as the reporter because colorimetric staining of LacZ by X-gal in whole-mount embryos or tissue sections allows highly sensitive detection of enhancer activity in situ.

Advantages

Easy generation of transgenic animals: The construct can be readily used to make transgenic embryos or live mice with high efficiency by conventional pronuclear injection.

Simple and sensitive readout: When using LacZ as the reporter, X-gal staining produces a vivid blue product that is readily detectible even at low expression levels, resulting in very sensitive readout of enhancer activity.

Disadvantages

Random integration into the host genome: When the vector is used to make transgenic mice by pronuclear injection, one or more copies of the vector can integrate randomly in the host genome. Neighboring genomic sequence at the integration site, coupled with copy number variation and varying degrees of chewing back of the integrated fragment, could influence the level and specificity of reporter gene expression. To overcome this, multiple transgenic lines are generally needed for a given construct, so as to identify the common expression pattern shared among the multiple lines, which is likely to be the true pattern rendered by the enhancer.

Key components

Enhancer: Your enhancer of interest is placed here.

Hsp68 minimal promoter: The minimal promoter sequence from mouse Hsp68 (heat shock protein 68kDa). This will drive transcription of the reporter if an enhancer element is present to activate it. In the absence of such enhancer activity, the minimal promoter will be almost completely inactive.

LacZ: The beta-galactosidase reporter gene. The encoded enzyme converts the colorless and soluble X-gal to an intensely blue insoluble product that stains the cells in which LacZ is expressed.

SV40 early pA: Simian virus 40 early polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

pET Bacterial Recombinant Protein Vector

Overview

The pET vector system is a powerful and widely used system for expressing recombinant proteins in E. coli. The gene of interest is cloned into the pET vector under the control of the strong bacteriophage T7 transcription and translation regulatory system. Activation of expression is achieved by providing T7 RNA polymerase within the cell. When the system is fully induced, nearly all of the cell’s resources are devoted to expressing the gene of interest. With just a few hours of induction, the recombinant protein could comprise nearly half of the cell’s total protein.

For further information about this vector system, please refer to the papers below.

ReferencesTopic
Methods Enzymol. 185:60-89 (1990)Use of T7 RNA polymerase to direct expression of cloned genes.
J Mol Biol. 219:45-59 (1993)Development of the T7lac promoter system.

Highlights

The gene of interest is initially cloned into the pET vector in a bacteria host that lacks the T7 RNA polymerase gene. This eliminates plasmid instability due to expression of proteins of interest that may be harmful to host cells. Afterwards, expression of the gene of interest can be initiated in two possible ways. The host cells can be infected with phage carrying the T7 RNA polymerase gene (e.g. λCE6 phage), or more commonly, the pET plasmid can be transferred into a bacteria host strain whose genome has been engineered to carry the T7 RNA polymerase gene under the control of theLacUV5 promoter. Expression of the T7 polymerase is induced by the addition of the lactose analog IPTG to the bacterial culture.

The pET vector exists as a low copy number plasmid in host E. coli, which reduces leaky expression before induction. The vector utilizes the T7lac promoter system for strong and tightly controlled gene expression. In this system, there is a T7 promoter that can be acted upon by T7 RNA polymerase to drive high-level expression of the gene of interest. Additionally, there is a lac operator (LacO) sequence just downstream of the T7 promoter that can be acted upon by the lac repressor (LacI) protein to block transcription of the T7 promoter. The plasmid also carries the natural promoter and coding sequence for LacI. The LacI protein acts at the LacUV5 promoter in the host cells to repress expression of the T7 RNA polymerase gene by the host polymerase, and also functions at the T7lac promoter on the pET vector to block transcription of the gene of interest by any T7 RNA polymerase that may be made due to leaky expression. Addition of IPTG blocks the inhibitory action of LacI, thereby inducing expression of T7 RNA polymerase and also removing LacI inhibition of the gene of interest.

Although the pET expression system is designed for high-level recombinant protein expression, the expression level can be reduced by decreasing the amount of IPTG supplied to host cells. This can be advantageous when expressing proteins with limited solubility. Additionally, the system is able to maintain the gene of interest in a transcriptionally silent state when T7 RNA polymerase is not present.

All custom pET vectors will be supplied in an E. coli strain designed to maximize plasmid integrity and lacking the T7 RNA polymerase gene (such as Stbl3). For recombinant protein production, we recommend transferring the vector to BL21(DE3) or HMS174(DE3) host bacteria strains, which carry chromosomal copies of the T7 RNA polymerase gene driven by the LacUV5 promoter. In cases when toxicity of the gene of interest is an issue in these expression host strains, the use of hosts carrying the pLysS or pLysE plasmids may be beneficial. These plasmids suppress basal expression of the gene of interest by producing T7 lysozyme, a T7 RNA polymerase inhibitor.

Advantages

Strong expression: The T7 transcription and translation regulatory system allows for very high-level production of proteins of interest, in many cases close to 50% of total protein in the culture.

Tightly controlled expression: The expression of the gene of interest is generally very strongly repressed in the absence of added IPTG, and this “off” state is very robust for most genes of interest in most host strains.

Disadvantages

Host requirements: Completed pET vectors should be maintained in an E. coli strain lacking the T7 RNA polymerase gene, and must be transferred to a separate host strain containing the T7 RNA polymerase gene before induction of protein expression.

Potential leaky expression in some hosts: Even in the absence of IPTG, there may be some low-level expression of T7 RNA polymerase from the LacUV5 promoter in some expression host strains, which could lead to bacterial toxicity for certain genes of interest in certain host strains.

Key components

T7 promoter: Drives high-level transcription of the gene of interest when T7 RNA polymerase is present. When placed immediately upstream of a LacO element, the entire cassette is known as the T7lac promoter.

LacO: Binding site for LacI. This element inhibits activity of the T7 promoter when LacI protein is present, preventing leaky expression of the gene of interest.

RBS: The ribosome-binding site and translation initiation element from T7 bacteriophage. This allows for efficient production of the protein of interest.

ORF: The open reading frame of your gene of interest is placed here.

T7 terminator: Signal sequence to terminate the transcript made from the gene of interest, preventing run-on transcription.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin as well as the Rop gene exist in low copy numbers in E. coli.

Rop: Repressor of primer. It encodes a small protein that regulates plasmid copy number. The presence of the Rop protein, in combination of pBR322 origin of replication on the plasmid, results in low copy numbers of the plasmid.

LacI: The E. coli natural promoter and coding sequence for the lac repressor. In the absence of induction of the system (i.e. without IPTG), the LacI protein represses transcription of the gene of interest from the T7lac promoter, as well as transcription of T7 RNA polymerase from the LacUV5 promoter in host strains used for recombinant protein production.

pBAD Bacterial Recombinant Protein Vector

Overview

The pBAD vector system is a reliable and controllable system for expressing recombinant proteins in bacteria. This system is based on the araBAD operon, which controls E. coli L-arabinose metabolism. The gene of interest is placed into the pBAD vector downstream of the araBAD promoter, which drives expression of the gene of interest in response to L-arabinose, and is inhibited by glucose. Precise control of expression levels makes this system ideal for producing problematic proteins, such as proteins with toxicity or insolubility issues.

For further information about this vector system, please refer to the papers below.

ReferencesTopic
J Bacteriol. 177:4121-30 (1995)Development of pBAD vectors

Highlights

The gene of interest is cloned into the pBAD vector and maintained in growth medium lacking L-arabinose. This reduces plasmid instability that could result from the expression of proteins of interest that may be harmful to host cells. Afterwards, expression of the gene of interest can be induced by the addition of L-arabinose to the medium.

Our pBAD vectors contain the bidirectional araBAD promoter, which drives expression of both the gene of interest and the regulatory protein AraC. In the absence of L-arabinose, AraC dimerizes to form a loop in the promoter region, blocking transcription. When L-arabinose is added, AraC changes conformation and binds to alternate sites in the promoter, activating transcription.

Addition of glucose to the growth medium can further suppress basal expression due to a reduction in cellular cAMP levels. In glucose-free medium, cAMP levels are high, and a cAMP-CRP (catabolite activator protein) complex binds to the pBAD promoter. This association is required for promoter activity, so addition of glucose will robustly repress expression of the gene of interest. This is particularly useful when the gene of interest is toxic or inhibits bacterial growth.

All custom pBAD vectors will be supplied in an E. coli strain designed to maximize plasmid integrity (such as Stbl3). To express the protein of interest, the plasmid can be transferred to a host strain lacking genes for L-arabinose catabolism (such as TOP10). This allows efficient and stable gene activation without reduction in the L-arabinose concentration over time. For genes of interest that require tight suppression of basal expression by glucose, the LMG194 host strain should be used. This strain is capable of growth on minimal defined medium (RM medium), allowing additional repression of the araBAD promoter by glucose.

Advantages

Very tightly controlled expression: Expression from pBAD vectors is more tightly controlled than from pET vectors. The expression of the gene of interest is at very low basal levels in the absence of L-arabinose, and is further repressed if glucose is present.

Strong induction: The araBAD operon system is highly inducible. It is possible to achieve >1000-fold induction upon removal of glucose and addition of L-arabinose.

Inexpensive induction: L-arabinose is inexpensive, making large-scale protein expression more economical.

Host requirements: Unlike pET vectors, pBAD can be maintained in the same E. coli strain that is used for induction, such as TOP10.

Disadvantages

Sub-maximal expression: pBAD vectors are generally not capable of achieving the very high level of expression possible with pET vectors.

Metabolism of inducer: Wild-type E. coli strains can catabolize L-arabinose. When expressing the protein of interest, host strains that are mutant for L-arabinose catabolism (such as TOP10 or LMG194) should be used to avoid inconsistent expression due to depletion of L-arabinose in the medium over time.

Key components

araBAD promoter: Drives transcription of the gene of interest when L-arabinose is present and glucose is absent. This promoter also controls AraC expression.

RBS: The ribosome-binding site and translation initiation element from T7 bacteriophage. This allows for efficient production of the protein of interest.

ORF: The open reading frame of your gene of interest is placed here.

rrnB terminator: Signal sequence to terminate the transcript made from the gene of interest, preventing run-on transcription.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin exist in medium copy numbers in E. coli.

araC: Encodes the regulatory protein of the E. coli araBAD operon. AraC inhibits expression from the araBAD promoter in the absence of L-arabinose or the presence of glucose, and activates transcription in the presence of L-arabinose and the absence of glucose.

pCS Bacterial Recombinant Protein Vector

Overview

The pCS vector system is an efficient system for expressing recombinant proteins in E. coli at low temperatures, typically 15°C. It is based on the E. coli cold-shock gene cspA, and also includes part of the lac operon regulatory system. It is considered to be complementary to the pET and pBAD systems that are typically used to express recombinant proteins at 37°C.

Temperature can have a significant impact on the folding of recombinant proteins, which in turn affects protein solubility and stability. As such, many proteins that exhibit poor solubility or stability at 37°C show improved properties at lower temperatures. When a recombinant protein is difficult to produce in pET or pBAD at 37°C, the pCS system could be a useful alternative to try.

The gene of interest is placed into the pCS vector downstream of the cspA promoter, lac operator (LacO), and the 5’ UTR of cspA. A short translation enhancing element (TEE) from the N-terminus of cspA is also included.

Transcription from the cspA promoter is very inefficient at 37°C, because of instability of the 5’ UTR at this temperature, but when the host bacteria is shifted to 15°C, the 5’ UTR adopts a highly stable secondary structure. The TEE sequence enhances translation initiation of the gene of interest through a ribosome trapping process.

The plasmid also carries the natural promoter and coding sequence for LacI. The LacI protein acts on the LacO sequence, adjacent to the cspA promoter, to block transcription of the gene of interest. In the absence of IPTG, this inhibition prevents any leaky expression of the downstream gene. Addition of IPTG blocks the inhibitory action of LacI. This, coupled with shifting of the culture from 37°C to 15°C, can efficiently induce gene expression.

For further information about this vector system, please refer to the papers below.

ReferencesTopic
Nature Biotechnology 22:877-82 (1995)Cold-shock induced high-yield protein production in E. coli
J. Biol. Chem. 274:10079-85TEE sequence translation enhancement

Highlights

E. coli containing the pCS vector is maintained in growth medium lacking IPTG at 37°C. Expression of the gene of interest can be induced by the addition of IPTG to remove LacI-based repression, combined with a temperature shift to 15°C, to activate the cspA promoter and stabilize the cspA 5’-UTR. This system can be used as an alternative when the recombinant protein of interest cannot be produced using the pET or pBAD systems. It is also recommended for expression of cold-stable proteins or proteins which are unstable at 37°C.

Advantages

Tightly controlled expression: Expression from pCS vectors is strongly repressed at 37°C in the absence of IPTG, and induced by the addition of IPTG and a temperature shift to 15°C.

Expression at low temperatures: The pCS vector system allows expression of recombinant proteins at low temperatures. This may be advantageous for many proteins of interest that cannot be efficiently achieved with other bacterial expression systems.

Host requirements: Unlike pET or pBAD vectors, which require specialized E. coli host strains for recombinant protein expression, pCS can be used to produce recombinant proteins in the same E. coli strain that is used for cloning (typically Stbl3), though the BL21 strain is recommended as the host for protein expression as this strain is engineered to reduce protein degradation.

Disadvantages

Sub-maximal expression: pCS vectors are generally not capable of achieving the very high level of expression that is possible with pET vectors.

Key Components

cspA promoter: Promoter of the cold-shock gene cspA. It drives transcription of the gene of interest at 15°C.

LacO: Binding site for LacI. This element inhibits activity of the cspA promoter when LacI protein is present, preventing leaky expression of the gene of interest.

cspA 5’ UTR: 5’ untranslated region of the cspA gene, which destabilizes the mRNA at 37°C and becomes highly stable at 15°C.

TEE: Translation enhancing element. This sequence is preferentially bound by ribosomes initiating translation, so once bound to the TEE, ribosomes are rarely available to translate other mRNAs.

ORF: The open reading frame of your gene of interest is placed here.

cspA 3’ UTR: 3’ untranslated region of the cspA gene.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

LacI: The E. coli natural promoter and coding sequence for the lac repressor. Without IPTG present, the LacI protein represses transcription of the gene of interest from the cspA promoter due to the LacO site adjacent to the cspA promoter.

S. cerevisiae Gene Expression Vector

Overview

Our S. cerevisiae gene expression vector system is based on the widely used pYES2 vector. This is a powerful and efficient system for expressing recombinant proteins in yeast, or for studying gene function in yeast via overexpression. The gene of interest is cloned into the vector under the control of a promoter selected by the user. Several standard promoters are available for selection on VectorBuilder. One of them is the strong inducible promoter from the yeast galactokinase (GAL1) gene, which is the most commonly used promoter in yeast recombinant protein expression systems.

In typical yeast laboratory strains (e.g. INVSc1), the transcriptional activity of the GAL1 promoter is responsive to the carbon source present in the medium. The presence of glucose represses transcription from the GAL1 promoter, while galactose activates the promoter. Therefore, induction of the gene of interest can be achieved by simply removing the glucose-containing medium from the cells and replacing with galactose-containing medium.

Alternatively, raffinose may be used as a carbon source. Raffinose neither represses nor induces transcription from the GAL1 promoter, and addition of galactose is sufficient to activate the GAL1 promoter even in the presence of raffinose. Induction of the GAL1 promoter by galactose is more rapid in cells maintained in raffinose-containing medium when compared to cells maintained in glucose-containing medium. However, since raffinose does not repress the GAL1 promoter, this methodology can result in “leaky” expression of the gene of interest prior to induction.

In general, recombinant proteins can be detected in ~4h after induction with galactose in cells that have been maintained in glucose, and in ~2h in cells that have been cultured in raffinose. We recommend that you perform a time course to optimize expression of your recombinant protein.

For further information about this vector system, please refer to the papers below.

ReferencesTopic
Science. 127:28-9 (1958) Mol. Cell. Biol. 4:1985-98 (1984) Mol. Cell. Biol. 4:2467-78 (1984)The GAL1 promoter
Cell 40:767-774 (1985)Induction of gene expression using the GAL1 promoter
Methods Enzymol. 194:1-863. (1991)Extensive information about gene expression in yeast

Highlights

This vector system is designed for constitutive or inducible gene expression in S. cerevisiae. In the case of inducible expression, the gene of interest is cloned into the vector under the control of the GAL1 promoter, which allows induction of the gene of interest by addition of galactose to the medium. The presence of glucose in the medium will repress expression of the gene of interest. Raffinose can also be used as a carbon source that neither activates nor represses expression from the GAL1 promoter.

Advantages

Strong expression: The inducible GAL1 promoter allows for very high-level expression of genes of interest.

Tightly controlled expression: Under the control of the GAL1 promoter, the expression of the gene of interest is generally very strongly repressed in the presence of glucose and strongly activated by galactose.

Rapid induction: Recombinant proteins expressed from the GAL1 promoter may be detectable in ~2 hours after induction in cells that have been cultured in raffinose.

Very large cargo space: Our transposon vectors can accommodate ~30 kb of total DNA. The plasmid backbone and transposon-related sequences only occupies about 3 kb, leaving plenty of room to accommodate the user's sequence of interest.

Disadvantages

Potential leaky expression: For protein expression driven by the GAL1 promoter, raffinose may be used as a carbon source instead of glucose or galactose. However, raffinose does not represses the GAL1 promoter, which can result in leaky expression of the gene of interest. Glucose needs to be used in the media for repression of the GAL1 promoter.

Key components of our vector

Promoter: The promoter that drives your gene of interest is placed here. When the inducible GAL1 promoter is used, galactose will induce high-level transcription of the gene of interest, while glucose will strongly repressed expression. Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here.

CYC1 terminator: Sequence which facilitates transcriptional termination and polyadenylation of mRNA in yeast.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

Marker: A yeast selectable marker is placed here. It allows the yeast cells successfully transformed with the vector to be selected. One commonly used marker is the orotidine-5'-phosphate decarboxylase (URA3) gene, which allows selection of yeast transformants in uracil or uridine deficient medium. Additionally, if 5-Fluoroorotic acid (5-FOA) is added to the media, the URA3 gene product will convert 5-FOA into 5-fluorouracil, which is a toxin that will cause cell death, thereby allowing selection against yeast carrying the plasmid.

2µ ori: Origin of replication which permits high-copy replication and maintenance in S. cerevisiae.

Baculovirus Recombinant Protein Expression Vectors

Overview

The baculovirus vector system is widely used for the expression of recombinant proteins in cultured insect cells. It is one of the most versatile and powerful systems for eukaryotic expression of recombinant proteins. This system is particularly advantageous for large-scale preparation of proteins that require expression in eukaryotic host cells. Many eukaryotic proteins undergo posttranslational modifications that can only take place in eukaryotic cells (e.g. glycosylation), or they need an eukaryotic cellular milieu for proper folding (e.g. membrane proteins). In these cases, prokaryotic expression systems are often inadequate and the baculovirus expression system could be a good alternative.

Baculovirus is a double-stranded DNA virus that commonly infects insects, particularly members of the order Lepidoptera (moths, butterflies and skippers). The cloning vector in our baculovirus expression system, pBV, is optimized for use with the baculovirus shuttle vector (known as bacmid) derived from the baculovirus strain AcMNPV (Autographa californica multicapsid nucleopolyhedrovirus), which has a 134 kb genome in its wildtype form.

The gene of interest is first cloned into the pBV vector under the control of a strong promoter. This entire expression cassette, along with a gentamicin resistance gene, is flanked by the Tn7 transposon terminal elements, Tn7L and Tn7R. This vector is then transformed into E. coli carrying the bacmid shuttle vector and a helper plasmid. The bacmid is essentially a very large plasmid containing the baculovirus genome modified to carry a lacZ gene and an attTn7 docking site inserted in the lacZ coding region. The helper plasmid expresses the Tn7 transposase. The transposase would then mediate the transposition of the region flanked by Tn7R and Tn7L on the pBV vector, which contains the expression cassette for the gene and interest and gentamicin resistance, into the attTn7 docking site of the bacmid. Colonies containing recombinant bacmids can be identified by gentamicin selection and blue/white screening (non-recombinant colonies are blue due to lacZ expression whereas recombinant colonies are white due to disruption of lacZ by transposon insertion). Purified bacmid DNA can then be used to transfect insect cells to generate live baculovirus, which can be used to produce the recombinant protein of interest.

The most commonly used cell line for expressing recombinant proteins from baculovirus vectors is Sf9. This clonal line was derived from ovarian tissue of Spodoptera frugiperda (fall armyworm). This cell line is adaptable to a variety of culture and media conditions, including suspension or monolater culture and serum-free media. Larvae and other Lepidoptera cell lines have also been used extensively, and there are some reports of baculovirus being an effective vector for mammalian cells.

For further information about this vector system, please refer to the papers below.

ReferencesTopic
J Virol. 67:4566-79 (1993Generation of recombinant baculovirus by site-specific transposon-mediated insertion
Meth. Mol Med. 13:213-35 (1998)Generation of recombinant baculovirus DNA in E.coli using a baculovirus shuttle vector
Nat Biotech. 23: 567–575 (2005)Baculovirus as versatile vectors for protein expression in insect and mammalian cells

Highlights

Our baculovirus recombinant protein expression vector system enables efficient production of recombinant proteins in insect cells. This system allows for expression of proteins with posttranslational processing characteristic of eukaryotic cells, and with good adaptability to large-scale applications.

Advantages

Eukaryotic system: Insect cells carry out posttranslational processing of proteins similar to that of mammalian cells. Our system is thus particularly suitable for expressing mammalian and other eukaryotic proteins whose function requires proper post-translational processing not present in prokaryotic expression systems, such as covalent modifications or membrane targeting.

Strong expression and good solubility: In most cases, the protein of interest is highly expressed, soluble, and can be easily recovered from infected cells.

Ease of scale-up: In our system, baculovirus obtained from initial transfection of insect cells can be used to infect more cells to further amplify viral titer. Protein production with our system can therefore be reproducibly scaled up.

Suspension culture: Sf9 and other Lepidoptera cell lines grow well in suspension cultures, allowing for the production of recombinant proteins in large-scale bioreactors.

Safety: Baculovirus cannot replicate outside of insect cells and are nonpathogenic to mammals and plants. Thus our expression system can be used in insect cell lines under minimal biosafety conditions.

Disadvantages

Technical complexity: Protein production using the baculovirus expression system requires multiple steps, including cloning the gene of interest into pBV, generating recombinant bacmid from pBV, and transfecting bacmid into insect cells. These procedures are technical demanding and time consuming relative to recombinant protein expression in bacterial systems. These demands can be alleviated by choosing our recombinant bacmid generation and baculovirus packaging services when ordering your vector.

Key components

PH promoter: AcMNPV polyhedrin promoter. It drives high-level expression of the gene encoding your recombinant protein.

ORF: The open reading frame of your gene of interest is placed here.

SV40 early pA: Simian virus 40 early polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

Tn7L: Tn7 transposon left terminal element. It is recognized by Tn7 transposase. DNA flanked by Tn7R and Tn7L can be transposed by Tn7 transposase into attTn7 docking sites.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Tn7R: Tn7 transposon right terminal element. It is recognized by Tn7 transposase. DNA flanked by Tn7R and Tn7L can be transposed by Tn7 transposase into attTn7 docking sites.

Gentamicin: Gentamicin resistance gene. It allows for drug selection of E. coli carrying recombinant bacmids.