Exam 9: Tools for Analyzing Gene Expression

Why is green fluorescent protein widely used as a reporter gene for investigation of tissue-specific gene expression and cellular localization or proteins?

What kinds of information can be obtained from a Western blot? Diagram the key steps in the method.

Consider a Northern blot: (a) Which strand of a DNA probe will hybridize to mRNA on the blot: the anti-sense strand, the sense strand, or both strands? (b) On the diagram of the DNA-RNA hybrid shown below, which bonds form when the DNA probe hybridizes to the mRNA?

Design an experiment to quantify transcript levels for a particular mRNA using RNase protection assay (RPA). Show sample results.

You are analyzing the tissue-specific expression of a poorly characterized gene in mice. Which type of gene would serve as the best positive control for your experiment?

Explain the molecular basis of an RNase protection assay. How can this method be used to map the transcription start site for a gene?

Western blotting is the process that is used to identify

You have received from a scientific collaborator some transgenic mice that have had the gene encoding the BRCA1 gene knocked out. Which method would help you to confirm the genotype of these mice most directly?

Production of recombinant proteins in bacteria involves the cloning of the cDNA encoding the desired protein into an "expression vector" that contains a(n)

Which of the following reporter genes allows one to visualize a protein in living cells? A) beta-galactosidase ( $\beta$ -Gal) B) green fluorescent protein (GFP) C) chloramphenicol acetyltransferase (CAT) D) luciferase (Luc)

You have identified a specific region in a virus that you suspect is bound by the host transcription factor Sp1. Which of the following methods could you use to test your hypothesis?

Compare and contrast the type of information that can be obtained from cryoelectron microscopy with atomic force microscopy.

The technique of in situ hybridization gives information about:

Diagram a cotransfection assay. What information can be obtained from this type of assay?

You want to confirm the interaction of a particular protein with a specific sequence of DNA within the context of a living cell. What technique would you use and why?

Compare and contrast the steps in production of polyclonal and monoclonal antibodies.

You have transfected siRNA against transcription factor A from Q. 59 into cells that normally express the transcription factor. (a) What would be the effect on CAT reporter gene expression? (b) How could you determine whether RNAi acts at the level of mRNA or translation in this case?