Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before insemination
Autor(a) principal: | |
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Data de Publicação: | 2022 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | por |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10174/35579 https://doi.org/Navarro-Serna, S, Dehesa-Etxebeste, M., Piñeiro-Silva, C., Romar, R., Lopes, J. S., Munaín, A. L., & Gadea, J. (2022). Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before insemination. Theriogenology. https://doi.org/10.1016/j.theriogenology.2022.04.012 https://doi.org/10.1016/j.theriogenology.2022.04.012 |
Resumo: | Limb girdle muscular dystrophy type R1 (LGMDR1) is an autosomal recessive myopathy described in humans resulting from a deficiency of calpain-3 protein (CAPN3). This disease lacks effective treatment and an appropriate model, so the generation of KO pigs by CRISPR-Cas9 offers a way to better understand disease ethology and to develop novel therapies. Microinjection is the main method described for gene editing by CRISPR-Cas9 in porcine embryo, but electroporation, which allows handling more embryos faster and easier, has also recently been reported. The objective of the current study was to optimize porcine oocyte electroporation to maximize embryo quality and mutation rate in order to efficiently generate LGMDR1 porcine models. We found that the efficiency of generating CAPN3 KO embryos was highest with 4 electroporation pulses and double sgRNA concentration than microinjection. Direct comparison between microinjection and electroporation demonstrated similar rates of embryo development and mutation parameters. The results of our study demonstrate that oocyte electroporation, an easier and faster method than microinjection, is comparable to standard approaches, paving the way for democratization of transgenesis in pigs. |
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Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before inseminationLimb girdle muscular dystrophy type R1 (LGMDR1) is an autosomal recessive myopathy described in humans resulting from a deficiency of calpain-3 protein (CAPN3). This disease lacks effective treatment and an appropriate model, so the generation of KO pigs by CRISPR-Cas9 offers a way to better understand disease ethology and to develop novel therapies. Microinjection is the main method described for gene editing by CRISPR-Cas9 in porcine embryo, but electroporation, which allows handling more embryos faster and easier, has also recently been reported. The objective of the current study was to optimize porcine oocyte electroporation to maximize embryo quality and mutation rate in order to efficiently generate LGMDR1 porcine models. We found that the efficiency of generating CAPN3 KO embryos was highest with 4 electroporation pulses and double sgRNA concentration than microinjection. Direct comparison between microinjection and electroporation demonstrated similar rates of embryo development and mutation parameters. The results of our study demonstrate that oocyte electroporation, an easier and faster method than microinjection, is comparable to standard approaches, paving the way for democratization of transgenesis in pigs.Elsevier2023-10-10T08:48:27Z2023-10-102022-04-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlehttp://hdl.handle.net/10174/35579https://doi.org/Navarro-Serna, S, Dehesa-Etxebeste, M., Piñeiro-Silva, C., Romar, R., Lopes, J. S., Munaín, A. L., & Gadea, J. (2022). Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before insemination. Theriogenology. https://doi.org/10.1016/j.theriogenology.2022.04.012http://hdl.handle.net/10174/35579https://doi.org/10.1016/j.theriogenology.2022.04.012porndndndndJordana.lopes@uevora.ptndnd369Navarro-Serna, SergioDehesa-Etxebeste, MartxelPiñeiro-Silva, CeliaRomar, RaquelLopes, Jordana SLopéz, AdolfoGadea, Joaquininfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-01-03T19:38:09Zoai:dspace.uevora.pt:10174/35579Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T01:23:30.101429Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before insemination |
title |
Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before insemination |
spellingShingle |
Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before insemination Navarro-Serna, Sergio |
title_short |
Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before insemination |
title_full |
Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before insemination |
title_fullStr |
Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before insemination |
title_full_unstemmed |
Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before insemination |
title_sort |
Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before insemination |
author |
Navarro-Serna, Sergio |
author_facet |
Navarro-Serna, Sergio Dehesa-Etxebeste, Martxel Piñeiro-Silva, Celia Romar, Raquel Lopes, Jordana S Lopéz, Adolfo Gadea, Joaquin |
author_role |
author |
author2 |
Dehesa-Etxebeste, Martxel Piñeiro-Silva, Celia Romar, Raquel Lopes, Jordana S Lopéz, Adolfo Gadea, Joaquin |
author2_role |
author author author author author author |
dc.contributor.author.fl_str_mv |
Navarro-Serna, Sergio Dehesa-Etxebeste, Martxel Piñeiro-Silva, Celia Romar, Raquel Lopes, Jordana S Lopéz, Adolfo Gadea, Joaquin |
description |
Limb girdle muscular dystrophy type R1 (LGMDR1) is an autosomal recessive myopathy described in humans resulting from a deficiency of calpain-3 protein (CAPN3). This disease lacks effective treatment and an appropriate model, so the generation of KO pigs by CRISPR-Cas9 offers a way to better understand disease ethology and to develop novel therapies. Microinjection is the main method described for gene editing by CRISPR-Cas9 in porcine embryo, but electroporation, which allows handling more embryos faster and easier, has also recently been reported. The objective of the current study was to optimize porcine oocyte electroporation to maximize embryo quality and mutation rate in order to efficiently generate LGMDR1 porcine models. We found that the efficiency of generating CAPN3 KO embryos was highest with 4 electroporation pulses and double sgRNA concentration than microinjection. Direct comparison between microinjection and electroporation demonstrated similar rates of embryo development and mutation parameters. The results of our study demonstrate that oocyte electroporation, an easier and faster method than microinjection, is comparable to standard approaches, paving the way for democratization of transgenesis in pigs. |
publishDate |
2022 |
dc.date.none.fl_str_mv |
2022-04-01T00:00:00Z 2023-10-10T08:48:27Z 2023-10-10 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10174/35579 https://doi.org/Navarro-Serna, S, Dehesa-Etxebeste, M., Piñeiro-Silva, C., Romar, R., Lopes, J. S., Munaín, A. L., & Gadea, J. (2022). Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before insemination. Theriogenology. https://doi.org/10.1016/j.theriogenology.2022.04.012 http://hdl.handle.net/10174/35579 https://doi.org/10.1016/j.theriogenology.2022.04.012 |
url |
http://hdl.handle.net/10174/35579 https://doi.org/Navarro-Serna, S, Dehesa-Etxebeste, M., Piñeiro-Silva, C., Romar, R., Lopes, J. S., Munaín, A. L., & Gadea, J. (2022). Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before insemination. Theriogenology. https://doi.org/10.1016/j.theriogenology.2022.04.012 https://doi.org/10.1016/j.theriogenology.2022.04.012 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.relation.none.fl_str_mv |
nd nd nd nd Jordana.lopes@uevora.pt nd nd 369 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.publisher.none.fl_str_mv |
Elsevier |
publisher.none.fl_str_mv |
Elsevier |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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1799136717120208896 |