RFA on Development and Improvement of Inbred ES Cell Lines for Use in Generation of Mouse Mutants (DA-06-009)

Letters of Intent Due: October 20, 2005

Applications Due: November 22, 2005

The purpose of this RFA is to develop C57BL/6 ES cell lines that are efficient for high-throughput gene targeting and in successfully generating mice that can transmit the targeted mutation through the germline, both steps being necessary to create knockout mice on a C57BL/6 background. The research objectives for this RFA are to develop one or more C57BL/6 ES cell line(s) that can be used efficiently in gene targeting and in generating mice that can efficiently transmit introduced mutations through the germline. The desired cell line(s) should have the following characteristics: (1) attain gene targeting efficiencies, using BAC targeting vectors, that will allow in terms of throughput and cost efficiency, the use of a C57BL/6 ES cell line in a large scale-mutant generation projects; (2)be feeder line-independent; (3) achieve a minimum of 25% germline transmission in mice derived from independently targeted ES lines such that no more than 4 clones per targeted gene need to be used to assure germline transmission, and this should be demonstrated with a minimum of fifty unique mutant genes; and (4) maintain totipotency, normal karyotype, and high germline efficiency through multiple passages, such that it would be possible to expand the mutant ES cell line–derivatives to complete the knockout of the mouse genome and provide a standard resource of mutant ES cells for many years to come.