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In order to target the RAD51 gene, a very specific experimental procedure was followed, and it is clearly outlined in this paper. This procedure was designed to produce RAD51 mutant clones, and this was done by causing a disruption to both of the alleles in the RAD51 gene…
This was done to obtain the RAD51+/- cells (EMBO, 1998). Then, the RAD51+/- cells have been transpected with conditional human Rad51 expression constructs to obtain RAD51+/- cells carrying the constructs at random sites on the chromosome (RAD51+/-/HsRAD51)." (, 1998) Finally, the RAD51 construct that contained the blasticidin was then transpected into "several RAD51+/-/HsRAD51 clones to isolate RAD51-/-/HsRAD51 clones." (EMBO, 1998)
The targeting process is shown in figures one and two. In order to target the necessary genes, a probe and southern blot analysis were used to indicate the knockout constructs. Samples of the cells and genetic material were loaded onto five different lanes and were combined with SDS-polyacrylamide gel. The three samples were the wild-type DT40, which was loaded onto lane 1, the RAD51+/-, which was loaded onto the second lane, a RAD51+/- clone that contained the human Rad51 transgene, which was loaded onto the third lane, #110 RAD51-/- clone was placed onto the fourth lane, and a human B lymphocyte line Ramos was loaded onto the fifth lane (EMBO, 1998).
The targeting probe was constructed of A chicken RAD51 (GdRAD51) cDNA, and this probe was used to isolate the genomic clones that were of the RAD51 locus. These clones were, in part, sequenced to determine the position of the exons. ...
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