title: Inducing goat pluripotent stem cells with four transcription factor mRNAs that activate endogenous promoters.
abstract: Traditional approaches for generating goat pluripotent stem cells (iPSCs) suffer from complexity and low preparation efficiency. Therefore, we tried to derive goat iPSCs with a new method by transfecting exogenous Oct4, Sox2, Klf4 and c-Myc mRNAs into goat embryonic fibroblasts (GEFs), and explore the mechanisms regarding the transcription regulation of the reprogramming factors in goat iPSCs induction.
  
  
  
  mRNAs of the four reprogramming factors were transfected into GEFs, and were localized in nucleus with approximately 90% transfection efficiency. After five consecutive transfections, GEFs tended to aggregate by day 10. Clones appeared on day 15-18, and typical embryonic stem cell -like clones formed on day 20. One thousand AKP staining positive clones were achieved in 104GEFs, with approximately 1.0% induction efficiency. Immunofluorescence staining and qRT-PCR detection of the ESCs markers confirmed the properties of the goat iPSCs. The achieved goat iPSCs could be cultured to 22nd passage, which showed normal karyotype. The goat iPSCs were able to differentiate into embryoid bodies with three germ layers. qRT-PCR and western blot showed activated endogenous pluripotent factors expression in the later phase of mRNA-induced goat iPSCs induction. Epigenetic analysis of the endogenous pluripotent gene Nanog revealed its demethylation status in derived goat iPSCs. Core promoter regions of the four reprogramming factors were determined. Transcription factor binding sites, including Elf-1, AP-2, SP1, C/EBP and MZF1, were identified to be functional in the core promoter regions of these reprogramming genes. Demethylation and deacetylation of the promoters enhanced their transcription activities.
  
  
  
  We successfully generated goat iPSCs by transfection of Oct4, Sox2, Klf4 and c-Myc mRNAs into GEFs, which initiated the endogenous reprogramming network and altered the methylation status of pluripotent genes. Core promoter regions and functional transcription binding sites of the four reprogramming genes were identified. Epigenetic regulation was revealed to participate in mRNA induced iPSCs formation. Our study provides a safe and efficient approach for goat. iPSCs generation.
authors.affiliation: Department of Orthopaedics, The Frist Affiliated Hospital of Soochow University, No. 188 Shizi Street, Suzhou, Jiangsu, 215006, People's Republic of China.
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, 88 South University Ave., Yangzhou, Jiangsu, 225009, People's Republic of 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, 88 South University Ave., Yangzhou, Jiangsu, 225009, People's Republic of China.
authors.forename: Yingjie
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Animal & Avian Sciences, University of Maryland, Baltimore, MD, 20741, USA.
authors.forename: Jiuzhou
authors.initials: J
authors.lastname: Song
authors.affiliation: Department of Orthopaedics, The Frist Affiliated Hospital of Soochow University, No. 188 Shizi Street, Suzhou, Jiangsu, 215006, People's Republic of China. suzhouspine@hotmail.com.
authors.forename: Huilin
authors.initials: H
authors.lastname: Yang
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, 88 South University Ave., Yangzhou, Jiangsu, 225009, People's Republic of 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, 88 South University Ave., Yangzhou, Jiangsu, 225009, People's Republic of China. yubcli@yzu.edu.cn.
authors.forename: Bichun
authors.initials: B
authors.lastname: Li
mesh_terms: D000818:Animals; D061249:Batch Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000235:genetics; D000067470:Cellular Reprogramming Techniques / Q000379:methods; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D006041:Goats / Q000235:genetics*; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000502:physiology*; D035683:MicroRNAs / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation / Q000502:physiology
chemical_list: D035683:MicroRNAs; D014157:Transcription Factors
keywords: Goat embryonic fibroblasts; Goat iPS cells; Reprogramming; mRNA