Producing Mutant Clones - Essay Example

Assignments - Propose a knock out construct for the genomic locus on the Next page. Show where you set the homolgy arms of the Construct. Designa probe and a restriction digest strategy to Distinguish WT and targeted allele by Southern blotting. Calculate the size of the resulting bands using the kb Standard. 30% The diagram is below as follows: ------------------------------ 2 x 4 6 8 10 12 14 18 - Describe the targeting strategy used in the following paper: EMBO J. 1998 Jan 15; 17(2):598-608. (250 words 20%) 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. In order for such a procedure to be done, two RAD51 targeting constructs that contained either neomycin or blasticidin first had to be prepared. Once these targeting constructs were prepared, the RAD51 neo constructs were transpected into DT40 cells, and heterozygous clones were isolated for 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. 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 furthering genetic research. While these tools are quite effective, and while there are several advantages to using these tools, there are also some disadvantages. In order to get a clearer picture of what these advantages and disadvantages of these tools, we will look at the advantages and disadvantages of each tool, which will enable one to determine which tool he or she wants to use. First, we will take a look at siRNA. SiRNA, which stands for Small Interfering RNA, is a relatively new technology that allows the researcher to not only explore the genes in depth, this technology enables the researcher to determine which is the target mRNA. SiRNA is believed to be used both as a technology that furthers research endeavors, as well as serves as a form of therapy (Ventura, 2004). According to Gene Link, siRNA enables the researcher to regulate the way that a gene functions without interaction with the gene. The results that are achieved depend upon the level of RNA that have matured, as well as to how many proteins have been translated (Nature Review Genetics, 2004) Another advantage with using siRNA is that the expression of genes can easily be suppressed, which enables the researcher to have more flexibility when working with them and analyzing them (Nature Review Genetics, 2004). The disadvantages of siRNA are that for one, it is still a relatively new technology, and there is still a lot that is unknown about it. Furthermore, unlike gene targeting, one is not yet able to quite target everything as effectively, though, experts do feel that siRNA is an innovative technology that will develop into something great in time. SiRNA is already being used in genetic research at present and will continue to be as it perfects (Ventura, 2004). Now, let's analyze Gene targeting. Gene targeting is a tool that enables genetic researchers to very thoroughly research and analyze genes by identifying them, targeting them, and exploring different reactions to various environments, such as the reaction of the particular gene when certain constructs are knocked out, as well as the reaction to deletion of genetic material. Gene targeting is also useful in the research of drugs and how they might react to humans who consume them (Case Transgenic and Targeting Facility 2009). One advantage to using this technology is that is more established than siRNA, and more is known about it. Another advantage is that the technology of gene targeting is more thorough. Hough this technology is one that is well established and well known, there are a couple of disadvantages. One disadvantage to using such a technology is that more is involved. Gene targeting is more labor intensive. Furthermore, it is also the more expensive of the tools, as much more is needed in this method of research. Both of these tools have their good points as well as their bad. Nevertheless, both are very innovative technologies, and they both serve their purpose well. Thursday, 17 December 2009 I 2 3 4 Kilobase standard 2 4 6 8 10 12 14 16 18 Not1 EcoRV Not1 Neomycin Selection marker You want to delete Exon 2 and 3 Your minimal arm length is 2kb Together both arms should cover >6 kb The probe should be approximately 0.5kb Your selection marker is Neomycin, Length of the cassette is 2.5kb Thursday, 17 December 2009 References Case Transgenic and Targeting Facility 2009, viewed 10 Jan, 2010, www.ko.cwru.edu/services/targeting.html. EMBO J. 1998 Jan 15, 'Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death'; 17(2):598-608. (250 words 20%) jura.wi.mit.edu 2009, viewed 10, Jan., 2010, jura.wi.mit.edu/bioc/siRNAext/keep/FAQ.html. Ventura, Beverly. 2004, 'Is siRNA the tool of the future for in vivo mammalian gene research The experts speak out' Physiological Genomics. 18: 252-254. Read More