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

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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.

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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.

Conflict of interest

The authors declare that there are no competing interests.

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Authors and Affiliations

  1. Principal Scientist, Embryo Biotechnology Lab, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132 001, India

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

  2. Environmental and Industrial Biotechnology Division, The Energy and Resources Institute (TERI), New Delhi, 110 003, India

    Archana Kumari

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  1. Ramakant Kaushik
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  2. Karn Pratap Singh
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Correspondence to Manmohan Singh Chauhan.

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11033_2014_3464_MOESM1_ESM.eps

Fig.S1. Schematic representation of the strategy used to mutate the human proinsulin cDNA. The primers INSF1, H10A1 and CA2, INSR2 were used for preparation of INS-1 and INS-4, fragments. The primes INSF1, BC1 and BC2, CA1 were used for preparation of INS-2 and INS-3 fragments. The fragment INS5 was obtained after INS-2+3 fragment ligation by overlapping extension PCR and was then ligated between INS-1 and INS-4 by overlapping extension PCR. The resulting fragment INS6 contained the entire mutated cDNA. ∆ indicates mutation at the His-10 site; □ indicates mutations at the junction of the B and C peptides; * indicates mutation at the junction of the C and A peptides. (EPS 298 kb)

11033_2014_3464_MOESM2_ESM.tif

Fig.S2. PCR amplification of all inserted fragments in pAcISUBC vector by different combination of primers: Lane 1- buBLG promoters (BLGP3F & BLGP3R primer), Lane 2- buBLG promoter + insulin fragment (BLGL1 & INSR primer). Lane 3- Human insulin gene, (INSF1 & INSR2 primer). Lane 4- Insulin + SV40polyA (INSF1 & SVR primer). Lane 5- Insulin + BLG 3’UTR fragment (INSF1 & UTRR primer). Lane 6- BLG 3’UTR (UTRF & UTRR primer). Lane 7- BLG3’UTR + CMV promoter (UTRF & CMV R primer). Lane 8- CMV promoter (CMVF & CMVR primer). (TIFF 233 kb)

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Kaushik, R., Singh, K.P., Kumari, A. et al. Construction of a recombinant human insulin expression vector for mammary gland-specific expression in buffalo (Bubalus bubalis) mammary epithelial cell line. Mol Biol Rep 41, 5891–5902 (2014). https://doi.org/10.1007/s11033-014-3464-3

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