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Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice

Genetically modified (GM) mice are essential tools in biomedical research. Traditional methods for generating GM mice are expensive and require specialized personnel and equipment. The use of clustered regularly interspaced short palindromic repeats (CRISPR) coupled with improved-Genome editing via...

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Autores principales: Melo-Silva, Carolina R., Knudson, Cory J., Tang, Lingjuan, Kafle, Samita, Springer, Lauren E., Choi, Jihae, Snyder, Christopher M., Wang, Yajing, Kim, Sangwon V., Sigal, Luis J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10177031/
https://www.ncbi.nlm.nih.gov/pubmed/37174743
http://dx.doi.org/10.3390/cells12091343
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author Melo-Silva, Carolina R.
Knudson, Cory J.
Tang, Lingjuan
Kafle, Samita
Springer, Lauren E.
Choi, Jihae
Snyder, Christopher M.
Wang, Yajing
Kim, Sangwon V.
Sigal, Luis J.
author_facet Melo-Silva, Carolina R.
Knudson, Cory J.
Tang, Lingjuan
Kafle, Samita
Springer, Lauren E.
Choi, Jihae
Snyder, Christopher M.
Wang, Yajing
Kim, Sangwon V.
Sigal, Luis J.
author_sort Melo-Silva, Carolina R.
collection PubMed
description Genetically modified (GM) mice are essential tools in biomedical research. Traditional methods for generating GM mice are expensive and require specialized personnel and equipment. The use of clustered regularly interspaced short palindromic repeats (CRISPR) coupled with improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) has highly increased the feasibility of producing GM mice in research laboratories. However, genetic modification in inbred mouse strains of interest such as C57BL/6 (B6) is still challenging because of their low fertility and embryo fragility. We have successfully generated multiple novel GM mouse strains in the B6 background while attempting to optimize i-GONAD. We found that i-GONAD reduced the litter size in superovulated pregnant females but did not impact pregnancy rates. Natural mating or low-hormone dose did not increase the low fertility rate observed in superovulated B6 females. However, diet enrichment had a positive effect on pregnancy success. We also optimized breeding conditions to increase the survival of small litters by co-housing i-GONAD-treated pregnant B6 females with synchronized pregnant FVB/NJ companion mothers. Thus, GM mice generation was increased by an enriched diet and shared pup rearing with highly fertile females such as FVB/NJ. In the present study, we generated 16 GM mice using a CRISPR/Cas system to target individual and multiple loci simultaneously or consecutively. We also compared homology-directed repair efficiency using different methods for LoxP insertion for conditional knockout mouse production. We found that a two-step serial LoxP insertion, in which each LoxP sequence was inserted individually in different i-GONAD procedures, was a low-risk high-efficiency method for generating floxed mice.
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spelling pubmed-101770312023-05-13 Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice Melo-Silva, Carolina R. Knudson, Cory J. Tang, Lingjuan Kafle, Samita Springer, Lauren E. Choi, Jihae Snyder, Christopher M. Wang, Yajing Kim, Sangwon V. Sigal, Luis J. Cells Article Genetically modified (GM) mice are essential tools in biomedical research. Traditional methods for generating GM mice are expensive and require specialized personnel and equipment. The use of clustered regularly interspaced short palindromic repeats (CRISPR) coupled with improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) has highly increased the feasibility of producing GM mice in research laboratories. However, genetic modification in inbred mouse strains of interest such as C57BL/6 (B6) is still challenging because of their low fertility and embryo fragility. We have successfully generated multiple novel GM mouse strains in the B6 background while attempting to optimize i-GONAD. We found that i-GONAD reduced the litter size in superovulated pregnant females but did not impact pregnancy rates. Natural mating or low-hormone dose did not increase the low fertility rate observed in superovulated B6 females. However, diet enrichment had a positive effect on pregnancy success. We also optimized breeding conditions to increase the survival of small litters by co-housing i-GONAD-treated pregnant B6 females with synchronized pregnant FVB/NJ companion mothers. Thus, GM mice generation was increased by an enriched diet and shared pup rearing with highly fertile females such as FVB/NJ. In the present study, we generated 16 GM mice using a CRISPR/Cas system to target individual and multiple loci simultaneously or consecutively. We also compared homology-directed repair efficiency using different methods for LoxP insertion for conditional knockout mouse production. We found that a two-step serial LoxP insertion, in which each LoxP sequence was inserted individually in different i-GONAD procedures, was a low-risk high-efficiency method for generating floxed mice. MDPI 2023-05-08 /pmc/articles/PMC10177031/ /pubmed/37174743 http://dx.doi.org/10.3390/cells12091343 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 Article
Melo-Silva, Carolina R.
Knudson, Cory J.
Tang, Lingjuan
Kafle, Samita
Springer, Lauren E.
Choi, Jihae
Snyder, Christopher M.
Wang, Yajing
Kim, Sangwon V.
Sigal, Luis J.
Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice
title Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice
title_full Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice
title_fullStr Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice
title_full_unstemmed Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice
title_short Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice
title_sort multiple and consecutive genome editing using i-gonad and breeding enrichment facilitates the production of genetically modified mice
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10177031/
https://www.ncbi.nlm.nih.gov/pubmed/37174743
http://dx.doi.org/10.3390/cells12091343
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