Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering.
The porcine pluripotent cells that can generate germline chimeras have not been developed. The Oct4 promoter-based fluorescent reporter system, which can be used to monitor pluripotency, is an important tool to generate authentic porcine pluripotent cells. In this study, we established a porcine Oct...
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Format: | Article |
Language: | English |
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Public Library of Science (PLoS)
2016-01-01
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Series: | PLoS ONE |
Online Access: | http://europepmc.org/articles/PMC4710570?pdf=render |
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author | Sisi Lai Shu Wei Bentian Zhao Zhen Ouyang Quanjun Zhang Nana Fan Zhaoming Liu Yu Zhao Quanmei Yan Xiaoqing Zhou Li Li Jige Xin Yangzhi Zeng Liangxue Lai Qingjian Zou |
author_facet | Sisi Lai Shu Wei Bentian Zhao Zhen Ouyang Quanjun Zhang Nana Fan Zhaoming Liu Yu Zhao Quanmei Yan Xiaoqing Zhou Li Li Jige Xin Yangzhi Zeng Liangxue Lai Qingjian Zou |
author_sort | Sisi Lai |
collection | DOAJ |
description | The porcine pluripotent cells that can generate germline chimeras have not been developed. The Oct4 promoter-based fluorescent reporter system, which can be used to monitor pluripotency, is an important tool to generate authentic porcine pluripotent cells. In this study, we established a porcine Oct4 reporter system, wherein the endogenous Oct4 promoter directly controls red fluorescent protein (RFP). 2A-tdTomato sequence was inserted to replace the stop codon of the porcine Oct4 gene by homogenous recombination (HR). Thus, the fluorescence can accurately show the activation of endogenous Oct4. Porcine fetal fibroblast (PFF) lines with knock-in (KI) of the tdTomato gene in the downstream of endogenous Oct4 promoter were achieved using the CRISPR/CAS9 system. Transgenic PFFs were used as donor cells for somatic cell nuclear transfer (SCNT). Strong RFP expression was detected in the blastocysts and genital ridges of SCNT fetuses but not in other tissues. Two viable transgenic piglets were also produced by SCNT. Reprogramming of fibroblasts from the fetuses and piglets by another round of SCNT resulted in tdTomato reactivation in reconstructed blastocysts. Result indicated that a KI porcine reporter system to monitor the pluripotent status of cells was successfully developed. |
first_indexed | 2024-12-11T20:34:38Z |
format | Article |
id | doaj.art-7af622d4ee674549b432f9e119a00b6a |
institution | Directory Open Access Journal |
issn | 1932-6203 |
language | English |
last_indexed | 2024-12-11T20:34:38Z |
publishDate | 2016-01-01 |
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series | PLoS ONE |
spelling | doaj.art-7af622d4ee674549b432f9e119a00b6a2022-12-22T00:51:42ZengPublic Library of Science (PLoS)PLoS ONE1932-62032016-01-01111e014656210.1371/journal.pone.0146562Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering.Sisi LaiShu WeiBentian ZhaoZhen OuyangQuanjun ZhangNana FanZhaoming LiuYu ZhaoQuanmei YanXiaoqing ZhouLi LiJige XinYangzhi ZengLiangxue LaiQingjian ZouThe porcine pluripotent cells that can generate germline chimeras have not been developed. The Oct4 promoter-based fluorescent reporter system, which can be used to monitor pluripotency, is an important tool to generate authentic porcine pluripotent cells. In this study, we established a porcine Oct4 reporter system, wherein the endogenous Oct4 promoter directly controls red fluorescent protein (RFP). 2A-tdTomato sequence was inserted to replace the stop codon of the porcine Oct4 gene by homogenous recombination (HR). Thus, the fluorescence can accurately show the activation of endogenous Oct4. Porcine fetal fibroblast (PFF) lines with knock-in (KI) of the tdTomato gene in the downstream of endogenous Oct4 promoter were achieved using the CRISPR/CAS9 system. Transgenic PFFs were used as donor cells for somatic cell nuclear transfer (SCNT). Strong RFP expression was detected in the blastocysts and genital ridges of SCNT fetuses but not in other tissues. Two viable transgenic piglets were also produced by SCNT. Reprogramming of fibroblasts from the fetuses and piglets by another round of SCNT resulted in tdTomato reactivation in reconstructed blastocysts. Result indicated that a KI porcine reporter system to monitor the pluripotent status of cells was successfully developed.http://europepmc.org/articles/PMC4710570?pdf=render |
spellingShingle | Sisi Lai Shu Wei Bentian Zhao Zhen Ouyang Quanjun Zhang Nana Fan Zhaoming Liu Yu Zhao Quanmei Yan Xiaoqing Zhou Li Li Jige Xin Yangzhi Zeng Liangxue Lai Qingjian Zou Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering. PLoS ONE |
title | Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering. |
title_full | Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering. |
title_fullStr | Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering. |
title_full_unstemmed | Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering. |
title_short | Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering. |
title_sort | generation of knock in pigs carrying oct4 tdtomato reporter through crispr cas9 mediated genome engineering |
url | http://europepmc.org/articles/PMC4710570?pdf=render |
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