You are a researcher at a small biotech company and your company has just obtain
ID: 177364 • Letter: Y
Question
You are a researcher at a small biotech company and your company has just obtained the license for use of a human GENOMIC DNA fragment putatively encoding a potentially novel protein, which is thought to regulate p53, the known tumor supressor protein. The scientists who originally cloned this GENE fragment HDM5 "claim" that HDM5 shares 90% DNA sequence homology with one of the HDM2 genes (refer to the review Levine & Oren, 2009). They propose that HDM5 may have HDM2-like properties and may be involved in regulating cell proliferation, and thus a good target to potentially develop as a cancer therapy. Your company has asked you to characterize the gene and gene products, as well as to provide an opinion as to its potential human therapeutic uses.
After convincing yourself that this gene is expressed in humans, you now wish to determine the potential function of this protein by preparing cells in culture which express the protein.
a. How would you construct the expression plasmid (what elements would you include?).
b. Would you use the genomic fragment or a cDNA, and how would you obtain this material for insertion to your plasmid? Explain.
c. How would you transfect this plasmid into cells? Include an explanation of the method you would use to introduce the DNA and
d. What cell types would you use, and why?
e. Would your proposed preparation of transfected cells be a primary cell population, a cell strain or a cell line? Explain.
f. How would you confirm expression of your cDNA? Explain.
Explanation / Answer
a.The elements that would be included are:
b. We would use a cDNA.This can be obtained by the help of reverse transcriptase which is a RNA dependent DNA polymerase.oligo dT is used to bind to polyA tails of mRNA and then specific primers against the specific mRNA is used to amplify the mRNA. The single DNA strand is bound to the RNA.RNAase -H can be used next that breaks the RNA into nicks that is used by DNA polymerase to synthesize the second strand.
c.The plasmid can be transfected into the cells by lipofection.In this process,a transfection reagent known as lipofectamine is used along with liposomes that are positively charged to bind to negatively charged plasmids, surround them and helps them merge into the cells.
d.We would use mammalian cell types.This is because the protein to be expressed is a human protein and so there would be a need for splicing of mRNA as it takes place in human.
e.It would be called a cell strain.Cell strain refers to those cell lines that are derived from a parent cell with some modifications such as genetic changes.
f.The confirmation of expression of cDNA can be done by visualisation of radiolabelled cells or quantification of activity of gene via functional assays.
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