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
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
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
Sense, sequencing the product.
No signal from null mutant – know gene is not expressed.
Housekeeping gene** most important control because COMPARING expression
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