Cre recombination efficiency often impacts activation of Cre/LoxP dependent transgenes. We have recently encountered problems with recombination in certain cell types in vivo, especially with inducible recombinase (CreERT2). The recombination rates range from very high (>90%) to barely detectable. Increasing Cre concentration/action time by prolonging TAM induction regimens seems to improve it, albeit very modestly. Our transgene is in the Rosa26 locus, which many regard as a safe harbor. Interestingly, the same Cre recombinases (Cx43-CreERT2, Calb2-CreERT2, were amongst the problematic ones) seem to perform much better in other loci. We are considering two possibilities:
- methylation of the gene - this may effect recombination (e.g., if LoxP sites are methylated), but may be also happening independently
- closed chromatin leading to reduced Cre access to the locus and/or reduced overall expression of the gene
In any case, I am toying with the idea of using an alternative STOP cassette, flanked by 3 LoxP sites on each side, to increase the efficacy of recombination. It may not help if the issue is not cre related, but I don't see any risk with this approach. A slightly higher chance for random recombination seems a small price to pay if using such LoxP triplets (all facing in the same direction) would help. I would appreciate feedback from someone more experienced, about pros and cons of this solution.
On a related note: I am curious about the relative efficiencies of Cre, Flp and PhiC31 on different target sequences (e.g., LoxP vs Lox2272, AttP-CT/AttB-CT vs AttP-TC/AttB-TC, etc.). I would especially interested in strategies to optimize Cre/LoxP and Flp/FRT system, but I am also considering PhiC31 for the same purpose (especially that the latter works in unidirectional way - I see this and an advantage - and this paper shows that PhiC31 may be superior to Flp). I am aware of the one report discussing PhiC31 causing (potential) DNA damage, but I would love to hear more opinions. Regrettably, the literature mentions the above mentioned issues are very sparse; I failed to find any relevant recent papers. I would be delighted to hear expert opinions of fellow researchers.
Just one remark: If you have 3 identical CRE sites on both sides, you may have problems with multiple inversions/deletions of the same locus
@ Christian:
Just one remark: If you have 3 identical CRE sites on both sides, you may have problems with multiple inversions/deletions of the same locus
Why do you think so? The sites will face the same direction.
Then not. I thought you are dealing with inversion. So I don't see any harm of using three flanking sites. I am not sure whether this will help, but may be worth a try.
May different TAM uptake or clearence of certain cell types be a problem, too?
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