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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...

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Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
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.
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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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