BAC Modification (Recombineering)

VectorBuilder can fulfill all your BAC modification needs with rapid turnaround and unbeatable prices. We can place reporters behind regulatory sequences on the BAC, introduce point mutations into genes of interest, transfer regions of the BAC onto a plasmid, and add drug-selection or visualization markers to the BAC backbone.
How to obtain BAC modification services?
If you know what changes you wish to make to your BAC, just submit your needs to us by clicking the Send Design Request button on the homepage. You can describe your needs in general terms such as "I want to put a lacZ reporter after the promoter of this gene", and our skilled scientists will design a strategy for achieving the modification, all for free. We will send you a link to a proposal that includes the strategy along with price and turnaround time. If you are satisfied with the proposal, you can put it in your shopping cart and follow instructions there to place an order.
Description of services
VectorBuilder’s experienced team of scientists will put together a detailed and highly effective strategy for achieving the BAC modification your need, all for free!
If you know the ID number of the BAC you need, just give us the number and we will buy it for you. If don’t know which specific BAC to use, we will find the appropriate one for you based on the gene you want to study. The great majority of BACs are available from the BACPAC Resource Center at CHORI, but some BACs have to be obtained from other suppliers. This step is not needed if you send us your own BAC in E. coli stock.
This modification can be used to introduce either major sequence changes such as placing a reporter behind a gene’s promoter to track gene expression (Figure 1), or minor changes such as point mutations (Figure 2).
In the case of a major change (Figure 1), a DNA fragment containing your gene of interest (GOI) such as LacZ or GFP reporter, plus a drug-selection cassette flanked by FRT sites, along with flanking homology regions, is used to replace an endogenous region on the BAC via homologous recombination. The drug-selection cassette facilitates the identification of successfully modified BAC clones. This cassette is then removed by Flp-mediated recombination, leaving behind one FRT site.

Figure 1. Schematic representation of one-step BAC modification for introducing a gene of interest (GOI) such as LacZ or GFP reporter behind the promoter of a gene.

In the case of a point mutation (Figure 2), a DNA fragment containing the mutated region, plus a drug-selection cassette flanked by FRT sites, along with flanking homology regions, is used to replace the corresponding endogenous region via homologous recombination. This cassette can then be removed by Flp-mediate recombination, leaving behind one FRT site. This leftover FRT is considered a “scar” sequence because the BAC now carries this leftover FRT in addition to the desired point mutation. In order not to affect gene function, this scar sequence should be placed in a nonfunctional region of the gene, such as an intron or UTR.

Figure 2. Schematic representation of one-step BAC modification for introducing a point mutation in the first exon of a gene along with an FRT scar sequence in the downstream intron.

The two-step modification method is used to introduce minor changes such as point mutations into a BAC without leaving behind the FRT scar sequence (Figure 3). This approach is preferred over the one-step approach when there is no place in the vicinity of the desired mutation for the leftover FRT scar sequence. For example, if the gene to be modified is a very large single-exon gene, and the desired point mutation is in the middle of the gene, then there is no good place near the point mutation to place the scar sequence. In this case, the two-step method should be used. This method relies on positive and negative selection using a suitable drug-selection cassette (Figure 3). In the first step, this cassette is used to replace the target region via homologous recombination, which is facilitated by positive selection. In the second step, the cassette is further replaced with the final sequence containing the desired mutation via homologous recombination, which is facilitated by negative selection.

Figure 3. Schematic representation of two-step BAC modification for introducing a point mutation in a large single-exon gene without leaving behind any scar sequence.

A drug-selection marker such as neomycin resistance or a visualization marker such as EGFP can be added to the BAC backbone. Most BACs contain loxP sites on their backbones. These sites will be utilized to insert a cassette expressing the marker of interest.
When cells are transfected with such a modified BAC, the presence of a marker on the BAC backbone can facilitate the selection or visualization of cells successfully transfected with the BAC. This is especially useful for isolating cells containing stably integrated BACs by drug selection.
We can transfer any region of a BAC (up to 60 kb) onto a plasmid. This would allow the region to be studied without the influence of flanking sequences. For example, if an entire BAC is used to create transgenic mice in order to study the function of a gene on the BAC, the presence of other genes on the same BAC could confound the interpretation of the resulting phenotype. Isolating the gene onto a plasmid and making transgenic mice using the plasmid would circumvent this problem. An added benefit is the technical ease of working with plasmids versus BACs.
The modified region of the BAC will be confirmed by sequencing. Sometimes, BACs can undergo deletions due to the presence of repetitive sequences. To mitigate this possibility, we will re-transform the modified BAC to obtain a single clone, and confirm that it has not undergone major deletions by performing PCR amplifications of multiple sites across the entire BAC.
Price and turnaround time
The final deliverable of this service is E. coli stock containing the modified and validated BAC. The cost is relatively high due to the amount of work involved. But outsourcing to us is still far more economical than doing it yourself. Due to the complexity and error-prone nature of the procedure, most people trying it on their own can spend up to a year gathering reagents, learning the protocol, and troubleshooting before they succeed. You can better spend your time elsewhere and leave the tedious work to us.
Price and turnaround time for BAC modification services:
Service Price Turnaround*
Design BAC modification strategy Free 1-4 days
Obtain BAC from vendor $195 1-2 weeks
One-step BAC modification (without removing drug-selection cassette) From $1,950** 2-4 weeks
Optional: Remove drug-selection cassette used in one-step BAC modification $350 1-2 weeks
Two-step BAC modification From $4,950** 4-8 weeks
Add drug-selection or visualization marker to BAC backbone*** From $950 1-2 weeks
Transfer region of BAC onto plasmid $2,550 2-4 weeks
Validation of modified BAC Free 2-3 days

* Turnaround time is defined as the time from when an order is accepted to when it is shipped. It does not include waiting time for any materials that the customer needs to supply (e.g. BAC clone).

** Price may vary depending on the complexity of the sequence being introduced.

*** This service is available only for BACs that contain a loxP site on their backbones. Most BACs from the BACPAC Resource Center contain loxP sites.