The Phenotypic Effects Observed With Either Mutant Alleles ( Forward Genetic Screen ) Or Knockdown Effects?

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Question 3
Based on the phenotypic effects observed with either mutant alleles (forward genetic screen) or knockdown effects (e.g. RNAi or morpholino), you determine that you would like to generate a conditional knockout animal (in this case, in a mouse model).
(3A) What is the procedure to generate a conditional knockout mouse and how would you use it?
1.Create targeting construct using recombinant DNA technology. The construct must contain the transgene DNA sequence and the promoter necessary for gene expression. The taransgene has to be flanked by long segments of DNA homologous to endogenous mouse genome. The targeting construct must also contain positive selection gene between the homology arms and a negative selection gene outside of the homology arms.

To create a conditional KO mouse carrying a null gene identified in answer A, I would use a Cre/lox binary expression system. I would have to generate two mouse lines, TH-Cre mouse and mouse with a floxed gene (same orientation) of interest, and then cross these mice to produce a mouse which has a knock out of gene identified in answer A in DA neurons only (Cre expressing neurons).

I could also generate an additional KO animal in which Cre expression is limited to E11.5-E18 by injection of tamoxifen in Cre-ER fusion paradigm to investigate the effect of gene knock out during the time frame in which DA projections develop (although, tamoxifen exposure of the fetus might affect other developmental processes).

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