Skip to main content
SpringerLink
Log in

Molecular cloning and production of caprine recombinant Oct4 protein for generation induced pluripotent stem cells

  • Short Communications
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. Schöler HR, Ruppert S, Suzuki N, Chowdhury K, Gruss P (1990) New type of POU domain in germ line-specific protein Oct-4. Nature 344:435–439

    Article  PubMed  Google Scholar 

  2. Rosner Rosner MH, Vigano MA, Ozato K, Timmons PM, Poirier F, Rigby PW, Staudt LM (1990) A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature 21:686–692

    Article  Google Scholar 

  3. Palmieri SL, Peter W, Hess H, Schöler HR (1994) Oct-4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. Dev Biol 166:259–267

    Article  CAS  PubMed  Google Scholar 

  4. Gu P, LeMenuet D, Chung AC, Mancini M, Wheeler DA, Cooney AJ (2005) Orphan nuclear receptor GCNF is required for the repression of pluripotency genes during retinoic acid-induced embryonic stem cell differentiation. Mol Cell Biol 25:8507–8519

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Schöler H, Smith A (1998) Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95:379–391

    Article  CAS  PubMed  Google Scholar 

  6. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676

    Article  CAS  PubMed  Google Scholar 

  7. Takahashi K, Okita K, Nakagawa M, Yamanaka S (2007) Induction of pluripotent stem cells from fibroblast cultures. Nat Protocols 2:3081–3089

    Article  CAS  PubMed  Google Scholar 

  8. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920

    Article  CAS  PubMed  Google Scholar 

  9. Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, Bernstein BE, Jaenisch R (2007) In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448:318–324

    Article  CAS  PubMed  Google Scholar 

  10. Maherali N, Sridharan R, Xie W, Utikal J, Eminli S, Arnold K, Stadtfeld M, Yachechko R, Tchieu J, Jaenisch R, Plath K, Hochedlinger K (2007) Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 1:55–70

    Article  CAS  PubMed  Google Scholar 

  11. Meissner A, Wernig M, Jaenisch R (2007) Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat Biotechnol 25:1177–1181

    Article  CAS  PubMed  Google Scholar 

  12. Lowry WE, Richter L, Yachechko R, Pyle AD, Tchieu J, Sridharan R, Clark AT, Plath K (2008) Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc Natl Acad Sci USA 105:2883–2888

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Okita K, Ichisaka T, Yamanaka S (2007) Generation of germline competent induced pluripotent stem cells. Nature 448:313–317

    Article  CAS  PubMed  Google Scholar 

  14. Park IH, Zhao R, West JA, Yabuuchi A, Huo H, Ince TA, Lerou PH, Lensch MW, Daley GQ (2008) Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451:141–146

    Article  CAS  PubMed  Google Scholar 

  15. Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K (2008) Induced pluripotent stem cells generated without viral integration. Science 322:945–949

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Shi G, Jin Y (2010) Role of Oct4 in maintaining and regaining stem cell pluripotency. Stem Cell Res Ther 1:39

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Shu J, Wu C, Wu Y, Li Z, Shao S, Zhao W, Tang X, Yang H, Shen L, Zuo X, Yang W, Shi Y, Chi X, Zhang H, Gao G, Shu Y, Yuan K, He W, Tang C, Zhao Y, Deng H (2013) Induction of pluripotency in mouse somatic cells with lineage specifiers. Cell 153:963–975

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Montserrat N, Nivet E, Sancho-Martinez I, Hishida T, Kumar S, Miquel L, Cortina C, Hishida Y, Xia Y, Esteban CR, Izpisua Belmonte JC (2013) Reprogramming of human fibroblasts to pluripotency with lineage specifiers. Cell Stem Cell 13:1–10

    Article  Google Scholar 

  19. Kim D, Kim CH, Moon JI, Chung YG, Chang MY, Han BS, Ko S, Yang E, Cha KY, Lanza R, Kim KS (2009) Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4:472–476

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Kim JB, Zaehres H, Wu G, Gentile L, Ko K, Sebastiano V, Araúzo-Bravo MJ, Ruau D, Han DW, Zenke M, Schöler HR (2008) Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature 454:646–650

    Article  CAS  PubMed  Google Scholar 

  21. Garg S, Dutta R, Malakar D, Jena MK, Kumar D, Sahu S, Prakash B (2012) Cardiomyocytes rhythmically beating generated from goat embryonic stem cell. Theriogenology 7:829–839

    Article  Google Scholar 

  22. Lengner CJ, Welstead GG, Jaenisch R (2008) The pluripotency regulator Oct4: a role in somatic stem cells? Cell Cycle 7:725–728

    Article  CAS  PubMed  Google Scholar 

  23. Niwa H, Miyazaki J, Smith AG (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24:372–376

    Article  CAS  PubMed  Google Scholar 

  24. Cheng X, Meng S, Deng J, Lai W, Wang H (2011) Identification and characterization of the Oct4 extended transcriptional regulatory region in Guanzhong dairy goat. Genome 54:812–818

    Article  CAS  PubMed  Google Scholar 

  25. Pan G, Li J, Zhou Y, Zheng H, Pei D (2006) A negative feedback loop of transcription factors that controls stem cell pluripotency and self-renewal. FASEB J 20:1730–1732

    Article  CAS  PubMed  Google Scholar 

  26. Zhou H, Wu S, Joo JY, Zhu S, Han DW, Lin T, Trauger S, Bien G, Yao S, Zhu Y, Siuzdak G, Schöler HR, Duan L, Ding S (2009) Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 8:381–384

    Article  Google Scholar 

Download references

Acknowledgments

We would like to thank Department of Biotechnology, Govt. of India for providing funds and University Grant Commission, India for providing financial help to Dr. Dinesh K. Singhal as UGC-Senior Research Fellowship for smooth running of research work.

Author information

Authors and Affiliations

  1. Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, India

    Dinesh K. Singhal, Raxita Singhal, Hruda N. Malik, Surender Singh, Sudarshan Kumar, Jai K. Kaushik, Ashok K. Mohanty & Dhruba Malakar

Authors
  1. Dinesh K. Singhal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Raxita Singhal
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hruda N. Malik
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Surender Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sudarshan Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jai K. Kaushik
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ashok K. Mohanty
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Dhruba Malakar
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dhruba Malakar.

Ethics declarations

Conflicts of Interest

The authors declare no conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 2038 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Singhal, D.K., Singhal, R., Malik, H.N. et al. Molecular cloning and production of caprine recombinant Oct4 protein for generation induced pluripotent stem cells. Mol Biol Rep 42, 1583–1591 (2015). https://doi.org/10.1007/s11033-015-3926-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-015-3926-2

Keywords

Search

Navigation