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Efficient Editing of the ZBED6-Binding Site in Intron 3 of IGF2 in a Bovine Model Using the CRISPR/Cas9 System
Background: Insulin-like growth factor 2 is a growth-promoting factor that plays an important role in the growth and development of mammals. A nucleotide substitution in intron 3 of IGF2—which disrupts the ZBED6-binding site—affects muscle mass, organ size, and fat deposition in pigs. The ZBED6-bind...
Autores principales: | , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9325003/ https://www.ncbi.nlm.nih.gov/pubmed/35885915 http://dx.doi.org/10.3390/genes13071132 |
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author | Zou, Huiying Yu, Dawei Yao, Shun Ding, Fangrong Li, Junliang Li, Ling Li, Xue Zhao, Shanjiang Pang, Yunwei Hao, Haisheng Du, Weihua Zhao, Xueming Dai, Yunping Zhu, Huabin |
author_facet | Zou, Huiying Yu, Dawei Yao, Shun Ding, Fangrong Li, Junliang Li, Ling Li, Xue Zhao, Shanjiang Pang, Yunwei Hao, Haisheng Du, Weihua Zhao, Xueming Dai, Yunping Zhu, Huabin |
author_sort | Zou, Huiying |
collection | PubMed |
description | Background: Insulin-like growth factor 2 is a growth-promoting factor that plays an important role in the growth and development of mammals. A nucleotide substitution in intron 3 of IGF2—which disrupts the ZBED6-binding site—affects muscle mass, organ size, and fat deposition in pigs. The ZBED6-binding site is also conserved in cattle. Methods: In the present study, we introduced mutations in the ZBED6-binding site in intron3 of IGF2 in bovine fetal fibroblasts using the CRISPR/Cas9 system, and investigated the effect of disruption of ZBED6 binding on IGF2 expression. Results: Eleven biallelic-mutant single-cell clones were established, three of which contained no foreign DNA residues. Single-cell clones 93 and 135 were used to produce cloned embryos. Dual-luciferase reporter assay in C2C12 cells demonstrated that the mutation in the ZBED6-binding site increases the promoter 3 activity of bovine IGF2. A total of 49 mutant cloned embryos were transplanted into surrogate cows. Unfortunately, all cloned embryos died before birth. IGF2 was found to be hypomethylated in the only fetus born (stillborn), which may have been due to the incomplete reprogramming. Conclusions: We efficiently constructed IGF2-edited cell lines and cloned embryos, which provided a theoretical basis and experimental materials for beef cattle breeding. |
format | Online Article Text |
id | pubmed-9325003 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-93250032022-07-27 Efficient Editing of the ZBED6-Binding Site in Intron 3 of IGF2 in a Bovine Model Using the CRISPR/Cas9 System Zou, Huiying Yu, Dawei Yao, Shun Ding, Fangrong Li, Junliang Li, Ling Li, Xue Zhao, Shanjiang Pang, Yunwei Hao, Haisheng Du, Weihua Zhao, Xueming Dai, Yunping Zhu, Huabin Genes (Basel) Article Background: Insulin-like growth factor 2 is a growth-promoting factor that plays an important role in the growth and development of mammals. A nucleotide substitution in intron 3 of IGF2—which disrupts the ZBED6-binding site—affects muscle mass, organ size, and fat deposition in pigs. The ZBED6-binding site is also conserved in cattle. Methods: In the present study, we introduced mutations in the ZBED6-binding site in intron3 of IGF2 in bovine fetal fibroblasts using the CRISPR/Cas9 system, and investigated the effect of disruption of ZBED6 binding on IGF2 expression. Results: Eleven biallelic-mutant single-cell clones were established, three of which contained no foreign DNA residues. Single-cell clones 93 and 135 were used to produce cloned embryos. Dual-luciferase reporter assay in C2C12 cells demonstrated that the mutation in the ZBED6-binding site increases the promoter 3 activity of bovine IGF2. A total of 49 mutant cloned embryos were transplanted into surrogate cows. Unfortunately, all cloned embryos died before birth. IGF2 was found to be hypomethylated in the only fetus born (stillborn), which may have been due to the incomplete reprogramming. Conclusions: We efficiently constructed IGF2-edited cell lines and cloned embryos, which provided a theoretical basis and experimental materials for beef cattle breeding. MDPI 2022-06-24 /pmc/articles/PMC9325003/ /pubmed/35885915 http://dx.doi.org/10.3390/genes13071132 Text en © 2022 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 Zou, Huiying Yu, Dawei Yao, Shun Ding, Fangrong Li, Junliang Li, Ling Li, Xue Zhao, Shanjiang Pang, Yunwei Hao, Haisheng Du, Weihua Zhao, Xueming Dai, Yunping Zhu, Huabin Efficient Editing of the ZBED6-Binding Site in Intron 3 of IGF2 in a Bovine Model Using the CRISPR/Cas9 System |
title | Efficient Editing of the ZBED6-Binding Site in Intron 3 of IGF2 in a Bovine Model Using the CRISPR/Cas9 System |
title_full | Efficient Editing of the ZBED6-Binding Site in Intron 3 of IGF2 in a Bovine Model Using the CRISPR/Cas9 System |
title_fullStr | Efficient Editing of the ZBED6-Binding Site in Intron 3 of IGF2 in a Bovine Model Using the CRISPR/Cas9 System |
title_full_unstemmed | Efficient Editing of the ZBED6-Binding Site in Intron 3 of IGF2 in a Bovine Model Using the CRISPR/Cas9 System |
title_short | Efficient Editing of the ZBED6-Binding Site in Intron 3 of IGF2 in a Bovine Model Using the CRISPR/Cas9 System |
title_sort | efficient editing of the zbed6-binding site in intron 3 of igf2 in a bovine model using the crispr/cas9 system |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9325003/ https://www.ncbi.nlm.nih.gov/pubmed/35885915 http://dx.doi.org/10.3390/genes13071132 |
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