Generation of GHR-modified pigs as Laron syndrome models via a dual-sgRNAs/Cas9 system and somatic cell nuclear transfer

Journal of Translational Medicine
Hong-Hao YuHong-Jiang Wei

Abstract

Laron syndrome is an autosomal disease resulting from mutations in the growth hormone receptor (GHR) gene. The only therapeutic treatment for Laron syndrome is recombinant insulin-like growth factor I (IGF-I), which has been shown to have various side effects. The improved Laron syndrome models are important for better understanding the pathogenesis of the disease and developing corresponding therapeutics. Pigs have become attractive biomedical models for human condition due to similarities in anatomy, physiology, and metabolism relative to humans, which could serve as an appropriate model for Laron syndrome. To further improve the GHR knockout (GHRKO) efficiency and explore the feasibility of precise DNA deletion at targeted sites, the dual-sgRNAs/Cas9 system was designed to target GHR exon 3 in pig fetal fibroblasts (PFFs). The vectors encoding sgRNAs and Cas9 were co-transfected into PFFs by electroporation and GHRKO cell lines were established by single cell cloning culture. Two biallelic knockout cell lines were selected as the donor cell line for somatic cell nuclear transfer for the generation of GHRKO pigs. The genotype of colonies, cloned fetuses and piglets were identified by T7 endonuclease I (T7ENI) assay and sequen...Continue Reading

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Citations

Aug 29, 2019·In Vitro Cellular & Developmental Biology. Animal·Rui WangXiang-Xing Zhu
Sep 27, 2020·In Vitro Cellular & Developmental Biology. Animal·Maki HirataTakeshige Otoi
Mar 13, 2019·Applied Biochemistry and Biotechnology·Juqing ZhangJinlian Hua
Apr 13, 2021·The Journal of Reproduction and Development·Fuminori TaniharaTakeshige Otoi
Jan 9, 2022·Biotechnology Letters·Xiang-Xing ZhuDong-Sheng Tang

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Methods Mentioned

BETA
transfection
PCR
cleavage assay
electrophoresis
enzyme-linked immunosorbent assay

Software Mentioned

SPSS
Ensmbl

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