) The purpose of the Gene Knockout Cell Line Core is to collect, produce, and propagate cell lines derived from various gene knockout mice, since utilizing various null cell lines is central to all the projects proposed in this application. Among cells that this facility will generate and maintain are p53-/-, p53/mdm2-/-, Rb-/-, p21-/-, JNK-/-, and BRCA1-/- cells. Additional cultures from KO mice will be sought, as they become available. The core is directed by Drs. Manfredi (5 percent) and Sassoon (5 percent). A Research Assistant (100 percent effort) with a strong background in tissue culture technique (Amy Ream) will be responsible for the daily operations of this Core. Among the functions shared between all program project participants include the following: The Core will be responsible for purchasing and collecting various cell lines derived from gene knockout mice if they are available. For null cells that are not available as a cell line, the Core will purchase or collect the knockout mice. The mutant mice will be bred to homozygosity. Embryonic fibroblast cell lines (monitored via PCR) cultures will be generated and provided to the respective scientists in a timely manner. Dr. Sassoon has extensive experience with knockouts. For the preparation of MEF from embryos Dr. Douglas Forrest of the Department of Genetics at MSSM will advise the Core facility. Dr. Forrest is experienced in these protocols and is currently collaborating with several investigators in our cancer center (letter enclosed). Attempts to generate immortalized cultures will be made. The Core?s budget includes funds to house and breed various knockout mice at the Mount Sinai Animal facility, as well as for the day to day maintenance indicated above for purpose of generating cell lines. The KO derived cell lines will be stored at the Derald Ruttenberg Cancer Center cryogen storage facility, which is part of DHRCC service to MSSM community. The core will be responsible for collecting and maintaining hybridoma cell lines to generate monoclonal antibodies commonly used in the projects, such as monoclonal antibodies against p53, mdm2, p21, and BRCA1. In addition, the core will serve as a source for normalized growth of frequently used cell lines at earliest possible passages, such as 293, 293T, CV-1, cos-7, saos-2, (10)1, and NIH3T3. Technology and DNA constructs for generating tet regulatable cell lines have been transferred from Project 1 to the Core. This allows the Core to establish tet regulatable cell lines for genes of specific interest to different projects. The Core will provide quality control for identifying proper batches of Fetal Bovine Serum used by Program Project scientists. Support by the Core of specific projects will be determined during the monthly meetings of investigators. Priority will be based on the interdisciplinary scope of the requests as well as scientific merit in advancing important aims within individual projects.
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