Figure 3.

Selective gene isolation from human genome by transformation-associated recombination (TAR) cloning. (A) The diagram shows TAR cloning of a gene of interest from total human genomic DNA with a linearized TAR vector containing two unique targeting sequences (hook 1 and hook 2 in green) homologous to the 5′ and 3′ ends of a gene of interest (also in green; Kouprina, & Larionov, 2008; Kouprina et al., 2021). The TAR vector DNA is linearized at a unique restriction site located between the hooks to expose targeting sequences. After transformation into the yeast Saccharomyces cerevisiae, recombination between targeting sequences in the TAR vector and the targeted sequences of the genomic DNA fragment carrying a gene of interest results in the rescue of the gene/region of interest as a circular TAR/YAC molecule. (B) Scheme of the pJBRV1 vector (Kim et al., 2011). This vector contains the URA3 yeast selectable marker, a BAC cassette containing the F′ factor origin of replication (F′), the chloramphenicol acetyltransferase (Cm^R) gene, and a 3′ HPRT-loxP-eGFP-Neo^R cassette allowing gene loading into a unique loxP site of the alphoid^tetO-HAC in Hprt-deficient Chinese hamster ovary (CHO) cells by Cre-loxP mediated recombination. The vector is linearized between two sequences, BRV1 and BRV2 (in yellow and in brown), which have homology to vector sequences in a TAR/YAC molecule. (C) Schematic representation of retrofitting a circular TAR/YAC carrying a gene of interest into a YAC/BAC. Recombination between targeting sequences in the pJBRV1 vector and homologous regions in the TAR/YAC molecule replaces the ColEI origin of replication by the F′ -factor origin of replication that allows propagation of a molecule in a BAC form in bacterial cells. Then, the YAC/BAC molecules along with a gene of interest can be moved to Escherichia coli by electroporation for further BAC DNA isolation. Figures 3A and 3C are adapted from Kononenko et al. (2014).