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Generation of GHR-modified pigs as Laron syndrome models via a dual-sgRNAs/Cas9 system and somatic cell nuclear transfer
BACKGROUND: 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...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828148/ https://www.ncbi.nlm.nih.gov/pubmed/29482569 http://dx.doi.org/10.1186/s12967-018-1409-7 |
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author | Yu, Honghao Long, Weihu Zhang, Xuezeng Xu, Kaixiang Guo, Jianxiong Zhao, Heng Li, Honghui Qing, Yubo Pan, Weirong Jia, Baoyu Zhao, Hong-Ye Huang, Xingxu Wei, Hong-Jiang |
author_facet | Yu, Honghao Long, Weihu Zhang, Xuezeng Xu, Kaixiang Guo, Jianxiong Zhao, Heng Li, Honghui Qing, Yubo Pan, Weirong Jia, Baoyu Zhao, Hong-Ye Huang, Xingxu Wei, Hong-Jiang |
author_sort | Yu, Honghao |
collection | PubMed |
description | BACKGROUND: 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. METHODS: 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 sequencing. The GHR expression in the fibroblasts and piglets was analyzed by confocal microscopy, quantitative polymerase chain reaction (q-PCR), western blotting (WB) and immunohistochemical (IHC) staining. The phenotype of GHRKO pigs was recapitulated through level detection of IGF-I and glucose, and measurement of body weight and body size. GHRKO F1 generation were generated by crossing with wild-type pigs, and their genotype was detected by T7ENI assay and sequencing. GHRKO F2 generation was obtained via self-cross of GHRKO F1 pigs. Their genotypes of GHRKO F2 generation was also detected by Sanger sequencing. RESULTS: In total, 19 of 20 single-cell colonies exhibited biallelic modified GHR (95%), and the efficiency of DNA deletion mediated by dual-sgRNAs/Cas9 was as high as 90% in 40 GHR alleles of 20 single-cell colonies. Two types of GHR allelic single-cell colonies (GHR(−47/−1), GHR(−47/−46)) were selected as donor cells for the generation of GHRKO pigs. The reconstructed embryos were transferred into 15 recipient gilts, resulting in 15 GHRKO newborn piglets and 2 fetuses. The GHRKO pigs exhibited slow growth rates and small body sizes. From birth to 13 months old, the average body weight of wild-type pigs varied from 0.6 to 89.5 kg, but that of GHRKO pigs varied from only 0.9 to 37.0 kg. Biochemically, the knockout pigs exhibited decreased serum levels of IGF-I and glucose. Furthermore, the GHRKO pigs had normal reproduction ability, as eighteen GHRKO F1 piglets were obtained via mating a GHRKO pig with wild-type pigs and five GHRKO F2 piglets were obtained by self-cross of F1 generation, indicating that modified GHR alleles can pass to the next generation via germline transmission. CONCLUSION: The dual-sgRNAs/Cas9 is a reliable system for DNA deletion and that GHRKO pigs conform to typical phenotypes of those observed in Laron patients, suggesting that these pigs could serve as an appropriate model for Laron syndrome. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12967-018-1409-7) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5828148 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-58281482018-02-28 Generation of GHR-modified pigs as Laron syndrome models via a dual-sgRNAs/Cas9 system and somatic cell nuclear transfer Yu, Honghao Long, Weihu Zhang, Xuezeng Xu, Kaixiang Guo, Jianxiong Zhao, Heng Li, Honghui Qing, Yubo Pan, Weirong Jia, Baoyu Zhao, Hong-Ye Huang, Xingxu Wei, Hong-Jiang J Transl Med Research BACKGROUND: 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. METHODS: 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 sequencing. The GHR expression in the fibroblasts and piglets was analyzed by confocal microscopy, quantitative polymerase chain reaction (q-PCR), western blotting (WB) and immunohistochemical (IHC) staining. The phenotype of GHRKO pigs was recapitulated through level detection of IGF-I and glucose, and measurement of body weight and body size. GHRKO F1 generation were generated by crossing with wild-type pigs, and their genotype was detected by T7ENI assay and sequencing. GHRKO F2 generation was obtained via self-cross of GHRKO F1 pigs. Their genotypes of GHRKO F2 generation was also detected by Sanger sequencing. RESULTS: In total, 19 of 20 single-cell colonies exhibited biallelic modified GHR (95%), and the efficiency of DNA deletion mediated by dual-sgRNAs/Cas9 was as high as 90% in 40 GHR alleles of 20 single-cell colonies. Two types of GHR allelic single-cell colonies (GHR(−47/−1), GHR(−47/−46)) were selected as donor cells for the generation of GHRKO pigs. The reconstructed embryos were transferred into 15 recipient gilts, resulting in 15 GHRKO newborn piglets and 2 fetuses. The GHRKO pigs exhibited slow growth rates and small body sizes. From birth to 13 months old, the average body weight of wild-type pigs varied from 0.6 to 89.5 kg, but that of GHRKO pigs varied from only 0.9 to 37.0 kg. Biochemically, the knockout pigs exhibited decreased serum levels of IGF-I and glucose. Furthermore, the GHRKO pigs had normal reproduction ability, as eighteen GHRKO F1 piglets were obtained via mating a GHRKO pig with wild-type pigs and five GHRKO F2 piglets were obtained by self-cross of F1 generation, indicating that modified GHR alleles can pass to the next generation via germline transmission. CONCLUSION: The dual-sgRNAs/Cas9 is a reliable system for DNA deletion and that GHRKO pigs conform to typical phenotypes of those observed in Laron patients, suggesting that these pigs could serve as an appropriate model for Laron syndrome. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12967-018-1409-7) contains supplementary material, which is available to authorized users. BioMed Central 2018-02-27 /pmc/articles/PMC5828148/ /pubmed/29482569 http://dx.doi.org/10.1186/s12967-018-1409-7 Text en © The Author(s) 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Yu, Honghao Long, Weihu Zhang, Xuezeng Xu, Kaixiang Guo, Jianxiong Zhao, Heng Li, Honghui Qing, Yubo Pan, Weirong Jia, Baoyu Zhao, Hong-Ye Huang, Xingxu Wei, Hong-Jiang Generation of GHR-modified pigs as Laron syndrome models via a dual-sgRNAs/Cas9 system and somatic cell nuclear transfer |
title | Generation of GHR-modified pigs as Laron syndrome models via a dual-sgRNAs/Cas9 system and somatic cell nuclear transfer |
title_full | Generation of GHR-modified pigs as Laron syndrome models via a dual-sgRNAs/Cas9 system and somatic cell nuclear transfer |
title_fullStr | Generation of GHR-modified pigs as Laron syndrome models via a dual-sgRNAs/Cas9 system and somatic cell nuclear transfer |
title_full_unstemmed | Generation of GHR-modified pigs as Laron syndrome models via a dual-sgRNAs/Cas9 system and somatic cell nuclear transfer |
title_short | Generation of GHR-modified pigs as Laron syndrome models via a dual-sgRNAs/Cas9 system and somatic cell nuclear transfer |
title_sort | generation of ghr-modified pigs as laron syndrome models via a dual-sgrnas/cas9 system and somatic cell nuclear transfer |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828148/ https://www.ncbi.nlm.nih.gov/pubmed/29482569 http://dx.doi.org/10.1186/s12967-018-1409-7 |
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