Cre-mediated, loxP independent sequential recombination of a tripartite transcriptional stop cassette allows for partial read-through transcription.

ABSTRACT

One of the widely used applications of the popular Cre-loxP method for targeted recombination is the permanent activation of marker genes, such as reporter genes or antibiotic resistance genes, by excision of a preceding transcriptional stop signal. The STOP cassette consists of three identical SV40-derived poly(A) signal repeats and is flanked by two loxP sites. We found that in addition to complete loxP-mediated recombination, limiting levels of the Cre recombinase also cause incomplete recombination of the STOP cassette. Partial recombination leads to the loss of only one or two of the three identical poly(A) repeats with recombination breakpoints always precisely matching the end/start of each poly(A) signal repeat without any relevant similarity to the canonical or known cryptic loxP sequences, suggesting that this type of Cre-mediated recombination is loxP-independent. Incomplete deletion of the STOP cassette results in partial read-through transcription, explaining at least some of the variability often observed in marker gene expression from an otherwise identical locus.