The C57BL/6 (B6) inbred strain of mice is the preferred strain of mouse geneticists, however, high throughput gene targeting/trapping has been possible to date only with 129 strain embryonic stem (ES) cells. 129 ES lines maintain self-renewal under a wide range of conditions even in absence of mouse embryonic fibroblast (MEF) feeders if supplied with Bone Morphogenetic Protein (BMP) and Leukemia Inhibitory Factor (LIF). In contrast, self-renewal of B6 ES cells is dependent on the presence of MEFs. In preliminary data, we have shown that 129-conditioned medium dramatically improves the growth characteristics of B6 ES cells. We will exploit this discovery to make B6 cells less dependent on feeder cells and, if possible completely feeder-cell independent. Importantly, our approaches make no prior assumption of the biological nature of the difference in growth characteristics between 129 and B6 cell lines. We will pursue our goals through five specific aims:
In Aim 1, we will evaluate 18 male B6 ES-cell lines and 6 male albino B6 lines for cytogenetic stability, doubling time, and germ line transmission.
In Aim 2, we will optimize the growth conditions for feeder-less growth of B6 ES-cell lines using both published additives as well as novel cytokines and/or growth factors. These new additives will be identified through expression profiling of 129 vs. the B6 ES lines, because conditioned medium from 129, but not B6 ES cells promotes growth and feeder-independence of B6 ES-cells.
In Aim 3 we will utilize the optimized conditions of feeder-less growth of B6 ES-cells for a final assessment of gene targeting capability by targeting five genes in each cell line.
In Aim 4, we will develop a novel cost-effective high throughput test that rapidly predicts germ line transmission of ES lines and with a high degree of accuracy. We will exploit expression profiling to identify a subset of 96 genes whose expression level is most indicative of self-renewal and germ line transmission. These genes will then be used to develop a qPCR-based screen to predict germ line transmission that is more rapid, accurate and less expensive than conventional cytogenetics.
In Aim 5, we will use BAG recombineering and our optimize growth conditions to derive null mutations in 50 mouse genes as selected by the KOMP steering committee. In summary, in reponse to RFA-DA-06-009 we will develop high throughput gene targeting with B6 ES-cells and derive a novel molecular assay to cost-effectively and rapidly monitor B6 ES cell lines and their targeted derivatives for germ-line competence. ? ? ? ?

National Institute of Health (NIH)
National Institute on Drug Abuse (NIDA)
Research Project--Cooperative Agreements (U01)
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Special Emphasis Panel (ZHG1-HGR-N (M1))
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Pollock, Jonathan D
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University of Pennsylvania
Schools of Medicine
United States
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