Interested in developing a skeletal-muscle tissue specific genetic knockout in a mouse model. Any relevant methods papers or resources would be appreciated!
Hi,
Indeed, you need a cre-lox system. To make a KO, you basically introduce (with CRISPR, for example) the loxP sites before and after an important exon(s) (deletion leads to translation into non-functional protein) of your gene of interest. In case of appearing of Cre-recombinase, you will get floxing of loxP sites and a KO as a result. To make it cell type specific, you need to locate the Cre sequence under the promoter of the cell type specific gene. For muscles the most convenient genes are Pax7 (the marker of muscle stem cells, so you will get a KO in them and muscle as their progeny), myod1/myf5 (the markers of activated muscle stem cells, which in 90% of cases end in the muscles, but 10% return to quiescence), Myog (the marker of differentiation, all the cells will end up as a muscle tissue).
If you want to have a knockout of gene from the very beginning, you may cross the mice with loxP sites in your gene of interest with Pax7-ICN (for example), which always express Cre under Pax7 promoter. Anyway, that's the way to go if you are interested in the early development. Due to the minor Pax7 expression in other cells during early development you may receive some unwanted effects on progeny. If you are interested in the effect of KO on the adult mouse, you may use the Cre-ERT system, which activate Cre expression only upon injection of Tamoxifen. This means that your mouse will develop normally (as WT) before the injection of Tam.
Best,
Michail
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