DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

108. New Vectors for TAR Cloning and Retrofitting of Mammalian Genes

Maxim Y. Koriabine, Gregory G. Solomon, Lois A. Annab, J. Carl Barrett, and Vladimir L. Larionov

Laboratory of Molecular Genetics and Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709

koriabi1@niehs.nih.gov

The recent development of TAR (Transformation-Associated Recombination) cloning strategy for the selective isolation of specific regions and genes from complex genomes greatly advanced YAC cloning technology. Over the last two years the new technique was successfully applied for isolation of different genes and specific regions from human and mouse genomes. In this study we describe construction of a second generation of TAR cloning vectors, pVC604 (HIS3-CEN6-pBR), pVC604-A (HIS3-CEN6-pBR-Alu) and pVC604-B (HIS3-CEN6-pBR-B1), for gene isolation from human and mouse genomes. New vectors greatly simplify replacement of targeting sequences and subsequent physical analysis of the cloned material. In order to help to mobilize the DNA inserts in YACs for a variety of studies, we also have designed a set of vectors that retrofit YACs with different mammalian selectable markers and permit their transferring into E. coli cells as circular YAC/BACs. The following vectors were constructed: BRV1-N [BAC-URA3-Neomycin phosphotransferase (Neo)], BRV2-H [BAC-URA3-Hygromycin phosphotransferase (Hyg)], BRV3-B [BAC-URA3-Blasticidin S deaminase (BSD)], BRV4-G [BAC-URA3-xanthine-guanine phosphoribosyl transferase (gpt)] and BRV5-C (BAC-URA3-Cytidine deaminase (codA)]. In this study we have shown that using these vectors YACs up to ~700 kb can be efficiently converted to YAC/BACs with mammalian selectable markers by in vivo recombination in yeast. We also show evidence that circular YAC/BACs of up to 300 kb can be subsequently transferred into E. coli cells by electroporation for further DNA isolation. The YAC retrofitting method is simple, and opens possibility to use the YACs generated by TAR cloning for structural and functional studies.