While utilizing regular plasmid vectors is a standard and the most common approach for expressing transgenes of interest in cell culture and animals, the application of such vectors is sometimes limited by their ability to carry DNA sequences of only up to 30 Kb in length. Bacterial artificial chromosomes (BACs) are DNA vectors suitable for cloning and stably maintaining large DNA inserts of up to 300 Kb in E. coli. BACs are based on the fertility (F) factor plasmid of E. coli which helps in the maintenance of one copy of the BAC per bacterial cell, thereby making them less susceptible to rearrangements and therefore, more stable compared to high copy number plasmids. Majority of BACs can be readily and inexpensively obtained from various bioresource centers such as the BACPAC Resource Center, the Arabidopsis Resource Center, The Sanger Institute and INRA BAC-YAC Resource Center as well as from certain commercial resources. These BACs can then be genetically manipulated relatively easily and quickly to carry any desired modification using a homologous recombination-based genetic engineering technique known as BAC recombineering.
Due to their ability to confer stability to large pieces of DNA and the relative ease with which they can be engineered, BACs have been widely used in genome sequencing projects of several model organisms including human, mice and Arabidopsis thaliana. Other common applications of BACs include utilizing BAC library screening for positional cloning of disease-associated genes, creating transgenic cell lines or transgenic mice harboring BACs carrying wildtype, mutated or reporter tagged versions of genes or regulatory elements to study their roles in various disease and molecular pathways and generating infectious BAC clones carrying genomes of DNA or RNA viruses such as herpesvirus for studying infectious diseases caused by such viruses.
VectorBuilder offers a variety of BAC modification services including placing reporters behind regulatory sequences on your BAC, introducing point mutations into genes of interest, transferring regions of the BAC onto a plasmid, and adding drug-selection or visualization markers to the BAC backbone. All you will need to do is send us a design request describing your experimental goal and we will take care of the rest for you starting from designing your BAC modification strategy and ordering your BAC to generating and validating your final modified BAC clones.
Please find the overview of our available services below. Click here to view the detailed description of these services.
|EGFP, mCherry, LacZ, GCaMP6s, Luciferase, DTR||$2,999||42-63 days||Drug-selection cassette used in one-step BAC modification is removed.|
|$4,098||63-84 days||Drug-selection cassette used in one-step BAC modification is removed. piggyBac ITRs are added to BAC backbone.|
|Cre, CreERT2||$7,179||98-119 days||Drug-selection cassette used in one-step BAC modification is removed. LoxP sites on BAC backbone are removed. piggyBac ITRs can be added to BAC backbone.|
|Custom DNA fragment||Please inquire.|
|Custom point mutation||$5,599||49-77 days||Two-step BAC modification.|
|$6,698||70-98 days||Two-step BAC modification. piggyBac ITRs are added to BAC backbone.|
|Removal of loxP sites on BAC backbone|
|pBACe3.6||$4,480||63-84 days||The loxP and loxP511 sites on pBACe3.6 backbone may interact with Cre to cause unwanted recombination in the genome. piggyBac ITRs can be added to BAC backbone.|
|Other BAC cloning vectors||Please inquire.|
|Transfer fragment from BAC to plasmid|
|pStart-K||$2,999||35-49 days||Typically, the transferred fragment should not exceed 30 kb. pStart-K-ITR plasmid backbone has piggyBac ITRs.|
|Other plasmids||Please inquire.|
|Add markers or other elements to BAC backbone|
|Neo, Puro, piggyBac ITRs||$1,399||35-42 days||These elements are added to the loxP site on BAC backbone.|
|Other elements||Please inquire.|
- 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).
- The above listed price includes acquiring BAC clones from BACPAC. Price may vary depending on the complexity of the sequence being introduced.
- This service is available only for BACs that contain a loxP site in their backbones. Most BACs from the BACPAC Resource Center contain loxP sites.
How do I 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 online to place the order.
Detailed 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 you 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 as a 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, the 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. One example where a two-step BAC modification is preferred is when the gene to be modified is a very large single-exon gene, and the desired point mutation is in the middle of the gene. Thus, there is no location near the point mutation to leave the scar sequence. The two-step 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 help 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 breakdown of services
The final deliverable of this service is an 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 attempting it themselves 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.
Prices and turnaround times for BAC modification services:
|Design BAC modification strategy||Free||1-4 days|
|Obtain BAC from vendor||$300||2 weeks|
|One-step BAC modification (without removing drug-selection cassette)||From $2,090**||2-4 weeks|
|Optional: Remove drug-selection cassette used in one-step BAC modification||$350||1-2 weeks|
|Two-step BAC modification||From $5,099**||4-8 weeks|
|Add drug-selection or visualization marker to BAC backbone***||From $1,099||2-3 weeks|
|Transfer region of BAC onto plasmid||$2,699||2-4 weeks|
|Validation of modified BAC||Free||1 week|
* Estimated turnaround is the time from production initiation to completion. It does not include waiting time for any customer-supplied materials (e.g. template DNA), QC of such materials, and transit time for shipping final deliverables to the customer.
** 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.