Question

You are growing a population of cells that appear to be resistant to G418, a chemical that is normally toxic to cells. Upon further analysis of the cells’ genome you find that it contains a genomic sequence that encodes the protein neomycin phosphotransferase. Outline an experimental plan that you would follow to test if this protein confers resistance to the G418 chemical.

Answer 1

The outline for experiments to check whether the neomycin phospsotransferase is responsible for the cell's resistant to G418 is as follows :-

a) Gene knockout of neomycin phospsotransferase gene by homologous recombination method.

Reasoning:- Using homologous recombination method, gene of interest is removed from the genome and another gene which work as selective marker can be used to replace it. Possible candidates which can be used for this are mFabl and Thymidine kinase.

The cells can be then selected on a media containing triclosan (in the case of mFabl as selective marker). Cells growing on triclosan will possibly knockout cells without Neomycin phospsotransferase gene.

b) Real-time fluorescence quantitative reverse transcription PCR.

Reasoning: This will give a direct result of mRNA present in the cells and will provide insight into the preteomics of it. Housekeeping genes mRNA can be used as positive control.

c) Immunoblotting of Neomycin phospsotransferase.

Reasoning: Knockout cells and normal cells can be lysed and their proteins can be extracted and transferred to a nitrocellulose membrane for Immunoblotting to confirm the absence of Neomycin phospsotransferase protein.

d) Growth of cells on G418 containing media.

Reasoning: This will confirm the role of neomycin phospsotransferase gene in conferring the cells resistancy to G418. If the cells are neomycin phospsotransferase knocked out and neomycin phospsotransferase play a role in G418 resistance, the knockout cells will be killed while the healthy, normal cells which were used as control will survive.

Thank you