shRNA (3+1) Virus Packaging

Common viral vectors used in biomedical research include lentivirus, adeno-associated virus (AAV) and adenovirus each with its advantages and disadvantages. The table below lists key factors that should be taken into consideration while selecting the right viral vector for your experiment.

After harvesting viral particles, if the viral vector carries a fluorescent reporter gene, we usually first check the quality of virus by transducing the virus into some common cell lines (e.g. 293T or 293A) to observe the expression of fluorescent protein. Different methods are then used to quantify the titer of virus depending on viral type. Occasionally, if there is a major discrepancy between fluorescence observation and quantitative measurement, we will perform re-measurement or additional validation to ensure that viruses manufactured by VectorBuilder are of high quality.

Lentivirus

We use p24 Elisa for measuring lentivirus titer. This method employs a sandwich immunoassay to measure the levels of the HIV-1 p24 core protein in lentiviral supernatants. The lentivirus samples are first added to a microtiter plate, the wells of which are coated with an anti-HIV-1 p24 capture antibody, to bind the p24 in the lentivirus samples. This is followed by the addition of a biotinylated anti-p24 secondary antibody, which in turn binds to the p24 captured by the first antibody on the plate. A streptavidin-HRP conjugate is then added for binding the biotinylated anti-p24 antibody due to the interaction between streptavidin and biotin. A substrate solution is ultimately added to the samples which produces color upon interaction with HRP. The intensity of the colored product is proportional to the amount of p24 present in each lentivirus sample, which is measured by the use of a spectrophotometer and is then precisely quantified by comparing against a recombinant HIV-1 p24 standard curve. The p24 value is then correlated with the viral titer of the corresponding lentivirus sample.

Adeno-associated virus (AAV)

We measure the physical titer of AAV by directly extracting viral genome from lysed viral particles, and then using qPCR to accurately quantify the copy number of viral genome (using the copy number of ITR region as a proxy) in the stock. AAV particles are very stable. In our AAV preparation, viral particles are essentially all alive and can remain functional at room temperature for many days. As such, the physical titer, though not measured in a way involving the transduction of cells, is very close to the functional titer.

Adenovirus

For adenovirus, we also measure the functional titer. After transducing serially diluted adenovirus into 293A cells, we use an immunocytochemistry-based approach to count the number of cells being successfully transduced via the detection of adenovirus-specific hexon protein, and each immunostained cell is considered as one infectious unit. Cells are infected at very low multiplicity of infection (MOI) to ensure that most transduced cells are each infected by a single viral particle. This assay shows good correlation with conventional plaque assay. For ultra-purified adenovirus, we directly measure the optical density (using OD260) of the viral particles to estimate titer, because there is a tight correlation between the optical density of ultra-purified adenovirus and functional titer. Adenovirus has very good stability. In our preparation, the viral particles are essentially all alive and can remain functional at room temperature for many days.

VectorBuilder applies rules similar to that used by the RNAi consortium (TRC) to design and score shRNAs. For each given RefSeq transcript, we search for all possible 21mers that are considered as candidate target sites. Candidates are excluded if they contain features thought to reduce knockdown efficiency/specificity or cloneability, including a run of ≥4 of the same base, a run of ≥7 G or C, GC content <25% or>60%, and AA at the 5’ end. Knockdown scores are penalized for candidates that contain internal stem-loop, high GC content toward the 3’ end, known miRNA seed sequences, or off-target matches to other genes. For genes with alternative transcripts, target sites that exist in all transcripts are given higher scores.

All scores are ≥0, with mean at ~5, standard deviation at ~5, and 95% of scores ≤15. An shRNA with a knockdown score about 15 is considered to have the best knockdown performance and cloneability, while an shRNA with a knockdown score of 0 has the worst knockdown performance or is hard to be cloned.

Please note that knockdown scores are only a rough guide. Actual knockdown efficiency could depart significantly from what the scores predict. Target sites with low scores may still work well. Also, please note that targeting 3’ UTR can be as effective as targeting coding region.

Based on our experience and feedback from our customers, we know that generally when 3 or 4 shRNAs are tested for any arbitrary gene, typically 2 or 3 produce reasonable to good knockdown. However, when using shRNAs, it is important to recognize the fact that not all shRNAs will work. Typically, ~50-70% of shRNAs have noticeable knockdown effect, and ~20-30% of them have strong knockdown. If you try a few shRNAs targeting a specific gene, it is possible that by chance, none will produce satisfactory knockdown. When this happens, the best approach is to try more shRNAs, especially the ones that have literature validation. Many researchers also use a “cocktail” of shRNAs (i.e. mixture of different shRNAs) targeting the same gene, which sometimes can improve knockdown efficiency.

The assay for validating the knockdown of your gene is not performed properly

The most common and sensitive assay to evaluate shRNA knockdown efficiency is RT-qPCR. Sometimes, you may need to try several pairs of primers, and then choose the most specific and efficient pair to use. In general, the RT-qPCR primers should span exon-exon junction if possible to avoid amplifying genomic DNA. When using a new pair of primers, we recommend that you run the PCR product on an agarose gel to verify the band, or even validate the PCR product by sequencing. You should always include minus-RT control in RT-qPCR to better estimate the level of genomic DNA contamination. You can use NCBI primer designing tool to help you better examine the quality of your primers in silico.

Knockdown efficiency can also be assessed by Western blot. However, Western blot is notoriously prone to false positive bands from non-specific antibody binding, which could mistakenly lead to the interpretation that there is no knockdown. Care must therefore be taken to make sure that the antibody used is indeed specific to the gene of interest.

The shRNA might only target a subset of transcript isoforms of your gene

When designing shRNA, we generally recommend those that can target as many transcript isoforms of the gene as possible, unless you are only interested in knocking down a particular isoform. VectorBuilder has created shRNA databases that contain optimized shRNAs for common species. If you design shRNA vectors on VectorBuilder, when you insert the shRNA component into the vector, you will have the option to search the target gene in our database. Then, you will see the detailed information of all the available shRNAs we designed for you, including a link to UCSC Genome Browser to view these shRNAs in the context of genomic sequence and all the transcript isoforms.