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Evidence-Based Guide to Using Artificial Introns for Tissue-Specific Knockout in Mice

Up until recently, methods for generating floxed mice either conventionally or by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 (CRISPR-associated protein 9) editing have been technically challenging, expensive and error-prone, or time-consuming. To circumvent these issues,...

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Detalles Bibliográficos
Autores principales: McBeath, Elena, Fujiwara, Keigi, Hofmann, Marie-Claude
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10299402/
https://www.ncbi.nlm.nih.gov/pubmed/37373404
http://dx.doi.org/10.3390/ijms241210258
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author McBeath, Elena
Fujiwara, Keigi
Hofmann, Marie-Claude
author_facet McBeath, Elena
Fujiwara, Keigi
Hofmann, Marie-Claude
author_sort McBeath, Elena
collection PubMed
description Up until recently, methods for generating floxed mice either conventionally or by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 (CRISPR-associated protein 9) editing have been technically challenging, expensive and error-prone, or time-consuming. To circumvent these issues, several labs have started successfully using a small artificial intron to conditionally knockout (KO) a gene of interest in mice. However, many other labs are having difficulty getting the technique to work. The key problem appears to be either a failure in achieving correct splicing after the introduction of the artificial intron into the gene or, just as crucial, insufficient functional KO of the gene’s protein after Cre-induced removal of the intron’s branchpoint. Presented here is a guide on how to choose an appropriate exon and where to place the recombinase-regulated artificial intron (rAI) in that exon to prevent disrupting normal gene splicing while maximizing mRNA degradation after recombinase treatment. The reasoning behind each step in the guide is also discussed. Following these recommendations should increase the success rate of this easy, new, and alternative technique for producing tissue-specific KO mice.
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spelling pubmed-102994022023-06-28 Evidence-Based Guide to Using Artificial Introns for Tissue-Specific Knockout in Mice McBeath, Elena Fujiwara, Keigi Hofmann, Marie-Claude Int J Mol Sci Review Up until recently, methods for generating floxed mice either conventionally or by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 (CRISPR-associated protein 9) editing have been technically challenging, expensive and error-prone, or time-consuming. To circumvent these issues, several labs have started successfully using a small artificial intron to conditionally knockout (KO) a gene of interest in mice. However, many other labs are having difficulty getting the technique to work. The key problem appears to be either a failure in achieving correct splicing after the introduction of the artificial intron into the gene or, just as crucial, insufficient functional KO of the gene’s protein after Cre-induced removal of the intron’s branchpoint. Presented here is a guide on how to choose an appropriate exon and where to place the recombinase-regulated artificial intron (rAI) in that exon to prevent disrupting normal gene splicing while maximizing mRNA degradation after recombinase treatment. The reasoning behind each step in the guide is also discussed. Following these recommendations should increase the success rate of this easy, new, and alternative technique for producing tissue-specific KO mice. MDPI 2023-06-17 /pmc/articles/PMC10299402/ /pubmed/37373404 http://dx.doi.org/10.3390/ijms241210258 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Review
McBeath, Elena
Fujiwara, Keigi
Hofmann, Marie-Claude
Evidence-Based Guide to Using Artificial Introns for Tissue-Specific Knockout in Mice
title Evidence-Based Guide to Using Artificial Introns for Tissue-Specific Knockout in Mice
title_full Evidence-Based Guide to Using Artificial Introns for Tissue-Specific Knockout in Mice
title_fullStr Evidence-Based Guide to Using Artificial Introns for Tissue-Specific Knockout in Mice
title_full_unstemmed Evidence-Based Guide to Using Artificial Introns for Tissue-Specific Knockout in Mice
title_short Evidence-Based Guide to Using Artificial Introns for Tissue-Specific Knockout in Mice
title_sort evidence-based guide to using artificial introns for tissue-specific knockout in mice
topic Review
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10299402/
https://www.ncbi.nlm.nih.gov/pubmed/37373404
http://dx.doi.org/10.3390/ijms241210258
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