A researcher is trying to develop a gene therapy treatment for muscular dystroph

Question

A researcher is trying to develop a gene therapy treatment for muscular dystrophy using a modified lentivirus carrying the dystrophin gene fused to GFP (green fluorescent protein). He starts off by trying to infect cells in culture. This is what we know: The dystrophin protein is 100 kilodaltons, the GFP protein is 15kd, primers for dystropin give a band of 400bp, primers for GFP give a band of 200bp, the dystropin gene is 20,000bp, and dystropin mRNA is 4,000bp.

{A.} How can he tell which muscle stem cells have been infected with the virus without killing the cells? (3pts)

{B.} The researcher took the cells infected with the lentivirus and put it into the muscles of mice suffering from muscular dystrophy. He took a biopsy of the muscle and performed a Southern Blot using a probe that recognizes part of the lentivirus and got the results below. Which of the mice should he use for the rest of his experiments to determine how well the gene therapy will work, and why did you choose these mice? (5 pts) lanes= cells without lentivirus, cells with lentivirus, mouse 1, mouse 2, mouse 3, mouse 4, mouse 5.

Explanation / Answer

A) The researcher has transfect the cells with the lentivirus that carries the dystrophin gene. He also fused a GFP (green fluorescent protein) to the dystropin gene. Now, if the transformation process is sucessful, then the construct will be inside the cell. So he can identify the cells which has been positively transformed by checking the GFP expression under the microsope. As he tagged GFP with dystrophin gene, once the construct are taken up by the cells GFP will express and can perform as a reporter gene for positive selection of the ransformants. So the researcher can identify which uscle stem cell get infected by lentivirus by GFP expression directly.

B) The souther blot has done with the probe which hybridize part of the lentivirus. So as expected in the negative control, there is no band and the positive control band represent part of the lentivirus hybridize with the probe. Now, if we look at the mouse, mouse 3, do not show the band means the lentivirus consruct is not there. Between mouse 1,2 ,4 and 5, mouse 1 and 4 showed the band pattern which is nearer to the positive band, meaning here the probe can hybridize the part of lentivirus which is present in the mouse. On the other hand mouse 2 and 5, showed very thin band representing that the probe can not bind the part of the lentivirus fully. Thus I will choose mouse 1 and 4 for further work.

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