You are a researcher at a small biotech company and your company has just obtain
ID: 179307 • 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 expression vector would be constructed with the following elements:
b. We would use a cDNA.This can be obtained by reverse transcription process.The RNA can be used to synthesize a DNA strand by use of primers specific to the RNA and using PCR technique.The first DNA strand remains attached to the RNA.This RNA can be degraded by RNAase H enzyme to form short fragments that can be amplified again by PCR using DNA polymerase I and DNA ligase to form a second DNA strand and a complete c DNA is thus synthesized.
c.This plasmid can be transfected into the cells via lipofection using lipofectamine.This is a method in which plasmids can be inserted to cells by use of liposomes that fuse through the cell membrane.
d.We would use mammalian cell types because human proteins synthesis may require splicing mechanisms and other post translational modifications to be carried out.
e.It would be a cell strain as the cells after being produced from their parent cells have undergone genetic changes by addition of plasmids that carry a cDNA.
f.The confirmation for the expression of cDNA can be done by expression of the reporter gene such as GFP that are expressed along with the protein.
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