International Mammalian Genome Society

The 15th International Mouse Genome Conference (2001)


POSTER 107 - RAPID MODIFICATION AND HIGH THROUGHPUT RESOLUTION OF BACTERIAL ARTIFICIAL CHROMOSOMES IN LIQUID FOR ANALYSIS OF EXPRESSED GENES

Dr. Shiaoching Gong
Rockefeller University
Box 339
1230 York Avenue
The Rockefeller University
New York
10021
USA

Co-Authors: W Yang, N Heintz
Institutions: Rockefeller University

Traditionally, overexpression of cDNA in transgenic mice has been widely used on the study of gene function and regulation. However, the cDNA itself is often missing the important regulatory elements which results in the failure of its expression. Bacterial artificial chromosome (BACs) and P-1 derived artificial chromosomes (PACs) are clonal stable, low chimerasm and easy in DNA manipulation. BAC and PAC transgenic technology has therefore become useful for studying gene function and regulation. We report here a novel and quick method for modifying and resolving BACs  in E coli.  To accomplish this, we built a shuttle vector containing a R6kr origin of replication and E.coli RecA gene. We found that although such origin could not replicate in the BAC host bacteria, the transient expression of the RecA gene from the shuttle vector is sufficient to support homologous recombination between the plasmid and the BAC, which results in high modification efficiency and low background. We have successfully introduced an IRESEGFP marker gene into 3'untranslated regions of ten murine genes in multiple BACs with two highly efficient and easy steps. A couple of BACs modified by this method have yield transgenic mice. This simple process can be readily expanded to large-scale to accelerate BAC-mediated functional studies.


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