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

Content Provider	Springer Nature Link
Author	Kaushik, Ramakant Singh, Karn Pratap Kumari, Archana Rameshbabu, K. Singh, Maj Kumar Manik, Radhey Shyam Palta, Prabhat Singla, Suresh Kumar Chauhan, Manmohan Singh
Copyright Year	2014
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 × 10$^{6}$ cells.
Starting Page	5891
Ending Page	5902
Page Count	12
File Format	PDF
ISSN	03014851
Journal	Molecular Biology Reports
Volume Number	41
Issue Number	9
e-ISSN	15734978
Language	English
Publisher	Springer Netherlands
Publisher Date	2014-06-27
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Diabetes Beta-lactoglobulin promoter Transgenic Furin Pro-insulin Animal Biochemistry Animal Anatomy / Morphology / Histology
Content Type	Text
Resource Type	Article
Subject	Genetics Medicine Molecular Biology

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