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Generating a tissue specific knockout of Rad51d in mice. (Molecular Biology and Genetics 02:00 PM, Saturday, April 5, 2003 Brewer/Frost Science 141 Dr. Beth Berger Pritts-Presiding).


When damage occurs to DNA, repair is crucial for normal cell function. Rad51d, which is part of the Rad51 family, is a gene that plays a vital role in repairing double strand breaks (DSB). The impairment of DSB repair mechanisms causes the loss of genomic stability, which is a forerunner of various cancers. In 2000 a complete knockout of Rad51d in mouse embryos was produced but absence of Rad51d resulted in embryo lethality. Therefore, in this study, a conditional disruption (CD) concerning the first four exons of the gene was employed for tissue specific studies. The Rad51d CD electroporation was utilized to transfect sixteen plates of [10.sup.7] mouse embryonic stem (ES) cells. Partial incorporation of the CD indicated correctly targeted ES cell colonies since these colonies did not take up Geneticin nor FIAU. DNA from these colonies was extracted and run on agarose gel. The DNA was then transferred onto a membrane and hybridized with a 51d 5'2 Forward/Reverse probe containing a P32 label. This membrane was then exposed on x-ray film to detect the presence of bands. A positive result was considered to be the presence of two bands with sizes of 9.4kb (the wild type allele) and 6.4kb (CD). Transfections yielded approximately 180 colonies post-selection, which produced gels that displayed large amounts of extracted DNA. Although all steps prior to Southern blotting had positive results, no bands were seen on the exposed film: therefore the presence and number of correctly targeted colonies could not be determined. Further experimentation will involve numerous manipulations until correctly targeted cell lines are created so that Rad51d can be studied in specific tissues.

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Author:Roach, Kylie A.
Publication:The Ohio Journal of Science
Article Type:Abstract
Geographic Code:1U3OH
Date:Mar 1, 2003
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