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Biology (Sci)
BIOL 301

Question 1 – Eyeless TF of regulation of eye development in Drosophila. Null mtant flies of eyeless don’t develop. You constructed 5 different RNAi constructs that target different portions of eyeless mRNA. These constructs are expressed in the eye using an eye specific promoter. Only three constructs show no eyes phenotype while two others produced smaller but visible eyes. How could you explain the discrepancy among 5 different constructs and how would you test it? Don’t forget to include controls. What is it asking you? RNAi – caveats – some are specific and some might not work EXPLAIN THIS DISCREPANCY AND HOW WOULD YOU TEST IT (not make new RNAi construct) Hypothesis: Maybe some constructs work better than others Which analysis: RT-PCR or northern blot to quantify the expression level (concise description of the technique) Need to collect RNA for those flies treated with those 5 different construct and then design antisense probe or primer depending on technique *Note important steps to designing these. Compare the intensity of different lanes Controls: Sense, sequencing the product. No signal from null mutant – know gene is not expressed. Housekeeping gene** most important control because COMPARING expression levels. Question 2 – Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem cells. Little is known about factors that induce this reprogramming. In order to address this question, you would like to know which genes are specifically expressed or repressed in these reprogrammed cells. Describe how you would go about doing this. Don’t forget to include controls for this analysis, including what you would do to double-check its results. What is it asking you to do? Microarray to check which genes are up or down- regulated. (don’t know which genes to look at therefore can’t do RT PCR etc) Asks you to compare expression profile of differentiated c
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