Q:

I observed high mortality rates in mES cells after Nucleofection™ with a Cre recombinase expression vector. How can I increase the viability?

A:
The High mortality after transfection might be due to the Cre recombinase itself, because the mammalian genome contains pseudo loxP sites (Thyagarajan B et al. (2000) Mammalian genomes contain active recombinase recognition sites. Gene. 244(1-2):47-54), which are nearly 100% active and lead to DNA damage and subsequent cell death (Loonstra A, et al. (2001) Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proc Natl Acad Sci U S A. 2001;98(16):9209-14). The DNA damage seems to be a matter of Cre recombinase concentration and is therefore strongly related to the promoter used in the expression vector. To overcome the toxic effects of Cre recombinase it might be sufficient to use a weaker promoter. The effect seems not to play a role in transient Cre expression (e.g. pPGKCre-bpA vector for excision of the "loxed"Âť selection marker.
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