title: Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering.
abstract: 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.
authors.affiliation: CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: Sisi
authors.initials: S
authors.lastname: Lai
authors.affiliation: CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: Shu
authors.initials: S
authors.lastname: Wei
authors.affiliation: CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: Bentian
authors.initials: B
authors.lastname: Zhao
authors.affiliation: CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Ouyang
authors.affiliation: CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: Quanjun
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: Nana
authors.initials: N
authors.lastname: Fan
authors.affiliation: CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: Zhaoming
authors.initials: Z
authors.lastname: Liu
authors.affiliation: CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: Quanmei
authors.initials: Q
authors.lastname: Yan
authors.affiliation: CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Zhou
authors.affiliation: CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.affiliation: Key Laboratory of Banna Mini-pig Inbred Line of Yunnan Province, Animal Science and Technology College, Yunnan Agricultural University, Kunming, China.
authors.forename: Jige
authors.initials: J
authors.lastname: Xin
authors.affiliation: Key Laboratory of Banna Mini-pig Inbred Line of Yunnan Province, Animal Science and Technology College, Yunnan Agricultural University, Kunming, China.
authors.forename: Yangzhi
authors.initials: Y
authors.lastname: Zeng
authors.affiliation: CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.|Jilin Provincial Key Laboratory of Animal Embryo Engineering, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, China.
authors.forename: Liangxue
authors.initials: L
authors.lastname: Lai
authors.affiliation: CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: Qingjian
authors.initials: Q
authors.lastname: Zou
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001755:Blastocyst / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics*; D065150:Cellular Reprogramming; D005333:Fetus / Q000166:cytology; D005347:Fibroblasts / Q000378:metabolism; D055879:Gene Knock-In Techniques; D017930:Genes, Reporter; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000378:metabolism; D016678:Genome; D005838:Genotype; D050814:Octamer Transcription Factor-3 / Q000235:genetics*; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D034421:Sus scrofa
chemical_list: D050814:Octamer Transcription Factor-3