Dataset Information

Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP.

ABSTRACT: Predictable, clean genetic modification (GM) in livestock is important for reliable phenotyping and biosafety. Here we reported the generation of isozygous, functional myostatin (MSTN) knockout cloned pigs free of selectable marker gene (SMG) by CRISPR/Cas9 and Cre/LoxP. CRISPR/Cas9-mediated homologous recombination (HR) was exploited to knock out (KO) one allele of MSTN in pig primary cells. Cre recombinase was then used to excise the SMG with an efficiency of 82.7%. The SMG-free non-EGFP cells were isolated by flow cytometery and immediately used as donor nuclei for nuclear transfer. A total of 685 reconstructed embryos were transferred into three surrogates with one delivering two male live piglets. Molecular testing verified the mono-allelic MSTN KO and SMG deletion in these cloned pigs. Western blots showed approximately 50% decrease in MSTN and concurrent increased expression of myogenic genes in muscle. Histological examination revealed the enhanced myofiber quantity but myofiber size remained unaltered. Ultrasonic detection showed the increased longissimus muscle size and decreased backfat thickness. Precision editing of pig MSTN gene has generated isozygous, SMG-free MSTN KO cloned founders, which guaranteed a reliable route for elite livestock production and a strategy to minimize potential biological risks.

SUBMITTER: Bi Y

PROVIDER: S-EPMC4987667 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP.

Bi Yanzhen Y Hua Zaidong Z Liu Ximei X Hua Wenjun W Ren Hongyan H Xiao Hongwei H Zhang Liping L Li Li L Wang Zhirui Z Laible Götz G Wang Yan Y Dong Faming F Zheng Xinmin X

Scientific reports 20160817

Predictable, clean genetic modification (GM) in livestock is important for reliable phenotyping and biosafety. Here we reported the generation of isozygous, functional myostatin (MSTN) knockout cloned pigs free of selectable marker gene (SMG) by CRISPR/Cas9 and Cre/LoxP. CRISPR/Cas9-mediated homologous recombination (HR) was exploited to knock out (KO) one allele of MSTN in pig primary cells. Cre recombinase was then used to excise the SMG with an efficiency of 82.7%. The SMG-free non-EGFP cells ...[more]

PMID: 27530319

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Project description:Bama minipigs are a local pig breed that is unique to China and has a high development and utilization value. However, its high fat content, low feed utilization rate, and slow growth rate have limited its popularity and utilization. Compared with the long breeding cycle and high cost of traditional genetic breeding of pigs, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) endonuclease 9 system (CRISPR/Cas9)-mediated gene editing can cost-effectively implement targeted mutations in animal genomes, thereby providing a powerful tool for rapid improvement of the economic traits of Bama minipigs. The iroquois homeobox 3 (IRX3) gene has been implicated in human obesity. Mouse experiments have shown that knocking out IRX3 significantly enhances basal metabolism, reduces fat content, and controls body mass and composition. This study aimed to knock out IRX3 using the CRISPR/Cas9 gene editing method to breed Bama minipigs with significantly reduced fat content. First, the CRISPR/Cas9 gene editing method was used to efficiently obtain IRX3-/- cells. Then, the gene-edited cells were used as donor cells to produce surviving IRX3-/- Bama minipigs using somatic cell cloning. The results show that the use of IRX3-/- cells as donor cells for the production of somatic cell-cloned pigs results in a significant decrease in the average live litter size and a significant increase in the average number of stillbirths. Moreover, the birth weight of surviving IRX3-/- somatic cell-cloned pigs is significantly lower, and viability is poor such that all piglets die shortly after birth. Therefore, the preliminary results of this study suggest that IRX3 may have important biological functions in pigs, and IRX3 should not be used as a gene editing target to reduce fat content in Bama minipigs. Moreover, this study shows that knocking out IRX3 does not favor the survival of pigs, and whether targeted regulation of IRX3 in the treatment of human obesity will also induce severe adverse consequences requires further investigation.

Dataset Information

Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP.

Publications

Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP.

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