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.
journal_name
Mol Biol Repjournal_title
Molecular biology reportsauthors
Kaushik R,Singh KP,Kumari A,Rameshbabu K,Singh MK,Manik RS,Palta P,Singla SK,Chauhan MSdoi
10.1007/s11033-014-3464-3subject
Has Abstractpub_date
2014-09-01 00:00:00pages
5891-902issue
9eissn
0301-4851issn
1573-4978journal_volume
41pub_type
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