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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Main Authors: 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
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2016-01-01
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.
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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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