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

Andreas M Bapst,Sophie L Dahl,Thomas Knöpfel,Roland H Wenger

doi:10.1016/j.bbagrm.2020.194568

Andreas M Bapst, Sophie L Dahl + Show 2 more

Open Access

https://doi.org/10.1016/j.bbagrm.2020.194568

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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.

Highlights

The Cre-loxP recombination system derived from bacteriophage P1 is one of the most popular site-specific recombination tools
Our observations demonstrate that the Cre recombinase can cause, or is at least involved in, partial recombination of this cassette by targeting the end-start junctions of the three identical poly(A) signal repeats, leaving one or two poly(A) sequences intact
The poly(A) sequence shows no significant similarity to the loxP sequence and we did not find any abnormality in the two loxP sites outside the STOP cassette: while only one loxP site remained following complete recombination, both loxP sites were still present following excision of one or two of the three poly(A) signal repeats

Summary

Introduction

The Cre-loxP recombination system derived from bacteriophage P1 is one of the most popular site-specific recombination tools. A popular variant of this approach is Cre expression profiling using Cre recombinase-mediated excision of a transcriptional STOP cassette, flanked by directly repeated loxP sites, which is placed between a strong promoter and a marker gene While this technique has originally been developed for targeted oncogene activation [7], it can be used to permanently activate detectable reporter genes. Knock-in strategies allowed for the insertion of such conditional reporter genes into the permissive Rosa locus [9], providing a highly versatile tool to visualize the cell-type specificity of virtually any kind of promoter/ Cre combination, following crossing of a Cre mouse strain with the reporter mouse strain Using this approach, Madisen et al [10] developed a set of Cre reporter mice, including the Ai14 strain, that employed a previously reported STOP cassette containing three identical 254 bp poly(A) signal repeats derived from the SV40 virus [11]

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