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Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning
Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transf...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Society for Reproduction and Development
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320426/ https://www.ncbi.nlm.nih.gov/pubmed/27725344 http://dx.doi.org/10.1262/jrd.2016-079 |
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author | GAO, Hanchao ZHAO, Chengjiang XIANG, Xi LI, Yong ZHAO, Yanli LI, Zesong PAN, Dengke DAI, Yifan HARA, Hidetaka COOPER, David K.C. CAI, Zhiming MOU, Lisha |
author_facet | GAO, Hanchao ZHAO, Chengjiang XIANG, Xi LI, Yong ZHAO, Yanli LI, Zesong PAN, Dengke DAI, Yifan HARA, Hidetaka COOPER, David K.C. CAI, Zhiming MOU, Lisha |
author_sort | GAO, Hanchao |
collection | PubMed |
description | Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications. |
format | Online Article Text |
id | pubmed-5320426 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | The Society for Reproduction and Development |
record_format | MEDLINE/PubMed |
spelling | pubmed-53204262017-02-27 Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning GAO, Hanchao ZHAO, Chengjiang XIANG, Xi LI, Yong ZHAO, Yanli LI, Zesong PAN, Dengke DAI, Yifan HARA, Hidetaka COOPER, David K.C. CAI, Zhiming MOU, Lisha J Reprod Dev Original Article Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications. The Society for Reproduction and Development 2016-10-08 2017-02 /pmc/articles/PMC5320426/ /pubmed/27725344 http://dx.doi.org/10.1262/jrd.2016-079 Text en ©2017 Society for Reproduction and Development This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License. (CC-BY-NC-ND 4.0: http://creativecommons.org/licenses/by-nc-nd/4.0/) |
spellingShingle | Original Article GAO, Hanchao ZHAO, Chengjiang XIANG, Xi LI, Yong ZHAO, Yanli LI, Zesong PAN, Dengke DAI, Yifan HARA, Hidetaka COOPER, David K.C. CAI, Zhiming MOU, Lisha Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning |
title | Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9
and handmade cloning |
title_full | Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9
and handmade cloning |
title_fullStr | Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9
and handmade cloning |
title_full_unstemmed | Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9
and handmade cloning |
title_short | Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9
and handmade cloning |
title_sort | production of α1,3-galactosyltransferase and cytidine monophosphate-n-acetylneuraminic acid hydroxylase gene double-deficient pigs by crispr/cas9
and handmade cloning |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320426/ https://www.ncbi.nlm.nih.gov/pubmed/27725344 http://dx.doi.org/10.1262/jrd.2016-079 |
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