2 replies to this topic

[vchu_sfva]

Hello,

I would greatly appreciate input on this topic. We are using the Cre-loxP recombination system in order to selectively KO the CXCR4 gene from collagen-expressing cells. I have been finding what seem to be impossible results when genotyping. In particular, I see three such results: a) animals with WT alleles (no loxP) expressing Cre and the excision product; animals with floxed alleles expressing excision product but not Cre; and c) animals with floxed alleles and Cre but without excision product.

I seem to be running into more and more of these irregularities. I am wondering if others have encountered these problems, and with what frequency. I would also love if someone could explain to me what could cause these results.

Thank you!

[Rsm]

I don't really understand your problem... my fault I guess.

So you've got floxed CXCR4 and Collagen-Cre animals, and you mate and screen the mouse. Some are floxed only, some are Cre only and some are double positive, right? And your question is, why do the double positives still express CXCR4?

What kind of cells have you checked? If you used ear punches, you'll have heterogeneous tissue, including cells which do not express Collagen-Cre, and are thus still positive for CXCR4. However, some are Cre positive (and CXCR4 negative), and that's where you get the Cre from. You've got some CXCR4 positive cells that are not expressing Collagen-Cre.

I hope I understood you right...

Cheers,

Minna

[vchu_sfva]

Hello,

Thanks for your reply!

Firstly, I should clarify that I am looking at genomic DNA and not expression. I am priming for three different things: the CXCR4 allele; Cre; and the excision product. For CXCR4, there are two possible products/bands- the lower molecular weight WT band or the higher molecular weight floxed band. I use this to determine whether my animals are fl-/-, fl+/-, or fl+/+. I prime for Cre to determine whether or not these mice have the Cre gene. Finally, I look for the CXCR4 excision product to determine whether or not the Cre has actively excised CXCR4. Hence, I determine successful excision with the excision product, and not from lack of CXCR4 expression.

My problem then is this- I am seeing animals that have the excision product when they lack Cre or floxed CXCR4, or animals that have floxed CXCR4 and Cre but lack the excision product. I have yet to determine a pattern in these anomalies, but they are occurring frequently enough that it difficult for me to brush this off as technical error on my part. From talking to other people utilizing Cre-loxP technology, it appears to me that others also run into these problems when genotyping but I have yet to hear a good explanation for why this occurs. This is very problematic as I can never be certain if I am seeing a false positive or a false negative.

If this helps, I have used both the Sigma RedExtract and QIAGEN Dneasy Blood & Tissue kits to perform my DNA extractions.

Please let me know if you have any input! I would so appreciate it. Thanks!