Construction of a recombinant human insulin expression vector for mammary gland-specific expression in buffalo (Bubalus bubalis) mammary epithelial cell line
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.
KeywordsDiabetes Beta-lactoglobulin promoter Transgenic Furin Pro-insulin
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.
- 2.Smeekens SP, Montag AG, Thomas G, Albiges-Rizo C, Carroll R, Bnig M, Phillips LA, Martin S, Ohagi S, Gardner P, Swir HH, Steiner DF (1992) Proinsulin processing by the subtilisin-related proprotein convertases furin, PC2, and PC3. Proc Natl Acad Sci USA 89:8822–8826PubMedCentralCrossRefPubMedGoogle Scholar
- 7.Groskreutz DJ, Sliwkowski MX, Gorman CM (1994) Genetically engineered proinsulin constitutively processed and secreted as mature, active insulin. J Biol Chem 25:6241–6245Google Scholar
- 23.Malewski T, Zwierzchowski L (2002) Genes expressed in the cow’s mammary gland computational analysis of 5′upstream sequences in search for factors conferring tissue and stage specific transcription. Anim Sci Papers Rep 20(1):5–20Google Scholar
- 24.Malewski T, Gajewska M, Zwierzchowski L (2005) Changes in DNA-binding activity of transcription factors in the differentiating bovine mammary gland. Anim Sci Pap Rep 23(2):75–84Google Scholar
- 32.Falqui L, Martinenghi S, Severini GM, Corbella P, Taglietti MV, Arcelloni C, Sarugeri E, Monti LD, Paroni R, Dozio N, Pozza G, Bordignon C (1999) Reversal of diabetes in mice by implantation of human fibroblasts genetically engineered to release mature human insulin. Hum Gene Ther 10:1753–1762CrossRefPubMedGoogle Scholar
- 35.Monzani PS, Sangalli JR, de Bem TH, Bressan FF, Fantinato-Neto P, Pimentel JR, Birgel-Junior EH, Fontes AM, Covas DT, Meirelles FV (2013) Breeding of transgenic cattle for human coagulation factor IX by a combination of lentiviral system and cloning. Genet Mol Res. 2013 Feb 28; 12 (AOP) [Epub ahead of print]Google Scholar
- 38.Shu JH, Zhang Y, Pan ZF, Peng SY, Cao JW, Li XC (2007) Construction of expression vector of human lactoferrin and its expression in bovine mammary epithelial cells. Belg J Zool 137(2):231–237Google Scholar
- 45.Lily C, Chao AJ, Kim SJ, Huang L, Martinson HG (1999) Assembly of the cleavage and polyadenylation apparatus requires about 10 seconds in vivo and is faster for strong than for weak poly(A) sites. Mol Cell Biol 19:5588–5600Google Scholar
- 52.Shen K, Qin X, Xiao H, Zhang X, Xu X, Han Z (2002) Mature insulin production by engineered non-beta cells. Chin Med J (Engl) 115(4):532–535Google Scholar