Question

1. You have recently read a paper that performed a RNA-seq on pancreas tissues comparing a mouse model of Type-2 diabetes and healthy mice. You are interested in studying one of the genes identified in the study, lots of progress (lop), that that you think may have a role in regulating insulin levels. This paper described that lop expression is increased 10-fold in mice with Type-2 diabetes compared to controls. Note: lop is a hypothetical gene that is being used as an example for this exam. You can assume that you have access to the same mice as the mentioned study and work in a lab with every technique discussed in class available to you. The questions are not based on any real studies.

1. You hypothesize that the protein lop encodes will be highly expressed by beta cells (the cells that produce insulin) in the pancreas of diabetic mice compared to healthy controls. Describe how you would test this hypothesis, include a description of technique(s), variables, controls and expected results. (5 points).

2. The results of your experiment support your hypothesis. LOP is highly expressed in beta cells of diabetic mice compared to controls where it is barely detectable. You now seek to determine the mechanism that links LOP to diabetes. Based on a conserved domain the DNA sequence of lop you suspect that it may be a DNA binding protein. You hypothesize that it is a negative regulator of genes that are involved in the production of insulin. Describe how you would test this hypothesis, include a description of technique(s), variables, controls and expected results. (5 points).

3. The results of the preceding experiment do not support your hypothesis. You cannot find evidence that LOP binds to any DNA sequences. Based on this result you alter your hypothesis and propose that LOP binds directly to insulin and stimulates its degradation. Describe how you would determine if a direct LOP-Insulin interaction occurs. Include a description of technique(s), variables, controls and expected results. (5 points).

Answer 1

a. To study the hypothesis on the protein lop encodes will be highly expressed by beta cells in pancreas of diabetic mice compared to healthy controls, the technique that I would be using will be:

1. Cell culturing of the beta cells of pancrease from both diabetic mice and healthy mice.
2. Expression of lop protein in both the cells by transfecting this protein into the cell culture.
3. Protein extraction from the cells.
4. Western blot of protein extracted from diabetic mice cells and healthy mice. As a positive control a purified lop protein will be kept. As a negative control healthy mice without transfection of lop genes will be kept.
5. Results will be that protein band in diabetic mice will be thicker when compared to the healthy mice cells.
6. Hence one can say that lop protein is highly expressed by beta cells.

b. To study that the lop is a DNA binding protein, the techniques that I would be using are:

1. Generation of potential DNA binding region of lop protein into a plasmid.
2. Performing the EMSA whereby one can study the DNA protein binding studies by including only the genes of interest in one lane and lop protein+dna in another lane. If the lop is interacting then a band will be formed at a position higher than the normal dna band position on the gel.

c. As the lop is not binding to the DNA, it may bind with insulin directly. This can be detected by labelling the lop protein with a fluorescent tag and then conducting a protein pull down assay and assaying for the insulin presence by conducting a western blot with insulin antibody. This is protein pull down assay. Also a florescent experiment can be done by labelling both insulin and lop protein and detecting there interaction in cell culture by fluorescence microscopy.

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