BAC編集（リコンビニアリング）受託サービス

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. BACs on the other hand, are 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 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.

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.

Adding a drug-selection marker such as neomycin resistance or a visualization marker such as EGFP to the BAC backbone facilitates 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. Most BACs contain loxP sites on their backbones which can be utilized to insert a cassette expressing the marker of interest.

Transferring a specific region of a BAC onto a plasmid allows the region to be studied without the influence of flanking sequences. For example, if an entire BAC is used to create transgenic mice for studying 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. We can transfer any region of a BAC (up to 60 kb) onto a plasmid.