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


Bapst, Andreas M; Dahl, Sophie L; Knöpfel, Thomas; Wenger, Roland H (2020). Cre-mediated, loxP independent sequential recombination of a tripartite transcriptional stop cassette allows for partial read-through transcription. Biochimica et Biophysica Acta. Gene Regulatory Mechanisms, 1863(8):194568.

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.

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.

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Contributors:Parmjit Jat, Inder Verma, Connie Cepko and Hongkui Zeng for the kind gifts of plasmids,, Patrick Spielmann for expert technical assistance
Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Life Sciences > Biophysics
Life Sciences > Structural Biology
Life Sciences > Biochemistry
Life Sciences > Molecular Biology
Life Sciences > Genetics
Uncontrolled Keywords:Biophysics, Genetics, Biochemistry, Molecular Biology, Structural Biology
Language:English
Date:22 April 2020
Deposited On:30 Apr 2020 16:24
Last Modified:29 Jul 2020 15:06
Publisher:Elsevier
ISSN:1874-9399
OA Status:Hybrid
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1016/j.bbagrm.2020.194568
Project Information:
  • : FunderSNSF
  • : Grant ID310030_184813
  • : Project TitleSingle cell variability of hypoxia-inducible gene expression

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