Mol Biol Rep DOI 10.1007/s11033-014-3464-3

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

Received: 16 February 2013 / Accepted: 14 June 2014 Ó Springer Science+Business Media Dordrecht 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 proinsulin 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 glandspecific 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

Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3464-3) contains supplementary material, which is available to authorized users. R. Kaushik  K. P. Singh  K. Rameshbabu  M. K. Singh  R. S. Manik  P. Palta  S. K. Singla  M. S. Chauhan (&) Principal Scientist, Embryo Biotechnology Lab, Animal Biotechnology Centre, National Dairy Research Institute, Karnal 132 001, India e-mail: [email protected] A. Kumari Environmental and Industrial Biotechnology Division, The Energy and Resources Institute (TERI), New Delhi 110 003, India

insulin at varying levels between 0.18 - 1.43 ng/ml/24 h/ 2.0 9 106 cells. Keywords Diabetes  Beta-lactoglobulin promoter  Transgenic  Furin  Pro-insulin

Introduction Diabetes mellitus is a major and increasing public health problem in the world. Globally, as of 2013, an estimated 382 million people have diabetes worldwide, with type 2 diabetes making up about 90 % of the cases. Secretary vesicles of pancreatic b-cells is containing endoprotease enzyme which is helping for conversion of proinsulin to mature insulin [1, 2]. In pancreatic b-cells, during the post translational modification, human proinsulin is converted into mature insulin by removing the C chain which located among the B and A chain by endoprotease enzyme. Nonneuroendocrine cell lines and primary cultures are not able to produce proinsulin to mature insulin because it contains only the constitutive pathway. These cell lines unable to any generalized, regulated secretary vesicles and also not have any specific proinsulin processing enzymes. Only two prohormone convertases enzymes are responsible for proinsulin to mature insulin conversion into the pancreatic b cells [3, 4]. Some reports indicated that the non-neuroendocrine cell lines are capable for proinsulin to mature insulin production after furin cleavage sites insertion between B–C and C–A junction [5–11]. Different organisms, including bacteria [12], yeast [13], fungi [14], mammalian cell cultures [15] and transgenic plants [16] have been used for production of recombinant human insulin but presently only Escherichia coli [12] and Saccharomyces cerevisiae [13] is widely using for

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commercial insulin production. Mammary gland of farm animals can be used as a bioreactor for production of pharmaceutical proteins in large scale because it provides correct post translational modification for production of recombinant protein and it can be easily isolated from animals. Here, we constructed an expression vector for human insulin production in mammary gland of farm animals for that buffalo beta-lactoglobulin (buBLG) promoter was used as a regulatory element.

Materials and methods Isolation of the insulin coding sequence Human insulin gene having three exons and two introns, among these only two exons (Exon 2 and 3) responsible for coding sequence (CDS) region. Both exons were isolated by PCR using two outer primer (INSF1, INSR2) and two overlapping primer (INSR1, INSF2). Outer primers contained XhoI restriction enzyme site. Amplified fragments of Exon 2 and 3, which had 9 bp overlapping ends, were mixed together after purification and 3 cycles of PCR were performed without primers using the PCR condition: 94 °C for 30 s. (denaturation), 54 °C for 30 s. (annealing), and 72 °C for 1 min. (extension). A human cDNA sequence encoding proinsulin was re-amplified using end-annealing primers with 32 cycle of PCR. The oligonucleotides used are given in table S1. In vitro mutagenesis of insulin coding sequence In proinsulin cDNA, three mutations were incorporated by overlap extension PCR, first mutation replaced His (10th position) to Asp of B peptide, second and third mutation inserted furin consensus sequence between B–C and A–C junction of proinsulin by replaced KTRR to RTKR amino acid and LQKR to RQKR amino acid as described in Fig. S1. PCR parameters were used as: 94 °C, for 30 s. (denaturation), 54 °C for 30 s. (annealing) and 72 °C for 1 min (extension). The oligonucleotides used are given in table S1. Buffalo beta-lactoglobulin promoter and 30 UTR amplification Beta lactoglobulin promoter (3.9 kb) was amplified from genomic DNA isolated from buffalo blood by using BLGP3F and BLGP3R primer containing NheI and XhoI restriction enzyme. PCR parameters were as: 94 °C, for 30 s. (denaturation), 54 °C for 30 s. (annealing) and 72 °C for 4 min (extension). The amplified 3.9 kb BLG promoter contained 3.1 kb of 50 flanking region, 0.8 kb of Exon 1, intron 1 and a small part of Exon 2. In buffalo BLG Exon 1

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contains two ATG translation-initiation codons which were replaced by non-initiating AAG and ATT sequence by overlap extension polymerase chain reaction (PCR) by using BLGL2, BLGL1 and BLGL3R, BLGL3F primers. The buBLG 30 UTR was amplified from buffalo genomic DNA by using UTRF and UTRR primer. Both primers contained restriction enzyme site XhoI (UTRF) and Cfr421 (UTRR). PCR were performed under the following conditions: 94 °C, for 30 s. (denaturation), 58 °C for 30 s. (annealing) and 72 °C for 4 min (extension).This BLG 30 UTR (1.5 kb) was comprised small parts of intron 6, Exon 7 and 30 flanking region. The oligonucleotides used are given in table S2. Beta-lactoglobulin promoter and 30 UTR sequence analyses Buffalo beta-lactoglobulin 50 UTR and 30 UTR sequences was submitted in GanBank and their accession numbers is HQ398854 and JF274007. BLAST program (http://www. ncbi.nlm.nih.gov/BLAST/) was used for sequence analysis of beta-lactoglobulin promoter and 30 UTR sequence. To establish a phylogenetic relationship of buBLG promoter and 30 UTR with other species, the ClustalW program (http://www.ebi.ac.uk/Tools/msa/clustalw2/) was used for alignment of buffalo sequence to other mammals. Based on the DNA sequences, the phylogenetic relationship was determined among buffalo and other species using Neighbor-Joining method with a bootstrap of 1,000 replicates in MEGA 4.0 software [17]. For transcription binding site search in 50 -flanking region of buBLG (promoter) we used Genomatix software (www.genomatix.de), TFSEARCH (www.cbrc.jp/research/db/TFSEARCH) and TRANSFAC software (www.gene-regulation.com). Construction of the hINS mammary gland-specific expression vector The mammalian expression vector pAcGFP-N1 (Clontech, CA) was used as a backbone for preparation of gene constructs. The buBLG promoter (3.9 kb) was inserted into MCS (Multiple cloning sites) of pAcGFP-N1 vector (4.7 kb) by digestion with NheI and XhoI restriction enzyme. The buBLG 30 UTR (1.5 kb) was inserted into MCS of pAc?pBLG vector by XhoI and Cfr421 restriction digestion and ligation. MluI restriction enzyme site was inserted into pAc?pBLG?30 UTR vector by CMV promoter (602 bp), amplified by CMVpF1 primer containing AseI and MluI restriction enzyme and CMVpR1 primer containing NheI enzyme. This CMV promoter was inserted into vector by digestion with AseI and NheI restriction enzyme and ligation. 29 b-Globin insulator (495 bp) was isolated from pBT268 expression vector (Addgene, Cambridge, USA) and inserted into pAc?pBLG?30 UTR vector by MluI and NheI

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Fig. 1 Diagrammatically presentation of the expression vector pAcISUBC: the pAcISUBC vector contain 494 bp 29 insulator, 3.9 kb of 50 flanking region including Exon 1 and intron 1 of BLG gene, 357 bp

human insulin cDNA sequence, 1.5 kb BLG 30 UTR including intron 6 and Exon 7 into a pAcGFP-N1 plasmid

restriction digestion and ligation. SV40polyA fragment (203 bp) was amplified from pAcGFP-N1 expression vector by SVF primer containing overlapping ends of human proinsulin and SVR primers containing XhoI enzyme. Mutated human proinsulin (357 bp) was re-amplified by INSF1 primer contained XhoI and INSV primer contained overlapping ends of SV40polyA fragment. Both fragments were joined together by overlapping PCR and inserted into pAc?pBLG?30 UTR?2xb vector by digestion with XhoI restriction enzyme and ligation. For eGFP expression, CMV promoter was amplified with CMVF and CMVR primer contained Cfr421 and AgeI restriction site and inserted into MCS of pAc?pBLG?30 UTR?2xb?INS?SV vector by restriction digestion and ligation. Finally, 11 kb GFP tag pAcISUBC expression vector was prepared for human insulin gene expression in mammary epithelial cells (Fig. 1). The oligonucleotides used are given in table S2. Recombinant plasmid pAcISUBC was confirmed by different set of primers PCR (Fig. S2).

Confluence BuMECs cells at passage 7 were used for transfection in 6-well plate. Expression vector was linearized by AflII restriction enzyme digation and Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) was used for transfection. Pure population of transfected cells was isolated after 3 weeks of G418 (500 lg/ml) (Gibco) selection (Fig. 2).

Cell culture, DNA transfection BuMECs were isolated from mammary tissue by digestion method as described earlier [18, 19]. Briefly, the mammary tissue was cut into very small pices and digested in DMEM medium containing 1 % FBS (Sigma-Aldrich), 2 mM Lglutamine (Sigma-Aldrich), 0.1 % collagenase (SigmaAldrich), 0.05 % hyaluronidase (Sigma-Aldrich), 0.03 % DNase-1 (Sigma-Aldrich), 0.25 % trypsin (Sigma-Aldrich) and 50 lg/ml penicillin G, 50 lg/ml streptomycin sulfate, 50 lg/ml gentamicin and 1 lg/ml fungizone (Gibco, Grand 89 Island, NY) with gentle agitation at 37 °C for 2 h. Undissociated tissue and debris was removed by filtration through stainless steel meshes (80 M) and filtered cells were isolated by centrifugation and then washed with culture medium for 4–6 times. The isolated cells were cultured in 35 mm collagen coated dishes with growth medium containing DMEM, 10 % fetal bovine serum, hydrocortisone (1 lg/ml; Sigma-Aldrich), insulin (5 lg/ ml; Sigma-Aldrich), epidermal growth factor (10 ng/ml; Sigma-Aldrich), prolactin (5 lg/ml; Sigma-Aldrich), 50 lg/ml streptomycin sulfate, 50 lg/ml penicillin G, 50 lg/ml gentamicin and fungizone and maintained at 37 °C in 5 % CO2.

RNA isolation and RT-PCR For milk protein gene expression analysis in transfected BuMEC cells (Passage-3) were embedded in collagen coated flask and cultured for 3 days with DMEM medium supplemented with 10 % fetal bovine serum and growth factors [insulin (5 lg/ml), hydrocortisone (1 lg/ml), prolactin (5 lg/ml), epidermal growth factor (10 ng/ml)]. After 3 days of plating the cell were cultured with serum free medium containing growth factor and without growth factors. We used the bovine pituitary extract (50 lg/ml BPE; sigma-aldrich) and 0.1 % BSA instead of FBS and IGF-1 (50 ng/ml; Gibco BRL) instead of insulin. After 24 h culture both cells were trypsinized and RNA was isolated from Trizol (Invitrogen) method. Any DNA contamination in RNA was removed by DNase I treatment (Fermentas, PA, USA) and DNase free RNA was used for cDNA synthesis by SuperScript III kit (Invitrogen). The expression of betacasein (CSN2), beta-lactoglobulin (BLG) and cytokeratin 18 (Cyto18) was analyzed by RT-PCR. Buffalo furin gene expression in BuMECs and human insulin gene expression in transfected and non-transfected BuMECs (Both cells were cultured with DMEM containing BPE, 0.1 % BSA, IGF-1, Hydrocortisone, prolactin and EGF as described previously in ‘‘RNA isolation and RTPCR’’ section) was analysed by RT-PCR. GAPDH gene was used as internal control. PCR parameters were as: 94 °C, for 30 s. (denaturation), 58 °C for 30 s. (annealing) and 72 °C for 1 min (extension). The oligonucleotides used are given in table S3. Immunocytochemistry Human insulin expression in transfected BuMECs was examined by immunostaining according to Zheng et al. [20] with slight modifications. The pAcISUBC expression vector was digested with Cfr421 and AflII restriction

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Fig. 2 EGFP expression in pAcISUBC transfected BuMECs: after 24 h transfection (9100) (a, b). Pure population of transfected BuMECs after G418 selection (9100) (c, d).View of cells under bright light (c, d) and under UV light (a, b)

enzyme for removing the pCMV?eGFP?SV40polyA fragment and remaining linearized expression vector was used for transfection into BuMECs. This transfected and non-transfected BuMECs at passage 3 were used for immunostaining. After 4 days of culture on 24 well culture plate (culture method and medium described previously in ‘‘RNA isolation and RT-PCR’’ section), media were removed and washed with 1 9 PBS. Cells were fixed into culture plate by incubated with 4 % paraformaldehyde (Sigma-Aldrich) for 20 min at 37 °C. Cells were blocked with 1x BSTX solution (19 PBS containing 10 % simple BSA, 0.1 % Triton X-100) by 30 min incubation. The cells were incubated with 1:100 diluted Guinea Pig Anti-human insulin primary antibody (Millipore, MA, USA) for 1 h. After incubation with primary antibody, cells were washed five times with 19 PBS and incubated with FITC labelled monoclonal anti-Guinea Pig IgG secondary antibody at 1:100 dilutions for 1 h. For nuclear staining, Hoechst stain (Sigma-Aldrich) was used and imaged by using phasecontrast microscope (Nikon eclipse Ti). Enzyme-linked immunosorbent assay (ELISA) Secreted recombinant human insulin into the culture media was quantified using the human insulin ELISA kit (Animal serum free) (Millipore, MA, USA). To assess secreted

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insulin, transfected and non-transfected BuMECs were then exposed to a serum-free medium containing DMEM, BPE, BSA, IGF-1, Hydrocortisone, prolactin and EGF for 24 h of incubation (described in previous ‘‘RNA isolation and RT-PCR’’ section). After 24 h incubation, cell culture medium was collected in fresh tube and cells debris was removed by centrifugation at 2,000 rpm for 5 min. These media proteins were concentrated using Protein Concentrators PES (Thermo Fisher Scientific, Rockford, IL USA) and proteins were quantified at 450 nm absorbance by using Automated ELISA reader (TECAN, Salzburg, Austria). Western blotting For human insulin protein expression analysis, we used a native polyacrylamide gel electrophoresis (native-PAGE) as described previously [21]. Secreted proteins into the culture media from the transfected and non-transfected BuMECs (culture method and medium has been described previously in ‘‘RNA isolation and RT-PCR’’ section), were concentrated using Protein Concentrators PES and concentrated proteins were separated on the native PAGE. After separation, protein were transferred onto a PVDF membrane (Millipore, USA) from native gel by using semidry blotting system (GeNei, Bangalore, India) at 80 mA for

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Fig. 3 In vitro mutagenesis of hINS sequence: insulin coding sequence synthesis by PCR amplification and point mutation inserted into hINS cDNA by overlapping PCR: lane 1 Exon 2 fragment, lane 2 Exon 3 fragment, lane 3 ligated Exon 2 and Exon 3 fragment, lane 4

INS1, lane 5 INS2, lane 6 INS3, lane 7 INS4, lane 8 INS5 fragment amplified after INS2 and INS3 fragments ligation, lane 9 INS6 fragment, after INS1 ? INS5 ? INS4 fragments ligation

1 h and 30 min. Membrane was blocked with blocking reagent contain TBS and skim milk for 1 h at room temperature. A Human insulin specific Guinea Pig polyclonal antibody (Millipore, USA) was used as a primary antibody and Horseradish peroxidase (HRP) conjugated anti Guinea Pig IgG antibody (Millipore, USA) was used as a secondary antibody. Finally, proteins were visualized using a DAB System (GeNei, Bangalore, India).

and found 210 bp product size of Exon 2 and 168 bp of Exon 3. Both amplified fragments having overlapping ends were joined together by ligation PCR and found to have 357 bp product size of insulin coding sequence. For introducing three mutations into the proinsulin fragment we used six overlapping primers in overlap extension PCR. For mutation insertion, Insulin gene was amplified in five fragments by overlapping primers. First fragment INS1 (119 bp) was amplified by using INSF1 and H10A1 primer for insertion of His-10 to Asp mutation in B chain. Second fragment INS2 (182 bp) was amplified by using INSF1 and BC1 primer for insertion of furin cleavage site in B chain. Third fragment INS3 (115 bp) was amplified by using BC2 and CA1 primer for insertion of furin cleavage site in C chain. Fourth fragment INS4 (103 bp) was amplified by using CA2 and INSR2 primer for insertion of furin cleavage site in A chain. INS5 (177 bp) fragment was generated by INS2 and INS3 fragment ligation through

Results Human proinsulin mutants Human insulin gene have three exons among these only two exons (Exon 2, 3) make full length CDS. Both fragments were amplified by using two outer primers (INSF1, INSR2) and two overlapping primers (INSLR1, INSLF1) Fig. 4 Sequencing result of hINS: mutated human proinsulin gene sequence and chromatogram: red and yellow boxes indicating mutation region. (Colour figure online)

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PCR using H10A2 and CA1 primer. Finally INS6 (357 bp) fragment was prepared by INS1, INS5 and INS4 fragment ligation through ligation PCR (Fig. 3). Sequencing result of mutated human proinsulin fragment showing in Fig. 4.

Fig. 7 50 -Flanking region of the buffalo BLG gene sequence and c their predicted transcription factor binding sites: The Exon 1 starting sequence was designated as ?1. Putative transcription factor binding sites (TFBS) were identified from BLG promoter sequence by transcription factor databases and the literature. TFBS are highlighted in gray boxes

Cloning and sequencing analysis of buBLG promoter and 30 UTR Mutated buffalo beta lactoglobulin promoter fragment was confirmed by DNA sequence analysis. Mutated translation initiation codon is showing in Fig. 5. The buBLG 50 flanking region amplified was 3.969 kbp in size and had 19.07 % A, 29.96 % C, 27.79 % G, 23.15 % T; A?T and G?C content were 42.23 and 57.75 %, respectively. The buBLG gene 30 UTR region amplified was 1.548 kbp and had 23.58 % A, 25.84 % C, 30.10 % G, 20.48 % T; A?T and G?C content were 44.06 and 55.94 %, respectively. BLAST analysis of 50 -flanking and 30 UTR region of BLG gene was showed similarity to other species, like bovine,

sheep and goat. Alignment result showed 95 % (bovine), 90 % (goat), 90 % (sheep) similarity of 50 -flanking region and 96 % (bovine), 93 % (goat), 87 % (sheep) similarity of 30 UTR respectively. The 50 -flanking and 30 UTR sequence of BLG gene was used for Phylogenetic tree analysis between buffalo, bovine, sheep and goat. The tree shows one cluster between buffalo and bovine and another cluster between goat and sheep (Fig. 6). This result indicated that bovine and buffalo might evolve from the same ancestor. Putative transcription factor binding sites (TFBS) were identified from buBLG promoter sequence by transcription factor databases and the literature [22–24]. Buffalo BLG promoter is sowing all essential TFBS which is responsible for gene regulation. The analysis showed the 50 flanking region of BLG gene contained putative binding sites for mammary gland specific-mammary cell activating factor (MAF), mammary gland specific nuclear factor (MGF), pregnancy specific mammary nuclear factor (PMF), as well as ubiquitous TFs-NF-kappa B binding site (NF-kB), CCAAT/enhancer binding protein (C/EBP), insulin response element (IRE), glucocorticoid response elements (GRE), yin and yang elements (YY1), CCAAT-binding factor/nuclear factor 1 (CTF/NF1), octamer 1 (Oct1), cAMP response element-binding protein (CERB) and stimulating protein 1 (Sp1) (Fig. 7). Milk protein gene expression in transfected BuMECs

Fig. 5 Chromatogram figure of mutated buffalo BLG gene region: the mutated translation initiation codon marked as red box. (Colour figure online)

Establishing of culture conditions of mammary epithelial cells is important for protein synthesis. We compared the protein synthesis ability of the transfected BuMECs cultured in growth medium and without growth medium by mRNA expression of the b-casein, b-lactoglobulin, Cyto18. The RT-PCR results showed that the transfected BuMECs cultured in growth medium was expressed all genes (Fig. 8a) and the transfected BuMECs cultured in without growth medium was expressed only Cyto18 (Fig. 8b). Identification of insulin production in BuMECs RT-PCR analysis

Fig. 6 Phylogenetic tree of BLG 50 UTR and 30 UTR sequences: (a) 50 UTR of BLG genes with other species and (b) 30 UTR of BLG genes with other species

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Before transfection of human insulin expression vector, furin mRNA expression was detected in BuMECs by RTPCR (Fig. 9a). cDNA was synthesized from transfected

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Immunocytochemical analysis Expression of insulin was determined by immunocytochemical analysis. Transfection of hINS cDNAs into mammary epithelial cells resulted in the translation of the corresponding proteins by the cells. BuMECs were seeded at 40 % confluency and tested for insulin expression using an insulin antibody. After observation in fluorescence microscop transfected cells cytoplasm showed green color and nucleus given blue signals but non-transfected cells (control) given only nuclear stain signal (Fig. 10). Western blot analysis Fig. 8 Milk protein gene expression was detected by RT-PCR in transfected BuMECs cultured in growth medium (a), without growth medium (b). Lane 1 b-casein, lane 3 b-lactoglobulin, lane 5 cytokeratin-18, lane 7 GAPDH and lanes 2, 4, 6, 8 shown negative PCR of respective gene

Culture media was collected after 24 h of culture in serum free media. Secretory proteins were concentrated from medium using protein concentrators PES (Thermo Fisher Scientific, Rockford, IL USA). Isolated proteins were separated in Native-PAGE and anti-insulin polyclonal antibody was used for blotting. Western blotting result showed that secreted human insulin in media from transfected BuMECs cultured in growth medium given positive signal but transfected BuMECs cultured in without growth medium and non-transfected BuMECs given negative signal of human insulin protein expression. Secreted human insulin protein (positive control) and transfected BuMECs cultured in growth medium given approximately 5.8 kDa band (Fig. 11). Enzyme-linked immunosorbent assays

Fig. 9 Buffalo furin and human insulin expression: (a) RT-PCR showing furin mRNA expression in buffalo mammary epithelial cells. Lane 1 furin gene, lane 2 negative PCR. (b) GAPDH as positive control. Lane 1 GAPDH, lane 2 negative PCR. (c) RT-PCR showing human insulin mRNA expression in transfected and non-transfected buffalo mammary epithelial cells. Lane 1 non transfected BuMECs, lane 2 pAcISUBC transfected BuMECs. (d) GAPDH as positive control. Lane 1 non transfected BuMECs, lane 2 pAcISUBC transfected BuMECs

Transfected and non-transfected BuMECs was grown in growth medium and these cells were washed three times with serum-free medium and incubated in 4 ml of serumfree medium for 24 h. After 24 h conditioned media was collected, and insulin levels were measured using a human insulin ELISA kit (animal serum free) (Millipore, MA, USA) that detects human insulin with no cross-reactivity to proinsulin or C-peptide. The pAcISUBC insulin-expressing clones secreted insulin at varying levels between 0.18 1.43 ng/ml/24 h/2.0 9 106 cells.

Discussion mammary epithelial cells by SuperScript III kit and human proinsulin expression was detected by PCR amplification. Insulin mRNA expression was detected in only transfected BuMECs but not in the non-transfected BuMECs (control) (Fig. 9c). The expression of human proinsulin was detected in pAcISUBC transfected cells. Amplification of GAPDH as an endogenous control in all cases, indicated that the mRNA was of required quality and that reverse transcription and PCR were conducted successfully (Fig. 9b, d).

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In pancreatic b-cells, during the post translational modification, human proinsulin is converted into mature insulin by removing the C chain which located among the B and A chain by endoprotease enzyme. Non-neuroendocrine cell lines and primary cultures are not able to produce proinsulin to mature insulin because it contains only the constitutive pathway. These cell lines not contain any generalized, regulated secretory vesicles and also not have

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Fig. 10 Human insulin expression in transfected BuMECs analyzed by immunocytochemistry. (a) BuMECs in bright light (9200). (b) BuMECs cytoplasm showed green color and nuclei labeled with

Fig. 11 Western blot analysis of human insulin protein: lane 1 secreted human insulin (positive control), lane 2 non-transfected cells (negative control), lane 3 transfected cells cultured in growth medium, lane 4 transfected cells cultured in without growth medium.

any specific proinsulin processing enzymes. There are two other members of the mammalian subtilisin enzymes which are widely expressed, furin [25–27] and PACE4 [28]. Furin is a propeptide-processing endoprotease in nonendocrine cells and has been demonstrated to be present in virtually all nonendocrine cells, including fibroblasts, epithelial cells, and hepatocytes. Furin cleaves the concensus processing site –Arg-4–X-3–Lys/Arg-2–Arg-1 ; X?1–. Previous study reported that the different types of nonendocrine cell lines contain different quantities of furin mRNA (NIH3T3 [ HepG2 [ COS [ CHO) [29]. When

Hoechst showed blue color (9200). (c) Control BuMECs in bright light (9200). (d) Control cell showed only nuclear stain with Hoechst (blue) (9200). (Colour figure online)

mutant insulin was transfected in these cell lines, the conversion of proinsulin to mature insulin was approximately 85 % in NIH3T3, 70 % in HepG2, 60 % in COS, and 50 % in CHO [29]. Kasten-Jolly et al. [30] demonstrated, the nonendocrine cells have not contained any b cell-specific endoproteases, therefore after transfection with wild type proinsulin these cells have not been produced mature insulin. Gros et al. [31] and Falqui et al. [32] was modified the human proinsulin fragment by inserted furin cleavage sequences between B–C and C–A junctions of proinsulin by site directed mutagenesis. This mutated proinsulin was transfected into hepatic and fibroblast cells and found biologically active mature insulin. Groskreutz et al. [7] reported mutation in B-chain (His B10 to Asp) of human insulin in hyperinsulinemia patients. This mutation allows 10- to over 100-fold more mature insulin production in the body. For production of mature insulin in BuMECs, we inserted His to Asp mutation at position 10 in the B-chain and furin cleavage sequence was incorporated at B–C and C–A junctions of human proinsulin. Our results demonstrate that the mutated human proinsulin is capable for producing biologically active mature insulin in nonendocrine cells. For generation of mammary gland as a bioreactor, selection of appropriate regulatory element is important and should express in only mammary tissue and high level

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expression of recombinant protein in transgenic animal. For detection of expression vector efficiency in mammary cells, we transfected liniarized pAcISUBC vector into in vitro cultured BuMECs and exogenous hINS expression was detected by immunocytochemistry, western blot assays and ELISA. Previously, Ilan et al. [33] was constructed a BLG/Human Serum Albumin (HSA) hybrid gene containing expression vector for recombinant HSA gene expression in ovine mammary epithelial cell line NISH. Monzani et al. [34] was successfully used b-casein promoter for EmGFP expression in bovine mammary epithelial cells. Monzani et al. [35] was used lantiviral vector containing b-casein promoter for hFIX expression in bovine mammary epithelial cells. Zhang et al. [36] was constructed a mammary gland-specific expression vector for hGCase gene and transfected into in vitro cultured HC11 MECs and biological activity was detected through mRNA expression and hGCase protein level. Liu et al. [37] was used bovine b-casein promoter for expression of HBD3 gene in GMEC lines and the mRNA expression was detected by RT-PCR and protein of HBD3 was detected by Western blot analysis in transgenic cells. Previously, beta lactoglobulin promoter is broadly used for transgene expression in mammary epithelial cells [38– 40]. A number of reports have been confirmed that the first intron plays a significant role in exogenous gene transcription and accurate splicing [41, 42]. When regulatory sequence of BLG gene with exon1 and intron 1 would used as a promoter for expression of recombinant protein, fused protein will produced because Exon 1 containing translation initiation codon. For prevent the fused protein formation we replaced first initiation codon ATG to AAG and second potential initiation codon ATG to ATT of exon1 of BLG gene. Reichenstein et al. [43] reported, the higher level of luciferase expression when positioned an additional UTR region (SV40PA) in the front of BLG native poly termination site in the mammary gland specific expression vector. Similarly, we have used the same combination of 30 UTR region and found the higher expression of insulin gene in mammary epithelial cells. It has been also reported that longer polyadenylation sequences result in higher levels of expression, irrespective of the reporter gene used [44], and numerous studies exemplify elements flanking the core poly A signal which are essential for full activity [45]. In buffalo, the 50 flanking region of BLG gene contain putative TATA box as described previously in bovine sequence [46]. Malewski [47] was found common motifs like C/EBP, CTF/NF1 and MGF in 28 mammary specific promoters by computer analysis. In the last several years the regulatory elements of milk protein genes has been widely studied, and the number of TFs involved in their induction has been characterized. These factors include

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C/EBP, CTF/NFI, GR, MAF, PMF, STAT5, YY1 [48, 49]. Malewski and Zwierzchowski [23] reported potential binding sequence of cow milk protein gene promoter for various TFs and found putative binding sites for mammary gland specific MAF, STAT5, PMF, as well as ever-present TFs-AP1, CREB, GR, NF-1, NF-kB, Oct1, Sp1, YY1. Malewski et al. [24] reported that the CREB, NFI, NF-jB, Oct1 and Sp1 transcription factors are present in the bovine mammary gland and are expressed at different levels during the lactation-reproduction cycle. Pregnancy is associated with changes in Sp1 DNA–protein binding activity. During transition from pregnancy to lactation changes take place in pattern of DNA–protein binding activity of transcription factors NFI, Oct1 and Sp1. Results from the present study indicated that pAcISUBC transfected BuMECs secreted mature human insulin. Immunocytochemical analysis of the translated proteins reveled that insulin was expressed in the cytoplasmic region of the BuMECs. Previous study also reported the same results in Vero cells [50], Human mesenchymal stem cells [51], LO2 cell [52], L-cell line [53] and COS-7 cells [54]. In the present study, we found the successful human insulin gene expression in BuMECs and recombinant human insulin expression was confirmed by western blotting and ELISA method. These results confirm that furin cleavage site inserted proinsulin is able to produce mature insulin in BuMECs. In contrast, control BuMECs not showing any protein band of human insulin in western blotting. Generally SDS and DTT is using as a reducing agent in PAGE [55]. Because of SDS and DTT separated the A- and B-chain of the insulin protein by removing the disulfide bonds from insulin protein. In addition, generally SDS-PAGE is using for[30 kDa protein separation [55]; therefore, separation of human insulin protein, native-PAGE is strongly recommended [21]. Previously many studies reported that mutated proinsulin is processed into products that co-migrate with the A and B chains of insulin, when the protein was run into SDS PAGE [7, 10, 56].

Conclusion In conclusion, we prepared mammary gland specific expression vector for high level human insulin expression in milk of farm animals. Additionally, this expression system can be used for producing other therapeutic important drug proteins in low cost. We have generated a BuMEC cell line that expressed human insulin under the control of the buBLG promoter. This expression system will used for production of transgenic donor cell for SCNT (somatic cell nuclear transfer) to produce transgenic animal.

Mol Biol Rep 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 interests.

The authors declare that there are no competing

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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.

The aim of the present study was construction of mammary gland specific expression vector for high level of human insulin (hINS) expression in transge...
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