Cargando…
CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus
Many researchers have focused on knock-in pigs for site-specific integration, but little attention has been given to genetically modified pigs with the targeted integration of multiple recombinant genes. To establish a multigene targeted knock-in editing system, we used the internal ribosome entry s...
Autores principales: | , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8305104/ https://www.ncbi.nlm.nih.gov/pubmed/34356043 http://dx.doi.org/10.3390/genes12071027 |
_version_ | 1783727494648561664 |
---|---|
author | You, Wenni Li, Mengjing Qi, Yilin Wang, Yanbing Chen, Yiwu Liu, Ying Li, Li Ouyang, Hongsheng Pang, Daxin |
author_facet | You, Wenni Li, Mengjing Qi, Yilin Wang, Yanbing Chen, Yiwu Liu, Ying Li, Li Ouyang, Hongsheng Pang, Daxin |
author_sort | You, Wenni |
collection | PubMed |
description | Many researchers have focused on knock-in pigs for site-specific integration, but little attention has been given to genetically modified pigs with the targeted integration of multiple recombinant genes. To establish a multigene targeted knock-in editing system, we used the internal ribosome entry site (IRES) and self-cleaving 2A peptide technology to construct a plasmid coexpressing the fatty acid desaturase (Fat-1) and porcine insulin-like growth factor-1 (IGF-1) genes at equal levels. In this study, pigs were genetically modified with multiple genes that were precisely inserted into the pRosa26 locus by using the clustered regularly spaced short palindrome repeat sequence (CRISPR)/CRISPR-related 9 (Cas9) system and somatic cell nuclear transfer technology (SCNT) in combination. Single copies of the Fat-1 and IGF-1 genes were expressed satisfactorily in various tissues of F0-generation pigs. Importantly, gas chromatography analysis revealed a significantly increased n-3 polyunsaturated fatty acid (PUFA) level in these genetically modified pigs, which led to a significant decrease of the n-6 PUFA/n-3 PUFA ratio from 6.982 to 3.122 (*** p < 0.001). In conclusion, the establishment of an editing system for targeted double-gene knock-in in this study provides a reference for the precise integration of multiple foreign genes and lays a foundation for the development of new transgenic pig breeds with multiple excellent phenotypes. |
format | Online Article Text |
id | pubmed-8305104 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-83051042021-07-25 CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus You, Wenni Li, Mengjing Qi, Yilin Wang, Yanbing Chen, Yiwu Liu, Ying Li, Li Ouyang, Hongsheng Pang, Daxin Genes (Basel) Article Many researchers have focused on knock-in pigs for site-specific integration, but little attention has been given to genetically modified pigs with the targeted integration of multiple recombinant genes. To establish a multigene targeted knock-in editing system, we used the internal ribosome entry site (IRES) and self-cleaving 2A peptide technology to construct a plasmid coexpressing the fatty acid desaturase (Fat-1) and porcine insulin-like growth factor-1 (IGF-1) genes at equal levels. In this study, pigs were genetically modified with multiple genes that were precisely inserted into the pRosa26 locus by using the clustered regularly spaced short palindrome repeat sequence (CRISPR)/CRISPR-related 9 (Cas9) system and somatic cell nuclear transfer technology (SCNT) in combination. Single copies of the Fat-1 and IGF-1 genes were expressed satisfactorily in various tissues of F0-generation pigs. Importantly, gas chromatography analysis revealed a significantly increased n-3 polyunsaturated fatty acid (PUFA) level in these genetically modified pigs, which led to a significant decrease of the n-6 PUFA/n-3 PUFA ratio from 6.982 to 3.122 (*** p < 0.001). In conclusion, the establishment of an editing system for targeted double-gene knock-in in this study provides a reference for the precise integration of multiple foreign genes and lays a foundation for the development of new transgenic pig breeds with multiple excellent phenotypes. MDPI 2021-07-01 /pmc/articles/PMC8305104/ /pubmed/34356043 http://dx.doi.org/10.3390/genes12071027 Text en © 2021 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 You, Wenni Li, Mengjing Qi, Yilin Wang, Yanbing Chen, Yiwu Liu, Ying Li, Li Ouyang, Hongsheng Pang, Daxin CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus |
title | CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus |
title_full | CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus |
title_fullStr | CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus |
title_full_unstemmed | CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus |
title_short | CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus |
title_sort | crispr/cas9-mediated specific integration of fat-1 and igf-1 at the prosa26 locus |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8305104/ https://www.ncbi.nlm.nih.gov/pubmed/34356043 http://dx.doi.org/10.3390/genes12071027 |
work_keys_str_mv | AT youwenni crisprcas9mediatedspecificintegrationoffat1andigf1attheprosa26locus AT limengjing crisprcas9mediatedspecificintegrationoffat1andigf1attheprosa26locus AT qiyilin crisprcas9mediatedspecificintegrationoffat1andigf1attheprosa26locus AT wangyanbing crisprcas9mediatedspecificintegrationoffat1andigf1attheprosa26locus AT chenyiwu crisprcas9mediatedspecificintegrationoffat1andigf1attheprosa26locus AT liuying crisprcas9mediatedspecificintegrationoffat1andigf1attheprosa26locus AT lili crisprcas9mediatedspecificintegrationoffat1andigf1attheprosa26locus AT ouyanghongsheng crisprcas9mediatedspecificintegrationoffat1andigf1attheprosa26locus AT pangdaxin crisprcas9mediatedspecificintegrationoffat1andigf1attheprosa26locus |