|Functional Genomics Section
DOE Human Genome Program Contractor-Grantee Workshop
134. YAC-ES (Y-ES) Cell Libraries for In Vivo Analysis of JGI Sequences
Yiwen Zhu, Veena Afzal, Jan-Fang
Cheng, and Edward Rubin
Libraries of the human genome, propagated in bacteria and somatic cells, have been an invaluable tool in the identification of genes based on in vitro assays. We have expanded upon this concept by creating a >10 Mb "in vivo" library of regions of the human genome sequenced by the JGI in the form of human YACs propagated in totipotent mouse embryonic stem (ES) cells.
Megabase human YACs from JGI sequenced regions were first characterized for integrity by Southern hybridization and STS content mapping. Appropriate YACs were then retrofitted with selectable markers and introduced individually into germ line transmitting ES cells by yeast spheroplast - ES cell fusion. The content and integrity of the human YACs in the ES cells were assayed and clones with intact human transgenes without detectable rearrangements have been cryopreserved to serve as publicly available reagents to explore the function of genes contained within the human sequences (for more information, visit our Web site at http://grail.lsd.ornl. gov/projects/jgi/fung.shtml). In our initial experiments, we have fused six 5q31 YACs and two 19q13.4 YACs into ES cells. The 19q13.4 Y-ES clones are being injected into mouse blastocysts to test their capacity to contribute to the germline. We have obtained good chimeras from one of the 19q13.4 Y-ES clones tested. We are now in the process of characterizing another 50 megaYACs in 5q region.
Possible uses of the Y-ES clones to biological researchers include: 1) ready made reagents for the investigation of expression/function of a human gene of interest; either in tissue culture or in transgenic mice derived from Y-ES clones; 2) as a reagent for fine mapping of mouse mutations based on functional in vivo complementation of the mutant mouse phenotype by YAC transgenes; and 3) for sifting through large candidate regions of the genome identified by human complex trait mapping studies using gene expression patterns or functional assays in mice propagating these regions.
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