title: CRISPR/Cas9-Mediated Deletion of C1EIS Inhibits Chicken Embryonic Stem Cell Differentiation Into Male Germ Cells (Gallus gallus).
abstract: We previously found that C1EIS is preferentially expressed in Chicken spermatogonial stem cells (SSCs) by RNA sequencing (RNA-seq), so our current study focused on C1EIS's role in Chicken embryonic stem cells (ESCs) differentiation into male germ cells. We constructed a CRISPR/Cas9 vector targeting C1EIS. T7 endonuclease I (T7EI) digestion method and sequencing of TA cloning were used to detect the knock-out efficiency of the Single guide RNA (sgRNA) after the cas9/gRNA vector transfected into D fibroblasts 1(DF-1), ESCs, and Chicken embryos. The results showed that CRISPR/Cas9 gene knockout efficiency is about 40%. Differentiation of the targeted ESCs into SSCs was inhibited at the embryoid body stage due to C1EIS deficiency. Immunofluorescent staining revealed that the mutagenized ESCs (RA (Retinoic Acid) with C1EIS Knock out) expressed lower levels of integrin α6 and integrin β1 compared to wild type cells. Quantitative real-time PCR (QRT-PCR) revealed Oct4 and Sox2 expression significantly increased, contrarily integrin β1 and Stra8 expression significantly decreased than RA induced group and RA with C1EIS Overexpression. During retinoic acid-induced differentiation, knockout of C1EIS in ESCs inhibited formation of SSC-like cells, suggesting C1EIS plays a vital role in promoting differentiation of avian ESCs to SSCs by regulating expression of multiple pluripotency-related genes. J. Cell. Biochem. 118: 2380-2386, 2017. © 2017 Wiley Periodicals, Inc.
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R. China.
authors.forename: Qisheng
authors.initials: Q
authors.lastname: Zuo
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R. China.
authors.forename: Kai
authors.initials: K
authors.lastname: Jin
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R. China.
authors.forename: Yingjie
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Animal and Avian Sciences, University of Maryland, College Park 20741, Maryland.
authors.forename: Jiuzhou
authors.initials: J
authors.lastname: Song
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R. China.
authors.forename: Yani
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R. China.
authors.forename: Bichun
authors.initials: B
authors.lastname: Li
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics / Q000502:physiology*; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D002642:Chick Embryo; D002645:Chickens; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005854:Germ Cells / Q000166:cytology* / Q000378:metabolism*; D007150:Immunohistochemistry; D019012:Integrin beta1 / Q000235:genetics / Q000378:metabolism; D008297:Male; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D019012:Integrin beta1; D050814:Octamer Transcription Factor-3; D055748:SOXB1 Transcription Factors; D003850:Deoxyribonuclease I
keywords: C1EIS; CRISPR/Cas9; EMBRYONIC STEM CELLS; SPERMATOGONIAL STEM CELLS