International Mammalian Genome Society

The 16th International Mouse Genome Conference (2002)


POSTER 65 - TARGETING OF LARGE TRANSGENIC SEQUENCES TO THE HPRT LOCUS IN MURINE ES CELLS

JD Heaney
Penn State College of Medicine

Bronson SK
Penn State College of Medicine

The technology exists to transfer large genomic fragments to both mouse ES cells and fertilized embryos. However, the transferred sequences are integrated randomly often with accompanying genetic alterations and variable expression of introduced genes.  We have chosen the hprt locus as a surrogate genomic site for the integration of both mouse and human bacterial artificial chromosomes (BAC) clones and have developed a method to transfer BAC clones in a single copy via a directly selectable homologous recombination event in ES cells. We designed a generic plasmid that will allow modification of virtually any BAC clone through in vitro Cre mediated loxP recombination. The modified clones can subsequently be introduced into one of several ES cell lines having a partial deletion of the hprt locus. The inclusion of sequences in the modified BAC that are able to complement the deletion adjacent to the downstream homology sequences allows for restoration of Hprt expression and survival in HAT medium for correctly targeted cells. An additional advantage to this system is that modification of the BAC clone yields a genomic insert flanked by loxP sequences permitting Cre recombinase-mediated conditional excision of the BAC transgene. Both human and mouse BAC clones from three different libraries have been successfully modified and are currently being introduced into the HM1 ES cell line.  The fidelity of the process is being monitored by diagnostic Southern analysis and PCR.


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