International Mammalian Genome Society

The 15th International Mouse Genome Conference (2001)


POSTER 109 - TARGETING OF LARGE TRANSGENIC SEQUENCES TO THE HPRT LOCUS IN MURINE ES CELLS

Mr. Jason Heaney
Penn State College of Medicine
Dept of Cellular & Molecular Physiology
The Penn State College of Medicine H166
The Milton S. Hershey Medical Center
500 University Drive
Hershey, PA
17033-0850
USA

Co-Authors: Bronson, SK
Institutions: Penn State College of Medicine, Dept. Cellular and Molecular Physiology

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 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 such that it can subsequently be introduced into one of several ES cell lines with a partial deletion of the hprt locus. The inclusion of sequences able to complement the deletion adjacent to the downstream homology sequences in the modified BAC allows restoration of Hprt expression and survival in HAT medium for correctly targeted cells. A prototype BAC has been modified using the Cre/loxP recombination system and is now being transferred to an ES cell line. The fidelity of the process is being monitored by diagnostic Southern analysis and PCR. The ability to transfer large pieces of DNA, in a precise fashion, will facilitate many types of genetic studies where correct interpretation of data is dependent on consistency of expression of the transferred genes, and the absence of unplanned rearrangements or deletions of endogenous DNA. 


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