QUESTION 16 You are studying the trafficking of a protein in mammalian cells and

Question

QUESTION 16 You are studying the trafficking of a protein in mammalian cells and want to find out whether the protein migrates from the cytoplasm to the nucleus in response to treatment. Available to you is a cDNA clone of the protein of interest, cloning vectors and a confocal/fluorescence microscope. Place the following steps in the order in which you would proceed in attempting to solve the problem Image cells with a confocal/fluorescence microscope and compare treated cells to untreated cells. Transfect a plasmid containing a fusion of the cDNA of interest to green fluorescent protein coding sequence into mammalian cells of interest. Treat some dishes with the substances of interest and treat some with the solvent to be used as controls Insert the cDNA in-frame with the coding sequence of green fluorescent protein in a suitable E.coli/mammalian cell shuttle vector using PCR, restriction enzymes and DNA ligase. 1. Transform a plasmid containing a fusion of the cDNA of interest to green fluorescent protein coding sequence into E. coli cells and select for transformants on solid medium containing the correct antibiotic in petri dishes Amplify a plasmid containing a fusion of the cDNA of interest to green fluorescent protein coding sequence by culturing transformed E. coli cells in liquid medium containing the correct antibiotics. Purify a plasmid containing a fusion of the cDNA of interest to green fluorescent protein coding sequence from E. coli cells.

Explanation / Answer

The correct sequence should be:

Step 1. Insert the cDNA in suitable E.coli/mammalian shuttle vector (plasmid) (option 3)

Step 2.Transform the plasmid containing cDNA (option 4)

Step 3. Amplify the plasmid in liquid media (option 5)

Step 4. Purify the plasmid from E.coli (option6)

Step 5. Transfect to mammalian cell of interest (option 2)

Step 6. Image the cells and compare treated and untreated cells (option1)

All the best :)

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