Creating targeted mutations in mouse embryonic stem cells using yeast artificial chromosomes.

Mammalian gene targeting involves construction of a vector containing a region of the gene of interest with a neo selectable marker cassette inserted into the coding region of the gene. The cassette can act as both the marker to select for integration and the introduced mutation. Selection for homologous recombination between the targeting vector and the endogenous mouse chromosome results in the disruption of the targeted locus leading to a null allele of this gene. Typically, gene targeting is performed in totipotent mouse embryonic stem (ES) cells that can give rise to any cell in a developing mouse embryo. A method for gene targeting using modified yeast artificial chromosomes (YACs) as targeting vectors has been developed. The YAC-based strategy required the generation of three categories of reagents and reliable protocols. The first component was the modification of YAC-cloned DNA using yeast recombination based methods. Prior to the modification, a long-range restriction enzyme map was generated of a specific YAC along with a lambda phage subclone library. The YAC was then altered to contain the mammalian selectable marker gene neo targeted to a specific region of the YAC insert. In addition, rapid modification of the cloned DNA was performed in yeast using the sup4-o selection system. The second component was the generation of a reproducible protocol for isolating intact, high molecular weight YAC DNA from the yeast host strain. This was achieved by the addition of high salt and polyethlyene glycol, which stabilizes large ($>$200 kb) DNA. Third, ES cell experiments were performed to optimize the transfection conditions for stable integration of YAC DNA in cultured cells. These experiments included the development of the Salmonella typhimurium hisD gene as a positive selectable marker for ES cells. Purified YAC DNA was used to transfect ES cells lines and confer resistance to the neomycin analogue G418. The results from the above tests are applicable to a generalized strategy for modifying YACs for reintroduction into the mammalian genome.PhDBiological SciencesMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/130384/2/9722112.pd