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Introduction of loxP sites by electroporation in the mouse genome; a simple approach for conditional allele generation in complex targeting loci

BACKGROUND: The discovery of the CRISPR-Cas9 system and its applicability in mammalian embryos has revolutionized the way we generate genetically engineered animal models. To date, models harbouring conditional alleles (i.e. two loxP sites flanking an exon or a critical DNA sequence of interest) are...

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Autores principales: Bernas, Guillaume, Ouellet, Mariette, Barrios, Andréa, Jamann, Hélène, Larochelle, Catherine, Lévy, Émile, Schmouth, Jean-François
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9097428/
https://www.ncbi.nlm.nih.gov/pubmed/35549895
http://dx.doi.org/10.1186/s12896-022-00744-8
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author Bernas, Guillaume
Ouellet, Mariette
Barrios, Andréa
Jamann, Hélène
Larochelle, Catherine
Lévy, Émile
Schmouth, Jean-François
author_facet Bernas, Guillaume
Ouellet, Mariette
Barrios, Andréa
Jamann, Hélène
Larochelle, Catherine
Lévy, Émile
Schmouth, Jean-François
author_sort Bernas, Guillaume
collection PubMed
description BACKGROUND: The discovery of the CRISPR-Cas9 system and its applicability in mammalian embryos has revolutionized the way we generate genetically engineered animal models. To date, models harbouring conditional alleles (i.e. two loxP sites flanking an exon or a critical DNA sequence of interest) are amongst the most widely requested project type that are challenging to generate as they require simultaneous cleavage of the genome using two guides in order to properly integrate the repair template. An approach, using embryo sequential electroporation has been reported in the literature to successfully introduce loxP sites on the same allele. Here, we describe a modification of this sequential electroporation procedure that demonstrated the production of conditional allele mouse models for eight different genes via one of two possible strategies: either by consecutive sequential electroporation (strategy A) or non-consecutive sequential electroporation (strategy B). This latest strategy originated from using the by-product produced when using consecutive sequential electroporation (i.e. mice with a single targeted loxP site) to complete the project. RESULTS: By using strategy A, we demonstrated successful generation of conditional allele models for three different genes (Icam1, Lox, and Sar1b), with targeting efficiencies varying between 5 and 13%. By using strategy B, we generated five conditional allele models (Loxl1, Pard6a, Pard6g, Clcf1, and Mapkapk5), with targeting efficiencies varying between 3 and 25%. CONCLUSION: Our modified electroporation-based approach, involving one of the two alternative strategies, allowed the production of conditional allele models for eight different genes via two different possible paths. This reproducible method will serve as another reliable approach in addition to other well-established methodologies in the literature for conditional allele mouse model generation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12896-022-00744-8.
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spelling pubmed-90974282022-05-13 Introduction of loxP sites by electroporation in the mouse genome; a simple approach for conditional allele generation in complex targeting loci Bernas, Guillaume Ouellet, Mariette Barrios, Andréa Jamann, Hélène Larochelle, Catherine Lévy, Émile Schmouth, Jean-François BMC Biotechnol Research Article BACKGROUND: The discovery of the CRISPR-Cas9 system and its applicability in mammalian embryos has revolutionized the way we generate genetically engineered animal models. To date, models harbouring conditional alleles (i.e. two loxP sites flanking an exon or a critical DNA sequence of interest) are amongst the most widely requested project type that are challenging to generate as they require simultaneous cleavage of the genome using two guides in order to properly integrate the repair template. An approach, using embryo sequential electroporation has been reported in the literature to successfully introduce loxP sites on the same allele. Here, we describe a modification of this sequential electroporation procedure that demonstrated the production of conditional allele mouse models for eight different genes via one of two possible strategies: either by consecutive sequential electroporation (strategy A) or non-consecutive sequential electroporation (strategy B). This latest strategy originated from using the by-product produced when using consecutive sequential electroporation (i.e. mice with a single targeted loxP site) to complete the project. RESULTS: By using strategy A, we demonstrated successful generation of conditional allele models for three different genes (Icam1, Lox, and Sar1b), with targeting efficiencies varying between 5 and 13%. By using strategy B, we generated five conditional allele models (Loxl1, Pard6a, Pard6g, Clcf1, and Mapkapk5), with targeting efficiencies varying between 3 and 25%. CONCLUSION: Our modified electroporation-based approach, involving one of the two alternative strategies, allowed the production of conditional allele models for eight different genes via two different possible paths. This reproducible method will serve as another reliable approach in addition to other well-established methodologies in the literature for conditional allele mouse model generation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12896-022-00744-8. BioMed Central 2022-05-12 /pmc/articles/PMC9097428/ /pubmed/35549895 http://dx.doi.org/10.1186/s12896-022-00744-8 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visithttp://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research Article
Bernas, Guillaume
Ouellet, Mariette
Barrios, Andréa
Jamann, Hélène
Larochelle, Catherine
Lévy, Émile
Schmouth, Jean-François
Introduction of loxP sites by electroporation in the mouse genome; a simple approach for conditional allele generation in complex targeting loci
title Introduction of loxP sites by electroporation in the mouse genome; a simple approach for conditional allele generation in complex targeting loci
title_full Introduction of loxP sites by electroporation in the mouse genome; a simple approach for conditional allele generation in complex targeting loci
title_fullStr Introduction of loxP sites by electroporation in the mouse genome; a simple approach for conditional allele generation in complex targeting loci
title_full_unstemmed Introduction of loxP sites by electroporation in the mouse genome; a simple approach for conditional allele generation in complex targeting loci
title_short Introduction of loxP sites by electroporation in the mouse genome; a simple approach for conditional allele generation in complex targeting loci
title_sort introduction of loxp sites by electroporation in the mouse genome; a simple approach for conditional allele generation in complex targeting loci
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9097428/
https://www.ncbi.nlm.nih.gov/pubmed/35549895
http://dx.doi.org/10.1186/s12896-022-00744-8
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