title: Molecular cloning and production of caprine recombinant Oct4 protein for generation induced pluripotent stem cells.
abstract: Oct4, pluripotency marker and transcription factor, expresses in embryonic stem cells. It plays a pivotal role in determination of stem cells fate. Up and down regulation of Oct4 causes differentiation of embryonic stem cells. It is one of the main transcription factors which remained concerned in every study related to induced pluripotent stem cell. Here, we report the production of goat Oct4 protein using plasmid and lentiviral based vectors. Firstly, Oct4 ORF was cloned in pAcGFP1-N1 plasmid vector and positive clones were screened with colony PCR. Oct4 was over-expressed in CHO-K1 cell line and expression was confirmed by observing green florescent protein expression in CHO-K1 cells. Secondly, Oct4 lentiviral expression construct has been prepared using pLenti-gw vector. Oct4 ORF was cloned into pLenti4/V5-DEST vector and viral particles were produced in 293FT cells. Oct4 viral particles were used to infect goat fibroblast cells. Oct4 expression was observed and confirmed in transfected goat fibroblast cells using RT-PCR. Detection of Oct4 protein in western blotting assay affirmed the capacity of over-expression of our Oct4 lentiviral vector. The lentiviral expression construct and recombinant Oct4 protein may be used for reprogramming of somatic cell into induced pluripotent stem cell.
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, India.
authors.forename: Dinesh K
authors.initials: DK
authors.lastname: Singhal
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, India.
authors.forename: Raxita
authors.initials: R
authors.lastname: Singhal
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, India.
authors.forename: Hruda N
authors.initials: HN
authors.lastname: Malik
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, India.
authors.forename: Surender
authors.initials: S
authors.lastname: Singh
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, India.
authors.forename: Sudarshan
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, India.
authors.forename: Jai K
authors.initials: JK
authors.lastname: Kaushik
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, India.
authors.forename: Ashok K
authors.initials: AK
authors.lastname: Mohanty
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, India. kdinesh777@gmail.com.
authors.forename: Dhruba
authors.initials: D
authors.lastname: Malakar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D003412:Cricetulus; D005822:Genetic Vectors; D006041:Goats / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells; D016086:Lentivirus / Q000235:genetics; D008969:Molecular Sequence Data; D050814:Octamer Transcription Factor-3 / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment
chemical_list: D050814:Octamer Transcription Factor-3; D011994:Recombinant Proteins
keywords: Induced pluripotent stem cells; Lentiviral vector; Oct4; Recombinant; Reprogramming