Bacterial Artificial Chromosome (BAC) Design and Cloning
Bacterial Artificial Chromosomes (BACs) are ultra-low copy vectors that can hold up to 300 kb of genomic fragments, making them ideal vectors for introduction of entire genes including the regulatory regions for disease modeling with transgenic animals. BACs are traditionally difficult to modify with restriction enzymes and ligases because of their large size. Applied StemCell uses modification of DNA using homologous recombination, (recombineering) to introduce precise changes into BACs for your specific experimental needs. Virtually any desired modification can be introduced into a BAC, including insertion of reporter genes, point mutations, Lox-STOP-Lox conditional modifications, and more. Our custom services include design, cloning, and validation of your genetic-sequence of interest into a BAC.
Applied StemCell can also help you create transgenic rats using BACs.
Case Study: Creation of GFP-Gene-of-Interest in a BAC
Goal: The purpose of this project was to generate a BAC targeting vector at the locus of GoI (Gene-of-Interest).
First, a pBT-GoI-GFP (GoI-GFP hereafter) donor vector was constructed by inserting two GoI fragments, the 3’-arm and 5’-arm, flanking a Neo cassette and GFP, into pBT vector. The features of the donor vector are illustrated in figure 1 and Appendix. The GoI locus in BAC was modified by inserting GFP cassette through homologous recombination between the 5’-, 3’- arms on the donor vector and the corresponding sequences at the GoI locus in BAC.
Figure 1. Schematic illustration of pBT-GFP-GoI donor vector
Figure 2. PCR screening and confirmation of the BAC-GoI vector. (a) PCR fragment using primer pairs RCMB1 and RCMB2 (lanes 1 to 4); RCMB3 and RCMB4 (lanes 5 to 8), and RCMB1 and RCMB4 (lanes 9 to 12); (b): GeneRuler 1kb DNA marker, Ms used in (a)
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