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Molecular cloning, sequence characterization and recombinant expression of Nanog gene in goat fibroblast cells using lentiviral based expression system

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Abstract

Nanog is a homeodomain containing protein which plays important roles in regulation of signaling pathways for maintenance and induction of pluripotency in stem cells. Because of its unique expression in stem cells it is also regarded as pluripotency marker. In this study goat Nanog (gNanog) gene has been amplified, cloned and characterized at sequence level with successful over-expression in CHO-K1 cell line using a lentiviral based system. gNanog ORF is 903 bp long which codes for Nanog protein of size 300 amino acids (aas). Complete nucleotide sequence shows some evolutionary mutation in goat in comparision to other species. Protein sequence of goat is highly similar to other species. Overall, gNanog nucleotide sequence and predicted protein sequence showed high similarity and minimum divergence with cattle (96 % identity/4 % divergence) and buffalo (94/5 %) while low similarity and high divergence with pig (84/15 %), human (81/23 %) and mouse (69/40 %) indicating evolutionary closeness of gNanog to cattle and buffalo. gNanog lentiviral expression construct was prepared for over-expression of Nanog gene in adult goat fibroblast cells. Lentiviral expression construct of Nanog enabled continuous protein expression for induction and maintenance of pluripotency. Western blotting revealed the expression of Nanog gene at protein level which supported that the lentiviral expression system is highly promising for Nanog protein expression in differentiated goat cell.

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Acknowledgments

University Grant Commission, Government of India has provided the Senior Research Fellowship to Dinesh K. Singhal, and Department of Biotechnology, Government of India has granted the fund for smooth running of this research work.

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Authors and Affiliations

  1. Cell and Molecular Biology Lab, National Dairy Research Institute, Karnal, 132001, Haryana, India

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

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  1. Dinesh K. Singhal
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  2. Raxita Singhal
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Correspondence to Dhruba Malakar.

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Singhal, D.K., Singhal, R., Malik, H.N. et al. Molecular cloning, sequence characterization and recombinant expression of Nanog gene in goat fibroblast cells using lentiviral based expression system. Mol Biol Rep 41, 1907–1915 (2014). https://doi.org/10.1007/s11033-013-2931-6

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  • DOI: https://doi.org/10.1007/s11033-013-2931-6

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