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1 year ago

Floxing mice with CRISPR

Hi everyone! I am planning on floxing mice with CRISPR but I am having trouble deciding which region to target. Do you have any tips on choosing?

Experiment: CRISPR Mouse - Deletion ATDC5 Nfatc1

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Hi Mario, I will try to walk you step by step on this. First you need to decide which part of the gene you will delete using loxP or Cre mediated recombination. Then you need to look up the mouse sequence. For this you can use something like Ensembl genome browser. You need to determine where the splice donor and acceptor sites are located. Afterwards, you should align your sequence with that of a different species and check for differences in the untranslated region. If you can identify stretches that are present in the other species but absent in mouse you should use those. You should select a species like rat which is similar to mouse because the stretches you selected are most likely not involved in gene regulation or splicing. If rat and mouse are too much alike then you can compare it to the human sequence and find some differences there. Hope this helps.

Answered 1 year ago


2 Comments


Floxing is a method to turn off or inactivate a specific DNA region (or well, move it etc, but most commonly it is used for knock out experiments I think), so you should target whatever thing you want to research? If you already have a chosen loci but want specific information on what places are best, maybe someone can help you (I wouldn't know). But at the moment your question seems to be very low on details. Of course if you want to know what exon is best to remove for knockout, you can look at some functions. I would also recommend to check if there are any genes on the opposite strand or any known regulatory elements, as I would personally try to knockout the exon that has the least amount of other effects (as far as we can predict it based on already existing information).

Answered 1 year ago


Hi Mario, I will try to walk you step by step on this. First you need to decide which part of the gene you will delete using loxP or Cre mediated recombination. Then you need to look up the mouse sequence. For this you can use something like Ensembl genome browser. You need to determine where the splice donor and acceptor sites are located. Afterwards, you should align your sequence with that of a different species and check for differences in the untranslated region. If you can identify stretches that are present in the other species but absent in mouse you should use those. You should select a species like rat which is similar to mouse because the stretches you selected are most likely not involved in gene regulation or splicing. If rat and mouse are too much alike then you can compare it to the human sequence and find some differences there. Hope this helps.

Answered 1 year ago


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