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Animal Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/labt20
MAC-T Cells as a Tool to Evaluate Lentiviral Vector Construction Targeting Recombinant Protein Expression in Milk Paulo S. Monzani f
a b c d
, Samuel Guemra
V. Meirelles & Matthew B. Wheeler
a b
, Paulo R. Adona
a b
e
, Otavio M. Ohashi , Flávio
c d
a
Centro de Ciencias Biológicas e da Saúde , Universidade Norte do Paraná , Londrina , Paraná , Brazil b
Agropecuária Laffranchi , Tamarana , Paraná , Brazil
c
Department of Animal Sciences , University of Illinois at Urbana-Champaign , Urbana , Illinois , USA d
Click for updates
Institute for Genomic Biology , University of Illinois at Urbana-Champaign , Urbana , Illinois , USA e
Instituto de Ciências Biológicas, Universidade Federal do Pará , Belém , Paraná , Brazil
f
Departamento de Ciências Básicas , Universidade de São Paulo , Pirassununga , São Paulo , Brazil Published online: 07 Nov 2014.
To cite this article: Paulo S. Monzani , Samuel Guemra , Paulo R. Adona , Otavio M. Ohashi , Flávio V. Meirelles & Matthew B. Wheeler (2015) MAC-T Cells as a Tool to Evaluate Lentiviral Vector Construction Targeting Recombinant Protein Expression in Milk, Animal Biotechnology, 26:2, 136-142, DOI: 10.1080/10495398.2014.941468 To link to this article: http://dx.doi.org/10.1080/10495398.2014.941468
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Animal Biotechnology, 26:136–142, 2015 Copyright # Taylor & Francis Group, LLC ISSN: 1049-5398 print=1532-2378 online DOI: 10.1080/10495398.2014.941468
MAC-T Cells as a Tool to Evaluate Lentiviral Vector Construction Targeting Recombinant Protein Expression in Milk Paulo S. Monzani,1,2,3,4 Samuel Guemra,1,2 Paulo R. Adona,1,2 Otavio M. Ohashi,5 Fla´vio V. Meirelles,6 and Matthew B. Wheeler3,4 1
Centro de Ciencias Biolo´gicas e da Sau´de, Universidade Norte do Parana´, Londrina, Parana´, Brazil Agropecua´ria Laffranchi, Tamarana, Parana´, Brazil 3 Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA 4 Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA 5 Instituto de Cieˆncias Biolo´gicas, Universidade Federal do Para´, Bele´m, Parana´, Brazil 6 Departamento de Cieˆncias Ba´sicas, Universidade de Sa˜o Paulo, Pirassununga, Sa˜o Paulo, Brazil
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Prior to generating transgenic animals for bioreactors, it is important to evaluate the vector constructed to avoid poor protein expression. Mammary epithelial cells cultured in vitro have been proposed as a model to reproduce the biology of the mammary gland. In the present work, three lentiviral vectors were constructed for the human growth hormone (GH), interleukin 2 (IL2), and granulocyte colony-stimulating factor 3 (CSF3) genes driven by the bovine b-casein promoter. The lentiviruses were used to transduce mammary epithelial cells (MAC-T), and the transformed cells were cultured on polystyrene in culture medium with and without prolactin. The gene expression of transgenes was evaluated by PCR using cDNA, and recombinant protein expression was evaluated by Western-blotting using concentrated medium and cellular extracts. The gene expression, of the three introduced genes, was detected in both induced and non induced MAC-T cells. The human GH protein was detected in the concentrated medium, whereas CSF3 was detected in the cellular extract. Apparently, the cellular extract is more appropriate than the concentrated medium to detect recombinant protein, principally because concentrated medium has a high concentration of bovine serum albumin. The results suggest that MAC-T cells may be a good system to evaluate vector construction targeting recombinant protein expression in milk. Keywords CSF-3; Growth hormone; Interleukin 2; Lentivirus; MAC-T cells; Protein expression
The production of recombinant proteins is an important achievement in biotechnology. Recombinant protein production has allowed increased utilization in several industrial sectors including pharmaceuticals, food, textiles, detergents, paper, cellulose, and others. Bacteria have been used extensively as bioreactors to produce heterologous proteins due to their ease of cultivation independent of the scale. However, their use is limited due to the lack of capacity to perform post-translational processing, which is necessary for various proteins to acquire active biological conformation (1). Yeast and filamentous fungi have also shown different patterns of post-translational processing in comparison to those found in insects and mammalian cells. Mammalian Address correspondence to Matthew B. Wheeler, Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 West Gregory Drive, Urbana, IL 61801, USA. E-mail: mbwheele@ illinois.edu Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/labt.
cells produce complex N-glycans containing terminal sialic acids, whereas insect cells produce simple N-glycans with terminal residues of mannose. These structural differences compromise the biologic activities and can induce immunogenic responses in patients (2). Mammalian cells perform complex post-translational processing, but the systems for production, on a large scale, have the highest cost for recombinant protein production (3). Transgenic animals are candidate sources of industrialscale recombinant protein production (4). In transgenic animals, milk is the preferred method of recombinant protein production, mainly because of the quantities of protein that can be produced using various mammary glandspecific promoters and the ease of production and purification (5). However, recombinant protein production is limited due the long interval from birth until the first lactation in domestic animals. Furthermore, the discontinuous lactation cycle and the substantial investments in time and material to produce transgenic animals must be considered as limiting factors (6).
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The mammary gland is arranged as a duct system that is bounded by glandular tissue and shows a cyclic process of development and regression through successive cycles during pregnancy and lactation. Reproduction in vitro of mammary gland function has been performed using explant cultures. It has been used to evaluate hormone action on the mammary gland, including the effects of leptin on milk protein and fat synthesis (7) and the influence of growth hormone on the b-casein gene expression (8). This technique has the advantage of keeping the integrity of the cellular and extracellular matrix composition intact as it was in the in vivo tissue; however, the results could be influenced by the physiologic condition of the tissue from the animal from which it was collected. Alternatively, the biology of the mammary gland can be reproduced using primary mammary epithelial cells (MEC), separated from the extracellular matrix, which will form a monolayer cell culture. Bovine MEC culture was established (9, 10), and it was verified that 1) cell cultures were able to synthetize and secret milk proteins, 2) cellular growth and differentiation are regulated by peptide and steroid hormones, and 3) cell-substrate and cell-cell interactions are intact (11–13). However, milk protein production and MEC differentiation into alveolar structures are dependent on the extracellular matrix on which they are cultured (14). Prior to generation of transgenic animals as bioreactors, it is important to evaluate the vector construction of the transgene for functionality of the promoter, its ability to respond to hormonal induction, and its capacity to express the recombinant protein of interest. Primary mammary epithelial cells may be an alternative to transgenic mouse models for evaluating the potential of gene constructs to be efficiently expressed in the mammary gland of transgenic farm animals (15). The MAC-T bovine mammary epithelial cell is derived from stable transfection with simian virus-40 large T-antigen resulting in an immortalized cell line where the typical morphology of epithelial cell is maintained as well its capacity to response to the lactogenic hormones and extracellular matrix (16). MAC-T cells have been used as a model for bovine in vitro lactation studies (16) and in a number of studies focused on host immune response (17) and breast cancer (18). The use of cell lines allows experiments to be conducted during the long period of time that the cells can be maintained in culture. Generally, primary cells from animals do not survive long in vitro passages in culture, so the use of immortalized cell lines that preserve the same characteristics of primary cells makes numerous passages possible that retain the original function throughout the experimental period. Herein, we have constructed three lentiviral vectors targeting the production of recombinant protein in milk. We have used MAC-T cells as a tool to analyze the lentiviral
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transgene constructs through transgene protein expression driven by a mammary-specific promoter regulated by the lactogenic hormones.
MATERIAL AND METHODS Lentivirus Vector Construction Three lentivirus vectors were constructed using the human growth hormone (GH), the human interleukin 2 (IL2), or the human granulocyte colony-stimulating factor 3 (CSF3) cDNAs. The bovine b-casein promoter drove the recombinant protein expression. The human growth hormone gene was amplified from genomic DNA previously purified from blood of a healthy person. Primers for GH were designed to include the sequence from the signal peptide until after the signal polyadenilation site, based on the GenBank Reference Sequence NG_011676.1 on chromosome 17. The fragment of 1.574-kb from the GH gene was amplified using 100 ng of gDNA. The commercial vectors containing the human CSF3 and IL2 cDNAs cloned (OriGene Technologies Inc.) were used as templates for amplification, and the NCBI Reference Sequences are NM_000759.2 and NM_000586.2, respectively. The amplicons included the cDNA of the CSF3 and IL2 genes, with are about 1.400-kb and 1.100-kb, respectively, from peptide signal sequence to and including the poly-A region of the human growth hormone gene contained in the commercial vector. The primers used are shown in the Table 1. The forward primers included the restriction site for BamHI, whereas the reverse primers contained the restriction site for XhoI. PCR amplification was done using the following cycling parameters: 3 min at 94 C; 35 cycles of 45 s at 94 C, 30 s at 60 C, and 1.5 min at 72 C; and a final 10 min elongation step at 72 C. The amplification products were recovered from a 1% agarose gel, cloned into the vector pGEM-T Easy, and used in the transformation of Escherichia coli DH5a CaCl2-competent cells. Transformant colonies were selected on the chromogenic substrate X-gal. PCR analysis and restriction mapping confirmed the sequence of the constructs. The lentivirus vector pLenti-Pbcas5-EmGFP previously constructed (19) in pGEM containing the human gene cDNAs were cleaved with the restriction enzymes BamHI and XhoI. The cleavage products were recovered and incubated with T4 DNA ligase overnight at 4 C, producing the vectors pLenti-Pbcas5-hGH, pLenti-Pbcas5-hCSF3, and pLenti-Pbcas5-hIL2. In this step, the EmGFP gene was excised from the vector, and the gene of interest =cDNAs were introduced. These vectors were used for E. coli DH-5a transformation and later purified. The construct was confirmed by restriction mapping and used for lentivirus production.
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TABLE 1 Primers used to amplify human gene=cDNAs Gene
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GH GH IL2 CSF3 CSF3=IL2
Primer
Sense
50 -GGATCCATGGCTACAGGTAAG-30 50 -CTCGAGTGATGCAACTTAATTTTATTAGGAC-30 50 -GGATCCATGTACAGGATGCAAC-30 50 -GGATCCATGGCTGGACCTG-30 50 -CTCGAGAATTCAACAGGCATCTAC-30
Foward Reverse Forward Forward Reverse
Lentiviral Production Lentiviral production was performed by lipofection of HEK 293FT cells using the vectors in the ViraPower Lentiviral Packaging Mix (Life Technologies, Grand Island, NY), as well as the constructed expression vectors, according to the manufacturer’s instructions. After 48 h of transfection, 10 mL of the culture media were filtered using PVDF syringe filters (0.45 mm, EMD Millipore, Billerica, MA), concentrated 100-fold using PEG-it Virus Precipitation Solution (System Biosciences, Mountain View, CA). The titer of the lentivirus was performed using 1 mL of concentrated lentivirus added into 19 mL of PBS buffer and analyzed by the Lentiviral Titer Test-Lenti-X GoStix kit (Clontech, Mountain View, CA). Two aliquots of 50 mL, containing more than 5 105 infectious units per mL=mL, were analyzed for each plate. The remaining concentrated lentivirus was frozen at 80 C until the transduction of MAC-T cells.
MAC-T Cells Transduction MAC-T cells were cultivated in 35-mm tissue culture plastic dishes until 30% confluence was reached, then the cells were transduced by incubation with 1.5 mL of medium plus 50 mL of the concentrated lentiviral solution and
6 mg=mL of hexadimethrine bromide. For each gene, two plates with appropriate cDNAs were transfected. After 24 h of incubation, the lentiviral solutions were replaced with normal growth medium and the cells were incubated for another 24 h. The growth medium was DMEM (high glucose, Sigma, St. Louis, MO), 5 mM ascorbic acid, 5 mM sodium acetate, 500 mg=mL a-lactose, 500 mg=mL of hydrolyzed lactalbumin, 5 mg=mL transferrin, 1 mg=mL of progesterone, 1 mg=mL of hydrocortisone, 10% FBS, and 100 units=mL penicillin with 0.1 mg=mL streptomycin. The selection of transformed cells was achieved by the addition of 8 mg=mL blasticidin into the culture medium for 20 days, with medium being changed every three days. The transgenes were confirmed by conventional PCR for the human gene=cDNAs, using the specific primers (Table 2), and the cycling parameters: 3 min at 94 C; 35 cycles of 45 s at 94 C, 30 s at 55 C and 30 s at 72 C, followed by a final 10 min elongation step at 72 C. The amplicons were visualized on a 3% agarose gel.
MAC-T Induction Transformed and non-transformed MAC-T cells were cultured in 25 cm2 tissue culture flasks with growth medium until about 90% confluence was reached. The medium was
TABLE 2 Primers used in the transgene and gene expression analysis Gene hGH hGH hIL2 hIL2 hCSF3 hCSF3 b-caseı´n b-caseı´n GAPDH GAPDH
Primer 50 50 50 50 50 50 50 50 50 50 -
AACTCACACAACGATGACGC-30 TGTCTCGACCTTGTCCATGT-30 TGTCACAAACAGTGCACCTAC-30 ATGTGAGCATCCTGGTGAGT-30 GTGAGTGAGTGTGCCACCTA-30 AAAAGGCCGCTATGGAGTTG-30 CCTCTGCTCCAGTCTTGGAT-30 AACAGGCAGGACTTTGGACT-30 GGCGTGAACCACGAGAAGTATAA-30 CCCTCCACGATGCCAAAGT-30
Sense Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
MAC-T AS TOOL TO EVALUATE VECTOR CONSTRUCTION
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then changed to milk protein induction medium, in which the FSB from growth medium was replaced by 668 mg= mL of bovine serum albumin and 1 mg=mL of prolactin. After seven days of induction, the cells were trypsinized, centrifuged at 200 g for 2 min, suspended in 200 mL of PBS, and separated into two aliquots that were used for gene and protein expression analysis. The medium from days 4 and 7 of induction were centrifuged at 16,000 g for 10 min, at 4 C. The supernatant was concentrated about 20-fold using a centrifugal filter device of 9 molecular weight cut-off (MWCO) (Pierce Protein Concentrators, Cat # 87748, Thermo Scientific, Rockford, IL) and used for protein expression analysis. MAC-T Gene Expression Analysis RNA from MAC-T cells was extracted using Trizol and treated with RNase-free DNase (Qiagen, Valencia, CA). RNA was purified again with the RNeasy Mini kit (Qiagen) and used for cDNA synthesis with the SuperScript III (Life Technologies) with the cycling parameters: 10 min at 25 C, 30 min at 50 C, and 5 min at 85 C. cDNA was treated with RNase H (Life Technologies) for 20 min at 37 C. Human GH, CSF3, and IL2 gene expression was analyzed by conventional PCR using the same primes and cycling parameters for the transgene analysis. As a control, we used the expression of the endogenous genes GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and b-casein at the same conditions for human gene=cDNAs. All primers used were shown in the Table 2. The amplicons were visualized at 3% agarose. MAC-T Protein Expression Analysis Transformed MAC-T cells and concentrated medium were used to evaluate the recombinant protein expression by Western-blotting. Goat polyclonal primary antibodies for human GH, CSF3, and IL2, alkaline phosphatase conjugated secondary antibody, and rabbit anti-goat IgGAP were acquired from Santa Cruz Biotechnology (Dallas, TX). The samples were separated by SDS-PAGE (12% gel) and transferred to PVDF membrane (Millipore) at 150 V for 1 h. Membranes were washed four times in wash buffer containing PBS at pH 7.4 and 0.05% Tween 20, then were blocked for 2 h with wash buffer plus 5% nonfat milk powder. The primary antibodies were diluted 1:100 in wash buffer supplemented with 1% nonfat milk powder, added to the PVDF membranes containing the separated protein products, and incubated overnight at 4 C. Following incubation, the blots were washed five times with wash buffer at room temperature for 10 min. The conjugated anti-goat IgG was diluted 1:5000 in wash buffer containing 1% nonfat milk powder and used as the second antibody during a 1 h incubation at room temperature. Following the second incubation, the blots were again washed five times with wash buffer at room temperature for 10 min. The alkaline
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phosphatase assays were performed using BCIP=NBT liquid substrate (Sigma) and washed with distilled water to stop the reactions.
RESULTS AND DISCUSSION The construction of the mammary gland-specific expression vectors for the human GH gene containing exons, introns, and polyadenilation signal, as well as for the human CSF3 and IL2 cDNAs containing the human growth hormone polyadenylation region, were performed using the lentiviral vector pLenti6.2=GW=EmGFP. We modified this vector as follows: 1) the EmGFP gene was changed to the human gene=cDNAs, and 2) the viral promoter PCMV was replaced by bovine b-casein promoter containing a fragment 5.335-kbp in length, which encompasses the 50 upstream region of the bovine b-casein gene and includes enhancers and the promoter region, and the 5’ untranslated region consisting of 43 bp of the first exon and 218-pb of first intron (20). The replacement of PCMV by the b-casein promoter was done so that we could evaluate the expression of the transgens under the control of a mammary-specific promoter that could be regulated by lactogenic hormones. The vector constructions were analyzed by restriction mapping, which is depicted in Fig. 1. Lentiviruses were produced and used to transduce MAC-T cells. After selection, using blasticidin, the transgene was confirmed through PCR using isolated gDNA from transfected cells (Fig. 2). The activity of the bovine b-casein promoter region cloned into lentiviral vector was evaluated by GH, CSF3, and IL2 gene and protein expression in transformed
FIG. 1. Restriction map of the constructed vectors. L, DNA ladder 1-kpb plus (Life Technologies); 1) pLenti-Pbcas5-hGH; 2) pLenti-Pbcas5-hCSF3; 3) pLenti-Pbcas5-hIL2. Size of the fragments: pLenti, 6.3-kb; Pbcas5, 5.3-kb; hGH, 1.5-kb; hCSF3, 1.4-kb; and hIL2, 1.1-kb.
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FIG. 2. Analysis of MAC-T transgene containing cells. L, DNA ladder 1-kbp plus (Life Technologies); NT, not transformed MAC-T; GH, MAC-T transformed with human GH; CSF3, MAC-T transformed with human CSF3; IL2, MAC-T transformed with human IL2. MAC-T cells cultured on plastic using growth and lactogenic induction media. The results of gene expression studies showed that all transgenes used in the experiments were expressed in the transfected MAC-T cultured in both media used (growth and lactation induction), see Fig. 3. The b-casein gene expression was not detected when it was evaluated using the same quantity of the cDNA as that used for GH, CSF3, IL2, and GAPDH (Fig. 3); however, it was detected using three-fold more cDNA than that initial concentrations (data not shown). The higher expression of the recombinant genes compared to the b-casein gene could be due the number of transgene insertion sites that may be higher than the b-casein gene, or the promoter used lacked a repressor sequence. The low expression of the b-casein gene by MAC-T cells cultured on plastic without prolactin induction has been reported, and when they were cultured on collagen without induction, a marked increase of the b-casein gene was observed, followed by highest expression when cultured on collagen when prolactin induction was performed (16). Mouse mammary epithelial cells grown on collagen had 3- to 10-fold more casein mRNA than cells
FIG. 3. Analysis of gene expression. L, DNA ladder 1-kbp plus (Life Technologies); NT, not transformed MAC-T induced as control for transgene; GH, transformed MAC-T with human GH; CSF, transformed MAC-T with human CSF3; IL2, transformed MAC-T with human IL2; BC, b-casein gene; GAP, endogenous gene GAPDH. Arrows appoint the material used from growing and lactogenic medium.
grown on plastic substrates (21). MAC-T cells have limited capacity to express milk proteins when compared to mammary tissue, particularly the caseins (22); however, expression and synthesis has been reported (23). Furthermore, we have shown that MAC-T cells can be grown on plastic substrates with b-casein gene expression detected apparently without the influence of prolactin. Similar data was reported when MAC-T cells were growing on collagen, but prolactin increased the b-casein gene expression (16). This suggests that MAC-T cells are able to exhibit basal expression of b-casein gene on plastic substrates, but b-casein gene expression can be substantially increased when cultured on collagen without induction with prolactin. The heterologous protein expression was evaluated by Western-blotting using the secreted protein in the concentrated culture medium and in the trypsinized MAC-T cells. Herein, we only made a qualitative evaluation of the capacity of MAC-T cells to promote recombinant protein expression driven by a mammary-specific promoter; therefore, the protein expression was not quantified. Human GH protein was detected in the concentrated medium and, apparently, was more highly expressed in induced cells than in noninduced cells (Fig. 4A). Initially, human GH was not detected in the cell extract but did appear when the medium was concentrated. Furthermore, human GH was not detected in the medium or cell extracts of the
Figure 4. Western-blotting analysis. 4A) Western-blotting for human GH using concentrated medium. 4B1) Western-blotting for human CSF3 using concentrated medium and cellular extract. 4B2) Western-blotting for human CSF3 using cellular extract of induced nontransformed and transformed MAC-T. 4C1) Western-blotting for human IL2 using cellular extract. 4C2) Western-blotting for human IL-2 using cellular extract of induced nontransformed and transformed MAC-T. MW, molecular weight; MC, concentrated growing medium; MI4, concentrated induced medium at 4th day of induction; MI7, concentrated induced medium at 7th day of induction; CC, cellular extract from MAC-T cultured in growth medium; IC, cellular extract from MAC-T cultured in inducing medium; NTC, cellular extract from nontransformed MAC-T cultured in inducing medium.
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MAC-T AS TOOL TO EVALUATE VECTOR CONSTRUCTION
nontransformed cells (data not showed). Human CSF3 was detected in the cellular extract and not in the concentrated medium (Fig. 4B1), and the analysis of the nontransformed cells did not show any similar band to the one found in the transformed cells (Fig. 4B2). The expression of human IL-2 was not definitively established as a similar size band was observed in the nontransformed cells. Further work will need to be done to establish human IL-2 protein expression in the transfected MAC-T cells. Even the CSF3 band, when compared to human IL2 band analyzed by Westernblotting, showed a differentially expressed band in the cellular extract but not in the concentrated medium (Fig. 4C1). The confirmation of the heterologous protein could not be performed once it was verified that a similar size band existed in the extract of the nontransformed cells (Fig. 4C2). Overall, the MAC-T cell extract was a more robust sample for analysis of recombinant protein than the concentrated medium. In the concentrated medium, a large quantity of BSA is present, which makes protein separation by gel electrophoresis and detection by Westernblotting difficult. However, the culture of MAC-T on collagen must improve the efficiency of the recombinant protein expression over that from culture on plastic, allowing the secreted protein to be more easily assessed. The usefulness of MAC-T cells for studying milk protein synthesis is controversial: it was reported that MAC-T cells cultured on plastic had limitations (22); whereas, others authors had proposed that MAC-T cells cultured on collagen were comparable to explant cultures when used to study mechanisms of milk protein synthesis and secretion (24). Transfected MAC-T for growth hormone receptor and STAT5 showed increased expression (16- to 117fold) of the four major milk protein genes when the medium culture was supplemented with growth hormone (23). The generation of a modified MAC-T cell line that expresses growth hormone receptor constitutively may be an interesting way to allow MAC-T cells to readily express recombinant proteins driven by milk protein promoters. MAC-T cells had been shown to be able to synthesize proteins that are difficult to express by other expression systems, such as recombinant spider silk proteins (25). These aspects make MAC-T cells an interesting system for studies of protein expression. In this work, gene and protein expression of the transgene driven by bovine b-casein promoter in the cells growing on plastic was obtained. Our results suggest that MAC-T cells may be used for analysis of milk protein promoter vector construction. Furthermore, previous results suggest that if MAC-T cells are grown on collagen substrates, protein production may be more efficient (24). In summary, MAC-T cells were shown to be effective as a system for the evaluation of vector construction that targets recombinant protein production in the milk, possibly being an alternative method to transgenic mouse model
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production for livestock transgene construct analysis. Vectors constructed were able to express the transgenes, suggesting that the b-casein promoter used was working. Under the control of lactogenic hormones, MAC-T cells growing on plastic substrate did not show the same capacity for milk protein expression that had previously been reported for MAC-T cells cultured on collagen. This suggests that the use of MAC-T cells as a model to recombinant milk protein expression should be improved when the cells are cultured on collagen. FUNDING This study was supported by University of Illinois at Urbana-Champaign (UIUC), Universidade Norte do Parana´ (UNOPAR), Agropecua´ria Laffranchi and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). Dr. Juan J. Loor of Department of Animal Science at UIUC provided the MAC-T cells. REFERENCES 1. Jana S, Deb JK. Strategies for efficient production of heterologous proteins in Escherichia coli. Appl Microbiol Biotechnol 2005; 67:289–298. 2. Harrison RL, Jarvis DL. Protein N-glycosylation in the baculovirus-insect cell expression system and engineering of insect cells to produce ‘‘mammalianized’’ recombinant glycoproteins. Adv Virus Res 2006; 68:159–191. 3. Andersen DC, Krummen L. Recombinant protein expression for therapeutic applications. Curr Opin Biotechnol 2002; 13:117–123. 4. Houdebine LM. Transgenic animal bioreactors. Transgenic Res 2000; 9:305–320. 5. Rudolph NS. Biopharmaceutical production in transgenic livestock. Trends Biotechnol 1999; 17:367–374. 6. Wall RJ, Kerr DE, Bondioli KR. Transgenic dairy cattle: Genetic engineering on a large scale. J Dairy Sci 1997; 80: 2213–2224. 7. Feuermann Y, Mabjeesh SJ, Shamay A. Leptin affects prolactin action on milk protein and fat synthesis in the bovine mammary gland. J Dairy Sci 2004; 87:2941–2946. 8. Yang J, Zhao B, Baracos VE, Kennelly JJ. Effects of bovine somatotropin on beta-casein mRNA levels in mammary tissue of lactating cows. J Dairy Sci 2005; 88:2806–2812. 9. MacKenzie DD, Forsyth IA, Brooker BE, Turvey A. Culture of bovine mammary epithelial cells on collagen gels. Tissue Cell 1982; 14:231–241. 10. MacKenzie DD, Brooker BE, Forsyth IA. Ultra structural features of bovine mammary epithelial cells grown on collagen gels. Tissue Cell 1985; 17:39–51. 11. Mehta NM, Ganguly N, Gangly R, Banerjee MR. Hormonal modulation of the casein gene expression in a mammogenesislactogenesis culture model of the whole mammary gland of the mouse. J Biol Chem 1980; 255:4430–4434. 12. Li ML, Aggeler J, Farson DA, Hatier C, Hassel J, Biessel MJ. Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in
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Animal Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/labt20
MAC-T Cells as a Tool to Evaluate Lentiviral Vector Construction Targeting Recombinant Protein Expression in Milk Paulo S. Monzani f
a b c d
, Samuel Guemra
V. Meirelles & Matthew B. Wheeler
a b
, Paulo R. Adona
a b
e
, Otavio M. Ohashi , Flávio
c d
a
Centro de Ciencias Biológicas e da Saúde , Universidade Norte do Paraná , Londrina , Paraná , Brazil b
Agropecuária Laffranchi , Tamarana , Paraná , Brazil
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Department of Animal Sciences , University of Illinois at Urbana-Champaign , Urbana , Illinois , USA d
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Institute for Genomic Biology , University of Illinois at Urbana-Champaign , Urbana , Illinois , USA e
Instituto de Ciências Biológicas, Universidade Federal do Pará , Belém , Paraná , Brazil
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Departamento de Ciências Básicas , Universidade de São Paulo , Pirassununga , São Paulo , Brazil Published online: 07 Nov 2014.
To cite this article: Paulo S. Monzani , Samuel Guemra , Paulo R. Adona , Otavio M. Ohashi , Flávio V. Meirelles & Matthew B. Wheeler (2015) MAC-T Cells as a Tool to Evaluate Lentiviral Vector Construction Targeting Recombinant Protein Expression in Milk, Animal Biotechnology, 26:2, 136-142, DOI: 10.1080/10495398.2014.941468 To link to this article: http://dx.doi.org/10.1080/10495398.2014.941468
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Animal Biotechnology, 26:136–142, 2015 Copyright # Taylor & Francis Group, LLC ISSN: 1049-5398 print=1532-2378 online DOI: 10.1080/10495398.2014.941468
MAC-T Cells as a Tool to Evaluate Lentiviral Vector Construction Targeting Recombinant Protein Expression in Milk Paulo S. Monzani,1,2,3,4 Samuel Guemra,1,2 Paulo R. Adona,1,2 Otavio M. Ohashi,5 Fla´vio V. Meirelles,6 and Matthew B. Wheeler3,4 1
Centro de Ciencias Biolo´gicas e da Sau´de, Universidade Norte do Parana´, Londrina, Parana´, Brazil Agropecua´ria Laffranchi, Tamarana, Parana´, Brazil 3 Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA 4 Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA 5 Instituto de Cieˆncias Biolo´gicas, Universidade Federal do Para´, Bele´m, Parana´, Brazil 6 Departamento de Cieˆncias Ba´sicas, Universidade de Sa˜o Paulo, Pirassununga, Sa˜o Paulo, Brazil
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Prior to generating transgenic animals for bioreactors, it is important to evaluate the vector constructed to avoid poor protein expression. Mammary epithelial cells cultured in vitro have been proposed as a model to reproduce the biology of the mammary gland. In the present work, three lentiviral vectors were constructed for the human growth hormone (GH), interleukin 2 (IL2), and granulocyte colony-stimulating factor 3 (CSF3) genes driven by the bovine b-casein promoter. The lentiviruses were used to transduce mammary epithelial cells (MAC-T), and the transformed cells were cultured on polystyrene in culture medium with and without prolactin. The gene expression of transgenes was evaluated by PCR using cDNA, and recombinant protein expression was evaluated by Western-blotting using concentrated medium and cellular extracts. The gene expression, of the three introduced genes, was detected in both induced and non induced MAC-T cells. The human GH protein was detected in the concentrated medium, whereas CSF3 was detected in the cellular extract. Apparently, the cellular extract is more appropriate than the concentrated medium to detect recombinant protein, principally because concentrated medium has a high concentration of bovine serum albumin. The results suggest that MAC-T cells may be a good system to evaluate vector construction targeting recombinant protein expression in milk. Keywords CSF-3; Growth hormone; Interleukin 2; Lentivirus; MAC-T cells; Protein expression
The production of recombinant proteins is an important achievement in biotechnology. Recombinant protein production has allowed increased utilization in several industrial sectors including pharmaceuticals, food, textiles, detergents, paper, cellulose, and others. Bacteria have been used extensively as bioreactors to produce heterologous proteins due to their ease of cultivation independent of the scale. However, their use is limited due to the lack of capacity to perform post-translational processing, which is necessary for various proteins to acquire active biological conformation (1). Yeast and filamentous fungi have also shown different patterns of post-translational processing in comparison to those found in insects and mammalian cells. Mammalian Address correspondence to Matthew B. Wheeler, Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 West Gregory Drive, Urbana, IL 61801, USA. E-mail: mbwheele@ illinois.edu Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/labt.
cells produce complex N-glycans containing terminal sialic acids, whereas insect cells produce simple N-glycans with terminal residues of mannose. These structural differences compromise the biologic activities and can induce immunogenic responses in patients (2). Mammalian cells perform complex post-translational processing, but the systems for production, on a large scale, have the highest cost for recombinant protein production (3). Transgenic animals are candidate sources of industrialscale recombinant protein production (4). In transgenic animals, milk is the preferred method of recombinant protein production, mainly because of the quantities of protein that can be produced using various mammary glandspecific promoters and the ease of production and purification (5). However, recombinant protein production is limited due the long interval from birth until the first lactation in domestic animals. Furthermore, the discontinuous lactation cycle and the substantial investments in time and material to produce transgenic animals must be considered as limiting factors (6).
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The mammary gland is arranged as a duct system that is bounded by glandular tissue and shows a cyclic process of development and regression through successive cycles during pregnancy and lactation. Reproduction in vitro of mammary gland function has been performed using explant cultures. It has been used to evaluate hormone action on the mammary gland, including the effects of leptin on milk protein and fat synthesis (7) and the influence of growth hormone on the b-casein gene expression (8). This technique has the advantage of keeping the integrity of the cellular and extracellular matrix composition intact as it was in the in vivo tissue; however, the results could be influenced by the physiologic condition of the tissue from the animal from which it was collected. Alternatively, the biology of the mammary gland can be reproduced using primary mammary epithelial cells (MEC), separated from the extracellular matrix, which will form a monolayer cell culture. Bovine MEC culture was established (9, 10), and it was verified that 1) cell cultures were able to synthetize and secret milk proteins, 2) cellular growth and differentiation are regulated by peptide and steroid hormones, and 3) cell-substrate and cell-cell interactions are intact (11–13). However, milk protein production and MEC differentiation into alveolar structures are dependent on the extracellular matrix on which they are cultured (14). Prior to generation of transgenic animals as bioreactors, it is important to evaluate the vector construction of the transgene for functionality of the promoter, its ability to respond to hormonal induction, and its capacity to express the recombinant protein of interest. Primary mammary epithelial cells may be an alternative to transgenic mouse models for evaluating the potential of gene constructs to be efficiently expressed in the mammary gland of transgenic farm animals (15). The MAC-T bovine mammary epithelial cell is derived from stable transfection with simian virus-40 large T-antigen resulting in an immortalized cell line where the typical morphology of epithelial cell is maintained as well its capacity to response to the lactogenic hormones and extracellular matrix (16). MAC-T cells have been used as a model for bovine in vitro lactation studies (16) and in a number of studies focused on host immune response (17) and breast cancer (18). The use of cell lines allows experiments to be conducted during the long period of time that the cells can be maintained in culture. Generally, primary cells from animals do not survive long in vitro passages in culture, so the use of immortalized cell lines that preserve the same characteristics of primary cells makes numerous passages possible that retain the original function throughout the experimental period. Herein, we have constructed three lentiviral vectors targeting the production of recombinant protein in milk. We have used MAC-T cells as a tool to analyze the lentiviral
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transgene constructs through transgene protein expression driven by a mammary-specific promoter regulated by the lactogenic hormones.
MATERIAL AND METHODS Lentivirus Vector Construction Three lentivirus vectors were constructed using the human growth hormone (GH), the human interleukin 2 (IL2), or the human granulocyte colony-stimulating factor 3 (CSF3) cDNAs. The bovine b-casein promoter drove the recombinant protein expression. The human growth hormone gene was amplified from genomic DNA previously purified from blood of a healthy person. Primers for GH were designed to include the sequence from the signal peptide until after the signal polyadenilation site, based on the GenBank Reference Sequence NG_011676.1 on chromosome 17. The fragment of 1.574-kb from the GH gene was amplified using 100 ng of gDNA. The commercial vectors containing the human CSF3 and IL2 cDNAs cloned (OriGene Technologies Inc.) were used as templates for amplification, and the NCBI Reference Sequences are NM_000759.2 and NM_000586.2, respectively. The amplicons included the cDNA of the CSF3 and IL2 genes, with are about 1.400-kb and 1.100-kb, respectively, from peptide signal sequence to and including the poly-A region of the human growth hormone gene contained in the commercial vector. The primers used are shown in the Table 1. The forward primers included the restriction site for BamHI, whereas the reverse primers contained the restriction site for XhoI. PCR amplification was done using the following cycling parameters: 3 min at 94 C; 35 cycles of 45 s at 94 C, 30 s at 60 C, and 1.5 min at 72 C; and a final 10 min elongation step at 72 C. The amplification products were recovered from a 1% agarose gel, cloned into the vector pGEM-T Easy, and used in the transformation of Escherichia coli DH5a CaCl2-competent cells. Transformant colonies were selected on the chromogenic substrate X-gal. PCR analysis and restriction mapping confirmed the sequence of the constructs. The lentivirus vector pLenti-Pbcas5-EmGFP previously constructed (19) in pGEM containing the human gene cDNAs were cleaved with the restriction enzymes BamHI and XhoI. The cleavage products were recovered and incubated with T4 DNA ligase overnight at 4 C, producing the vectors pLenti-Pbcas5-hGH, pLenti-Pbcas5-hCSF3, and pLenti-Pbcas5-hIL2. In this step, the EmGFP gene was excised from the vector, and the gene of interest =cDNAs were introduced. These vectors were used for E. coli DH-5a transformation and later purified. The construct was confirmed by restriction mapping and used for lentivirus production.
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TABLE 1 Primers used to amplify human gene=cDNAs Gene
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GH GH IL2 CSF3 CSF3=IL2
Primer
Sense
50 -GGATCCATGGCTACAGGTAAG-30 50 -CTCGAGTGATGCAACTTAATTTTATTAGGAC-30 50 -GGATCCATGTACAGGATGCAAC-30 50 -GGATCCATGGCTGGACCTG-30 50 -CTCGAGAATTCAACAGGCATCTAC-30
Foward Reverse Forward Forward Reverse
Lentiviral Production Lentiviral production was performed by lipofection of HEK 293FT cells using the vectors in the ViraPower Lentiviral Packaging Mix (Life Technologies, Grand Island, NY), as well as the constructed expression vectors, according to the manufacturer’s instructions. After 48 h of transfection, 10 mL of the culture media were filtered using PVDF syringe filters (0.45 mm, EMD Millipore, Billerica, MA), concentrated 100-fold using PEG-it Virus Precipitation Solution (System Biosciences, Mountain View, CA). The titer of the lentivirus was performed using 1 mL of concentrated lentivirus added into 19 mL of PBS buffer and analyzed by the Lentiviral Titer Test-Lenti-X GoStix kit (Clontech, Mountain View, CA). Two aliquots of 50 mL, containing more than 5 105 infectious units per mL=mL, were analyzed for each plate. The remaining concentrated lentivirus was frozen at 80 C until the transduction of MAC-T cells.
MAC-T Cells Transduction MAC-T cells were cultivated in 35-mm tissue culture plastic dishes until 30% confluence was reached, then the cells were transduced by incubation with 1.5 mL of medium plus 50 mL of the concentrated lentiviral solution and
6 mg=mL of hexadimethrine bromide. For each gene, two plates with appropriate cDNAs were transfected. After 24 h of incubation, the lentiviral solutions were replaced with normal growth medium and the cells were incubated for another 24 h. The growth medium was DMEM (high glucose, Sigma, St. Louis, MO), 5 mM ascorbic acid, 5 mM sodium acetate, 500 mg=mL a-lactose, 500 mg=mL of hydrolyzed lactalbumin, 5 mg=mL transferrin, 1 mg=mL of progesterone, 1 mg=mL of hydrocortisone, 10% FBS, and 100 units=mL penicillin with 0.1 mg=mL streptomycin. The selection of transformed cells was achieved by the addition of 8 mg=mL blasticidin into the culture medium for 20 days, with medium being changed every three days. The transgenes were confirmed by conventional PCR for the human gene=cDNAs, using the specific primers (Table 2), and the cycling parameters: 3 min at 94 C; 35 cycles of 45 s at 94 C, 30 s at 55 C and 30 s at 72 C, followed by a final 10 min elongation step at 72 C. The amplicons were visualized on a 3% agarose gel.
MAC-T Induction Transformed and non-transformed MAC-T cells were cultured in 25 cm2 tissue culture flasks with growth medium until about 90% confluence was reached. The medium was
TABLE 2 Primers used in the transgene and gene expression analysis Gene hGH hGH hIL2 hIL2 hCSF3 hCSF3 b-caseı´n b-caseı´n GAPDH GAPDH
Primer 50 50 50 50 50 50 50 50 50 50 -
AACTCACACAACGATGACGC-30 TGTCTCGACCTTGTCCATGT-30 TGTCACAAACAGTGCACCTAC-30 ATGTGAGCATCCTGGTGAGT-30 GTGAGTGAGTGTGCCACCTA-30 AAAAGGCCGCTATGGAGTTG-30 CCTCTGCTCCAGTCTTGGAT-30 AACAGGCAGGACTTTGGACT-30 GGCGTGAACCACGAGAAGTATAA-30 CCCTCCACGATGCCAAAGT-30
Sense Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
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then changed to milk protein induction medium, in which the FSB from growth medium was replaced by 668 mg= mL of bovine serum albumin and 1 mg=mL of prolactin. After seven days of induction, the cells were trypsinized, centrifuged at 200 g for 2 min, suspended in 200 mL of PBS, and separated into two aliquots that were used for gene and protein expression analysis. The medium from days 4 and 7 of induction were centrifuged at 16,000 g for 10 min, at 4 C. The supernatant was concentrated about 20-fold using a centrifugal filter device of 9 molecular weight cut-off (MWCO) (Pierce Protein Concentrators, Cat # 87748, Thermo Scientific, Rockford, IL) and used for protein expression analysis. MAC-T Gene Expression Analysis RNA from MAC-T cells was extracted using Trizol and treated with RNase-free DNase (Qiagen, Valencia, CA). RNA was purified again with the RNeasy Mini kit (Qiagen) and used for cDNA synthesis with the SuperScript III (Life Technologies) with the cycling parameters: 10 min at 25 C, 30 min at 50 C, and 5 min at 85 C. cDNA was treated with RNase H (Life Technologies) for 20 min at 37 C. Human GH, CSF3, and IL2 gene expression was analyzed by conventional PCR using the same primes and cycling parameters for the transgene analysis. As a control, we used the expression of the endogenous genes GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and b-casein at the same conditions for human gene=cDNAs. All primers used were shown in the Table 2. The amplicons were visualized at 3% agarose. MAC-T Protein Expression Analysis Transformed MAC-T cells and concentrated medium were used to evaluate the recombinant protein expression by Western-blotting. Goat polyclonal primary antibodies for human GH, CSF3, and IL2, alkaline phosphatase conjugated secondary antibody, and rabbit anti-goat IgGAP were acquired from Santa Cruz Biotechnology (Dallas, TX). The samples were separated by SDS-PAGE (12% gel) and transferred to PVDF membrane (Millipore) at 150 V for 1 h. Membranes were washed four times in wash buffer containing PBS at pH 7.4 and 0.05% Tween 20, then were blocked for 2 h with wash buffer plus 5% nonfat milk powder. The primary antibodies were diluted 1:100 in wash buffer supplemented with 1% nonfat milk powder, added to the PVDF membranes containing the separated protein products, and incubated overnight at 4 C. Following incubation, the blots were washed five times with wash buffer at room temperature for 10 min. The conjugated anti-goat IgG was diluted 1:5000 in wash buffer containing 1% nonfat milk powder and used as the second antibody during a 1 h incubation at room temperature. Following the second incubation, the blots were again washed five times with wash buffer at room temperature for 10 min. The alkaline
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phosphatase assays were performed using BCIP=NBT liquid substrate (Sigma) and washed with distilled water to stop the reactions.
RESULTS AND DISCUSSION The construction of the mammary gland-specific expression vectors for the human GH gene containing exons, introns, and polyadenilation signal, as well as for the human CSF3 and IL2 cDNAs containing the human growth hormone polyadenylation region, were performed using the lentiviral vector pLenti6.2=GW=EmGFP. We modified this vector as follows: 1) the EmGFP gene was changed to the human gene=cDNAs, and 2) the viral promoter PCMV was replaced by bovine b-casein promoter containing a fragment 5.335-kbp in length, which encompasses the 50 upstream region of the bovine b-casein gene and includes enhancers and the promoter region, and the 5’ untranslated region consisting of 43 bp of the first exon and 218-pb of first intron (20). The replacement of PCMV by the b-casein promoter was done so that we could evaluate the expression of the transgens under the control of a mammary-specific promoter that could be regulated by lactogenic hormones. The vector constructions were analyzed by restriction mapping, which is depicted in Fig. 1. Lentiviruses were produced and used to transduce MAC-T cells. After selection, using blasticidin, the transgene was confirmed through PCR using isolated gDNA from transfected cells (Fig. 2). The activity of the bovine b-casein promoter region cloned into lentiviral vector was evaluated by GH, CSF3, and IL2 gene and protein expression in transformed
FIG. 1. Restriction map of the constructed vectors. L, DNA ladder 1-kpb plus (Life Technologies); 1) pLenti-Pbcas5-hGH; 2) pLenti-Pbcas5-hCSF3; 3) pLenti-Pbcas5-hIL2. Size of the fragments: pLenti, 6.3-kb; Pbcas5, 5.3-kb; hGH, 1.5-kb; hCSF3, 1.4-kb; and hIL2, 1.1-kb.
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FIG. 2. Analysis of MAC-T transgene containing cells. L, DNA ladder 1-kbp plus (Life Technologies); NT, not transformed MAC-T; GH, MAC-T transformed with human GH; CSF3, MAC-T transformed with human CSF3; IL2, MAC-T transformed with human IL2. MAC-T cells cultured on plastic using growth and lactogenic induction media. The results of gene expression studies showed that all transgenes used in the experiments were expressed in the transfected MAC-T cultured in both media used (growth and lactation induction), see Fig. 3. The b-casein gene expression was not detected when it was evaluated using the same quantity of the cDNA as that used for GH, CSF3, IL2, and GAPDH (Fig. 3); however, it was detected using three-fold more cDNA than that initial concentrations (data not shown). The higher expression of the recombinant genes compared to the b-casein gene could be due the number of transgene insertion sites that may be higher than the b-casein gene, or the promoter used lacked a repressor sequence. The low expression of the b-casein gene by MAC-T cells cultured on plastic without prolactin induction has been reported, and when they were cultured on collagen without induction, a marked increase of the b-casein gene was observed, followed by highest expression when cultured on collagen when prolactin induction was performed (16). Mouse mammary epithelial cells grown on collagen had 3- to 10-fold more casein mRNA than cells
FIG. 3. Analysis of gene expression. L, DNA ladder 1-kbp plus (Life Technologies); NT, not transformed MAC-T induced as control for transgene; GH, transformed MAC-T with human GH; CSF, transformed MAC-T with human CSF3; IL2, transformed MAC-T with human IL2; BC, b-casein gene; GAP, endogenous gene GAPDH. Arrows appoint the material used from growing and lactogenic medium.
grown on plastic substrates (21). MAC-T cells have limited capacity to express milk proteins when compared to mammary tissue, particularly the caseins (22); however, expression and synthesis has been reported (23). Furthermore, we have shown that MAC-T cells can be grown on plastic substrates with b-casein gene expression detected apparently without the influence of prolactin. Similar data was reported when MAC-T cells were growing on collagen, but prolactin increased the b-casein gene expression (16). This suggests that MAC-T cells are able to exhibit basal expression of b-casein gene on plastic substrates, but b-casein gene expression can be substantially increased when cultured on collagen without induction with prolactin. The heterologous protein expression was evaluated by Western-blotting using the secreted protein in the concentrated culture medium and in the trypsinized MAC-T cells. Herein, we only made a qualitative evaluation of the capacity of MAC-T cells to promote recombinant protein expression driven by a mammary-specific promoter; therefore, the protein expression was not quantified. Human GH protein was detected in the concentrated medium and, apparently, was more highly expressed in induced cells than in noninduced cells (Fig. 4A). Initially, human GH was not detected in the cell extract but did appear when the medium was concentrated. Furthermore, human GH was not detected in the medium or cell extracts of the
Figure 4. Western-blotting analysis. 4A) Western-blotting for human GH using concentrated medium. 4B1) Western-blotting for human CSF3 using concentrated medium and cellular extract. 4B2) Western-blotting for human CSF3 using cellular extract of induced nontransformed and transformed MAC-T. 4C1) Western-blotting for human IL2 using cellular extract. 4C2) Western-blotting for human IL-2 using cellular extract of induced nontransformed and transformed MAC-T. MW, molecular weight; MC, concentrated growing medium; MI4, concentrated induced medium at 4th day of induction; MI7, concentrated induced medium at 7th day of induction; CC, cellular extract from MAC-T cultured in growth medium; IC, cellular extract from MAC-T cultured in inducing medium; NTC, cellular extract from nontransformed MAC-T cultured in inducing medium.
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MAC-T AS TOOL TO EVALUATE VECTOR CONSTRUCTION
nontransformed cells (data not showed). Human CSF3 was detected in the cellular extract and not in the concentrated medium (Fig. 4B1), and the analysis of the nontransformed cells did not show any similar band to the one found in the transformed cells (Fig. 4B2). The expression of human IL-2 was not definitively established as a similar size band was observed in the nontransformed cells. Further work will need to be done to establish human IL-2 protein expression in the transfected MAC-T cells. Even the CSF3 band, when compared to human IL2 band analyzed by Westernblotting, showed a differentially expressed band in the cellular extract but not in the concentrated medium (Fig. 4C1). The confirmation of the heterologous protein could not be performed once it was verified that a similar size band existed in the extract of the nontransformed cells (Fig. 4C2). Overall, the MAC-T cell extract was a more robust sample for analysis of recombinant protein than the concentrated medium. In the concentrated medium, a large quantity of BSA is present, which makes protein separation by gel electrophoresis and detection by Westernblotting difficult. However, the culture of MAC-T on collagen must improve the efficiency of the recombinant protein expression over that from culture on plastic, allowing the secreted protein to be more easily assessed. The usefulness of MAC-T cells for studying milk protein synthesis is controversial: it was reported that MAC-T cells cultured on plastic had limitations (22); whereas, others authors had proposed that MAC-T cells cultured on collagen were comparable to explant cultures when used to study mechanisms of milk protein synthesis and secretion (24). Transfected MAC-T for growth hormone receptor and STAT5 showed increased expression (16- to 117fold) of the four major milk protein genes when the medium culture was supplemented with growth hormone (23). The generation of a modified MAC-T cell line that expresses growth hormone receptor constitutively may be an interesting way to allow MAC-T cells to readily express recombinant proteins driven by milk protein promoters. MAC-T cells had been shown to be able to synthesize proteins that are difficult to express by other expression systems, such as recombinant spider silk proteins (25). These aspects make MAC-T cells an interesting system for studies of protein expression. In this work, gene and protein expression of the transgene driven by bovine b-casein promoter in the cells growing on plastic was obtained. Our results suggest that MAC-T cells may be used for analysis of milk protein promoter vector construction. Furthermore, previous results suggest that if MAC-T cells are grown on collagen substrates, protein production may be more efficient (24). In summary, MAC-T cells were shown to be effective as a system for the evaluation of vector construction that targets recombinant protein production in the milk, possibly being an alternative method to transgenic mouse model
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production for livestock transgene construct analysis. Vectors constructed were able to express the transgenes, suggesting that the b-casein promoter used was working. Under the control of lactogenic hormones, MAC-T cells growing on plastic substrate did not show the same capacity for milk protein expression that had previously been reported for MAC-T cells cultured on collagen. This suggests that the use of MAC-T cells as a model to recombinant milk protein expression should be improved when the cells are cultured on collagen. FUNDING This study was supported by University of Illinois at Urbana-Champaign (UIUC), Universidade Norte do Parana´ (UNOPAR), Agropecua´ria Laffranchi and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). Dr. Juan J. Loor of Department of Animal Science at UIUC provided the MAC-T cells. REFERENCES 1. Jana S, Deb JK. Strategies for efficient production of heterologous proteins in Escherichia coli. Appl Microbiol Biotechnol 2005; 67:289–298. 2. Harrison RL, Jarvis DL. Protein N-glycosylation in the baculovirus-insect cell expression system and engineering of insect cells to produce ‘‘mammalianized’’ recombinant glycoproteins. Adv Virus Res 2006; 68:159–191. 3. Andersen DC, Krummen L. Recombinant protein expression for therapeutic applications. Curr Opin Biotechnol 2002; 13:117–123. 4. Houdebine LM. Transgenic animal bioreactors. Transgenic Res 2000; 9:305–320. 5. Rudolph NS. Biopharmaceutical production in transgenic livestock. Trends Biotechnol 1999; 17:367–374. 6. Wall RJ, Kerr DE, Bondioli KR. Transgenic dairy cattle: Genetic engineering on a large scale. J Dairy Sci 1997; 80: 2213–2224. 7. Feuermann Y, Mabjeesh SJ, Shamay A. Leptin affects prolactin action on milk protein and fat synthesis in the bovine mammary gland. J Dairy Sci 2004; 87:2941–2946. 8. Yang J, Zhao B, Baracos VE, Kennelly JJ. Effects of bovine somatotropin on beta-casein mRNA levels in mammary tissue of lactating cows. J Dairy Sci 2005; 88:2806–2812. 9. MacKenzie DD, Forsyth IA, Brooker BE, Turvey A. Culture of bovine mammary epithelial cells on collagen gels. Tissue Cell 1982; 14:231–241. 10. MacKenzie DD, Brooker BE, Forsyth IA. Ultra structural features of bovine mammary epithelial cells grown on collagen gels. Tissue Cell 1985; 17:39–51. 11. Mehta NM, Ganguly N, Gangly R, Banerjee MR. Hormonal modulation of the casein gene expression in a mammogenesislactogenesis culture model of the whole mammary gland of the mouse. J Biol Chem 1980; 255:4430–4434. 12. Li ML, Aggeler J, Farson DA, Hatier C, Hassel J, Biessel MJ. Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in
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