Construction of a recombinant human insulin expression vector for mammary gland-specific expression in buffalo (Bubalus bubalis) mammary epithelial cell line

Ramakant Kaushik, Karn Pratap Singh, Archana Kumari, K. Rameshbabu, Manoj Kumar Singh, Radhey Shyam Manik, Prabhat Palta, Suresh Kumar Singla, Manmohan Singh Chauhan

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5 Scopus citations

Abstract

The aim of the present study was construction of mammary gland specific expression vector for high level of human insulin (hINS) expression in transgenic buffalo for therapeutic use. We have constructed mammary gland specific vector containing human insulin gene and there expression efficiency was checked into in vitro cultured buffalo mammary epithelial cells (BuMECs). Human pro-insulin coding region was isolated from human genomic DNA by intron skipping PCR primer and furin cleavage site was inserted between B-C and C-A chain of human insulin by overlap extension PCR. A mammary gland-specific buffalo beta-lactoglobulin promoter was isolated from buffalo DNA and used for human insulin expression in BuMEC cells. The construct was transfected into BuMECs by lipofection method and positive transgene cell clones were obtained by G418 selection after 3 weeks. Expression of hINS in transfected cells were confirmed by RT-PCR, Immunocytochemistry, Western Blotting and ELISA. The pAcISUBC insulin-expressing clones secreted insulin at varying levels between 0.18 - 1.43 ng/ml/24 h/2.0 × 106 cells.

Original languageEnglish (US)
Pages (from-to)5891-5902
Number of pages12
JournalMolecular Biology Reports
Volume41
Issue number9
DOIs
StatePublished - Sep 2014
Externally publishedYes

Bibliographical note

Funding Information:
Acknowledgments This work was supported by Grant BT/ PR15035/AAQ/01/462/2011 from the Department of Biotechnology, Government of India, New Delhi, India. Author’s thanks to Ms. Neha Saini and Dr. Prashant Kadam for providing language help.

Keywords

  • Beta-lactoglobulin promoter
  • Diabetes
  • Furin
  • Pro-insulin
  • Transgenic

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