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. Approximately "5.5 kb of the GdRAD51 locus was then amplified by long-range PCR using genomic DNA from DT40 as a template." (EMBO, 1998)
Targeting events were determined by using southern blot analysis. From the targeting process, we also have found out that when RAD51 is deleted, a buildup of cells occurs in the g2/m phase, and the cells then die (EMBO, 1998).
- Propose an alternative conditional targeting strategy for the
Same paper (250 words 20%)
Another tactic could have been used to target the RAD51 gene and could possibly achieve the same results in the experiment. This is known to researchers as siRNA. Though this technology is fairly new, it is effective at targeting certain genes, nonetheless. According to a particular FAQ concerning siRNA, it is stated that siRNA is an effective technology in knocking out genes, as well as testing resistance or sensitivities to certain drugs. Just like the method of gene targeting, certain gene sequences can be achieved in humans or in mice, so long as these genes are correctly aligned (
While a bit less labor intensive, the same results can possibly be achieved in the experiment using siRNA. After all, the technology has been designed to reach a common goal. This goal is to experiment and further the research in genetics.
- Discuss advantages and disadvantages of siRNA versus Gene
Targeting as tools for Reverse Genetics (500 words 30%)
When working with reverse genetics, there are two tools that are known for their effectiveness. These tools are siRNA and gene targeting. Both of these tools use in depth technologies to aid in