We easily confirm that our mice are homozygous for the loxP-flanked allele. We also easily detect DNA recombination/excision in our mouse line Fl/Fl;vav-iCre. However, no Cre is detected (mouse tails). How can this happen?
See that even a positive PCR control is fine (we use a cDNA reaction from RNA taken from spleen sample from a single animal), we see no amplification of the iCre in the DNA samples in the genotyping procedures. We have reordered new batch of primers, changed PCR conditions (temperature), but nothing seems to solve that issue.
In addition, by means of RT-qPCR, we have noticed that deletion efficiency is dropping down in target organs (e.g., spleen).
Any ideas will be much appreciated.
this link is useful
Article Non-contact positions impose site selectivity on Cre recombinase
regards
If no Cre was expressed and deletion ccurred, the deletion could be due to homologous recombination (HR) between the two loxP sites. HR occurs between two identical recombination sties (from a site-specific recombination or SSR system) can loop out the gene inside the two recombination sites.
One example: researchers have placed two lambda phage attP sites (site-specific recombination system) to flank a DNA fragment; they found at no expression of lambda recombinase, the DNA in-between the two attP sites were deleted/excised.
Article Intrachromosomal recombination between attP regions as a too...
By the way, did you know whether iCre has been segregated away through breeding? If the allele (excised)-of-interest and the iCre gene are not linked, the iCre can be segreated away in the progeny population (and only the excised allele locus present). Did you ask your advisor whether the mice you have had been gone through breeding?
If the iCre has been segregated away, that was purely accidental. The mice were not bred on this purpose. Regarding to your previous answer, if the remaining one single loxP underwent HR (considering that target exons were excised), why do we see some mRNA levels yet? These levels are highly variable among mice with an average of 58% knock-down compared to WT, and we can confirm DNA recombination by PCR.
1. If your mice originally have the Cre gene, and there were no breeding process to segregate this gene (cre) away, then there is no reasons this cre gene would disappear in the thin air without a trace. right? Are you sure that your PCR cannot detect cre gene is because the gene is not there? Could it be some kinds of PCR inhibitors prevent PCR amplification?
2. No, I think you misunderstood my previous answer. NOT the 'remaining single loxP' go through HR. I meant that you have a -loxP-exons-loxP- construct in the mouse (and assume no Cre gene present for some reasons, as you mentioned), and through HR (w/o Cre protein), HR occured and looped out exons in-between the 2 loxP sites. HR probably not as efficient, so not 100% of the cells have excised the allele, so you could still detect some mRNA of your allele-of-interest.
By the way, when you PCRed to test the cre gene from gDNA isolated from mouse tail, did you include an internal-gene control (a positive control). The internal-gene positive control amplifed from the same gDNA sample should give you a band on gel. If no band, then probably your gDNA sample had a problem or the PCR conditions setup had a problem.
The vav-iCre mouse from The Jacson Lab is a line to produce Cre enzyme. How did your lab generate a transgenic mouse with FI/FI + vav-iCre?
Yuan-Yeu Yau, 1) OK. I understood the HR issue now. Thanks. Could you indicate a paper (or some) that have come accross with a similar issue in mice? 2) PCR's are currently running just fine to detect either the Flox or recombined allele, but iCre, in the same samples. 3) The vav-iCre is indeed from Jackson lab. The male mouse was crossed with the flox/flox female several years ago, and PCR’s for the iCre were running fine for genotyping. Of course that iCre allele shouldn’t simply fly away. That is my intriguing question. This is really puzzling. See that we have now crossed the “not-so-sure” fl/fl;vav-iCre with a Tomato reporter mouse, and we see none red fluorescence in the offspring. It seems to be a specific issue for this fl/fl;vav-icre.
@ Aristóbolo Mendes Silva,
1. Yes, the paper attached at my very first answer demonstrates that. Please look into it.
2. I checekd 'vav-iCre'-transfomred mouse line description from Jackson Lab.. The Cre is tissue-specifically expressed. So Cre-mediated deletion/excision of the 'flox' allele should be found in a specific organs or some specific organs, but not in others. That could the reason you detected both deletion and undeleted gene product. Please double-check for that.
3. From your description, "The male mouse was crossed with the flox/flox female several years ago, and PCR’s for the iCre were running fine for genotyping." The progeny from male homo (iCre/iCre) x female homo (flox/flox) parents could have crossed with other mouse line and segregated away the iCre gene. You need to check the lab book sfor the pedigree history of your own lab mouse breeding program.
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