Cre/loxP conditional knockdown ??

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Cre/loxP conditional knockdown ??

Postby esbena » Dec 16 2017 1:18 pm

Hi.

I have a problem with our credit/loxP system. We have confirmed Cre/loxP conditional knockdown of a tissue (kidney) specific gene mRNA, but still se protein bands on tissue homogenate samples on western blot.

What is going on here??

Thanks
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Re: Cre/loxP conditional knockdown ??

Postby 29yrsExperience » Dec 19 2017 6:05 pm

Hi esbena,
More information would be helpful. Have you made transgenic mice with a flox-interrupted promoter controlling an RNAi for knockdown and bred them with mice expressing Cre only in the Kidney? How much protein do you see relative to controls? How stable is the protein (high or low turnover rate)?
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Re: Cre/loxP conditional knockdown ??

Postby esbena » Dec 19 2017 8:48 pm

thanks..

The male (cre -/-, knockout protein +/+) is crossed with a female (cre -/+, knockout protein +/-)

The offspring are genotyped for the cre and knockout protein

The cre is under the Wnt4 promoter in the kidney
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Re: Cre/loxP conditional knockdown ??

Postby 29yrsExperience » Dec 20 2017 3:20 pm

OK, so it sounds as though you used a targeting vector to replace the endogenous gene-of-interest (GOI) with a floxed (conditional deletion) allele. And you’re doing a knockout, not a knockdown. You’ve confirmed the integration site was where the endogenous gene was located? How stable is the protein (high or low turnover rate)? The more stable it is, the more delayed the phenotypic effects. Also, Cre is never 100% effective at removing the floxed cassette, so that can affect your results. See Kwan 2002, Conditional alleles in mice: Practical considerations for tissue-specific knockouts. Genesis 32:49-62, DOI 10.1002/gene.10068. (page 54, the paragraph on mosaicism.) I can’t tell you exactly what is going on, but these are things to consider. Good luck. If anyone else has some ideas, please jump in!
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Re: Cre/loxP conditional knockdown ??

Postby esbena » Jan 02 2018 7:33 am

Thank you very much.

Yes, the GOI has been replaced by a floxed allele. Concerning the protein content, we are not sure. The floxed allele is under the activity of the Wnt4 promoter, which should give us a conditional knockout in the kidney. However, we are having trouble with finding a specific antibody which works. We see specific knockout of the floxed allele mRNA in the kidney. However, the specific antibody gives protein bands on western blotting at the same location as from WT control mice samples

?????
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