In vitro and ex vivo green fluorescent protein expression in alveolar mammary epithelial cells and mammary glands driven by the distal 5′-regulative sequence and intron 1 of the goat β-casein gene
Hsi-Tien Wu A B , Chich-Sheng Lin C and Mu-Chiou Huang A D
+ Author Affiliations
- Author Affiliations
A Institute of Animal Science, National Chung-Hsing University, 250 Kao-Kung Road, Taichung 402, Taiwan.
B National Laboratory Animal Center, National Applied Research Laboratories, PO Box 1-86, Nankang, Taipei 115, Taiwan.
C Department of Biological Science and Technology, National Chiao Tung University, 75 Po-Ai Street, Hsinchu 300, Taiwan.
D To whom correspondence should be addressed. email: mchuang@mail.nchu.edu.tw
Reproduction, Fertility and Development 15(4) 231-239 https://doi.org/10.1071/RD01050
Submitted: 28 May 2002 Accepted: 2 May 2003 Published: 2 May 2003
Abstract
The 5′-regulative sequence and intron 1 of the goat β-casein gene from −4044 to +2123 bp was cloned and fused with the reporter gene of green fluorescent protein (GFP) to create a plasmid termed pGB562/GFP. To detect GFP expression, pGB562/GFP was transfected in vitro via liposomes into the mammary epithelial cell line NMuMG. Cells could not express GFP unless the transfected NMuMG cells lined up to create functional alveoli. These functional cells were cultured with lactogenic hormones, including insulin, dexamethasone and prolactin, and were grown on a layer of the extracellular matrix Matrigel. Green fluorescent protein expression levels in NMuMG cells were 25-, 55- and 42-fold those in the control group at 24, 48, and 72 h after pGB562/GFP transfection respectively. In addition, pGB562/GFP was transfected ex vivo by electroporation into mammary gland fragments and cells were then cultured in vitro with a supplement of lactogenic hormones. Strong GFP expression localized in fragments of the mammary gland was observed 24 h after gene transfer. The novel strategy of ex vivo gene transfer into mammary tissue using GFP as a reporter gene to detect the function of a tissue-specific promoter is efficient and convenient. The data obtained herein reveal that the 5′-regulative sequence and intron 1 of the 6.2 kb goat β-casein gene can enhance the efficiency of transgene expression. Thus, the GB562 sequence may act as a good promoter and effectively elevate the production of exogenous protein in mammary glands.
Acknowledgments
We thank Dr San-Chi Liang, former Director, and Dr Ming-Hseng Wang, present Director of the National Laboratory Animal Center, for their support, encouragement and critical comments. We also thank Dr Yu-Tien Chu for help with the laser scanning confocal microscopy and Miss Nei-Chi Lian for assistance with interpretation of the SEM results.
References
Altiok, S. , and Groner, B. (1993). Interaction of two sequence-specific single-stranded DNA-binding proteins with an essential region of the β-casein gene promoter is regulated by lactogenic hormones Mol. Cell. Biol. 13, 7303–10.
| PubMed |
Bischoff, R. , Degryse, E. , Perraud, F. , Dalemans, W. , Ali-Hadji, D. , Thepot, D. , Devinoy, E. , Houdebine, L. M. , and Pavirani, A. (1992). A 17.6 kbp region located upstream of the rabbit WAP gene directs high level expression of a functional human protein variant in transgenic mouse milk FEBS Lett. 305, 265–8.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Bleck, G. T. , and Bremel, R. D. (1994). Variation in expression of a bovine alpha-lactalbumin transgene in milk of transgenic mice J. Dairy Sci. 77, 1897–904.
| PubMed |
Buhler, T. A. , Bruyere, T. , Went, D. F. , Stranzinger, G. , and Burki, K. (1995). Rabbit β-casein promoter directs secretion of human interleukin-2 into the milk of transgenic rabbits Bio/Technology 8, 140–3.
Burdon, T. , Sankaran, L. , Wall, R. J. , Spencer, M. , and Hennighausen, L. (1991). Expression of a whey acidic protein transgene during mammary development. Evidence for different mechanisms of regulation during pregnancy and lactation J. Biol. Chem. 266, 6909–14.
| PubMed |
Cerdan, M. G. , Young, J. I. , Zino, E. , Falzone, T. L. , Otero, V. , Torres, H. N. , and Rubinstein, M. (1998). Accurate spatial and temporal transgene expression driven by a 3.8-kilobase promoter of the bovine β-casein gene in the lactating mouse mammary gland Mol. Reprod. Dev. 49, 236–45.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Doppler, W. , Groner, B. , and Ball, R. K. (1989). Prolactin and glucocorticoid hormones synergistically induce expression of transfected rat β-casein gene promoter constructs in a mammary epithelial cell line Proc. Natl Acad. Sci. USA 86, 104–8.
| PubMed |
Doppler, W. , Hock, W. , Hofer, P. , Groner, B. , and Ball, R. K. (1990). Prolactin and glucocorticoid hormones control transcription of the beta-casein gene by kinetically distinct mechanisms Mol. Endocrinol. 4, 912–19.
| PubMed |
Doppler, W. , Welte, T. , and Philipp, S. (1995). CCAAT/enhancer-binding protein isoforms β and α are expressed in mammary epithelial cells and bind to multiple sites in the β-casein gene promoter J. Biol. Chem. 270, 17 962–9.
| PubMed |
Ebert, K. M. , DiTullio, P. , Barry, C. A. , Schindler, J. E. , Ayres, S. L. , Smith, T. E. , Pellerin, L. J. , Meade, H. M. , Denman, J. , and Roberts, B. (1994). Induction of human tissue plasminogen activator in the mammary gland of transgenic goats Biotechnology 12, 699–702.
| PubMed |
Faerman, A. , Barash, I. , Puzis, R. , Nathan, M. , Hurwitz, D. R. , and Shani, M. (1995). Dramatic heterogeneity of transgene expression in the mammary gland of lactating mice: a model system to study the synthetic activity of mammary epithelial cells J. Histochem. Cytochem. 43, 461–70.
| PubMed |
Glass, R. H. , Aggeler, J. , Spindle, A. , Pedersen, R. , and Werb, Z. (1983). Degradation of extracellular matrix by mouse trophoblast outgrowths: a model for implantation J. Cell Biol. 96, 1108–16.
| PubMed |
Groner, B. , and Gouilleux, F. (1995). Prolactin-mediated gene activation in mammary epithelial cells Curr. Opin. Genet. Dev. 5, 587–94.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Gutierrez, A. , Meade, H. M. , DiTullio, P. , Pollock, D. , Harvey, M. , Jimenez-Flores, R. , Anderson, G. B. , Murray, J. D. , and Medrano, J. F. (1996). Expression of a bovine kappa-CN cDNA in the mammary gland of transgenic mice utilizing a genomic milk protein gene as an expression cassette Transgenic Res. 5, 271–9.
| PubMed |
Houdebine, L. M. (2000). Transgenic animal bioreactors Transgenic Res. 9, 305–20.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Huang, M. C. , and Lin, C. S. (1996). Cloning and restriction mapping of goat β-casein gene J. Agric. Forest. 45, 83–94.
Huang, Y. T. , Chang, Y. H. , Liang, C. T. , Chou, C. K. , Hong, C. C. , and Wu, M. F. (1997). Immunologic and ultrastructural characteristics of T-cell lymphoblastic lymphoma associated with mast cell infiltration in Wistar rat Lab. Anim. Sci. 47, 209–11.
| PubMed |
Kang, Y. K. , Lee, C. S. , Chung, A. S. , and Lee, K. K. (1998). Prolactin-inducible enhancer activity of the first intron of the bovine β-casein gene Mol. Cell 8, 259–65.
Kim, S. J. , Cho, Y. Y. , Lee, K. W. , Yu, D. Y. , Lee, C. S. , Han, Y. M. , and Lee, K. K. (1994). Expression of human lactoferrin in milk of transgenic mice by using bovine β-casein/human lactoferrin cDNA fusion gene Mol. Cell 4, 355–60.
Lee, K. F. , DeMayo, F. J. , Atiee, S. H. , and Rosen, J. M. (1988). Tissue-specific expression of the rat β-casein gene in transgenic mice Nucleic Acids Res. 16, 1027–41.
| PubMed |
Li, M. L. , Aggeler, J. , Farson, D. A. , Hatier, C. , and Hassell, J. (1987). Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells Proc. Natl Acad. Sci. USA 84, 136–40.
| PubMed |
Maga, E. A. , Anderson, G. B. , and Murray, J. D. (1995). The effect of mammary gland expression of human lysozyme on the properties of milk from transgenic mice J. Dairy Sci. 78, 2645–52.
| PubMed |
Maschio, A. , Brickell, P. M. , Kioussis, D. , Mellor, A. L. , Katz, D. , and Craig, R. K. (1991). Transgenic mice carrying the guinea-pig α-lactalbumin gene transcribe milk protein genes in their sebaceous glands during lactation Biochem. J. 275, 459–67.
| PubMed |
National Institutes of Health (1996).
Nemir, M. , Bhattacharyya, D. , Li, X. , Singh, K. , Mukherjee, A. B. , and Mukherjee, B. B. (2000). Targeted inhibition of osteopontin expression in the mammary gland causes abnormal morphogenesis and lactation deficiency J. Biol. Chem. 275, 969–76.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Petitclerc, D. , Attal, J. , Théron, M. C. , Bearzotti, M. , Bolifraud, P. , Kann, G. , Stinnakre, M. G. , Pointu, H. , Puissant, C. , and Houdebine, L. M. (1995). The effect of various introns and transcription terminators on the efficiency of expression vectors in various cultured cell lines and in the mammary gland of transgenic mice J. Biotechnol. 40, 169–78.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Provot, C. , Persuy, M. A. , and Mercier, J. C. (1995). Complete sequence of the ovine β-casein-encoding gene and interspecies comparison Gene 154, 259–63.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Rijnkels, M. and Pieper, F. R. (1998). Casein gene-based mammary gland-specific transgene expression In
Rijnkels, M. , Kooiman, P. M. , Krimpenfort, P. J. A. , de Boer, H. A. , and Pieper, F. R. (1995). Expression analysis of the individual bovine β -, αs2- and κ-casein genes in transgenic mice Biochem. J. 311, 929–37.
| PubMed |
Roberts, B. , DiTullio, P. , Vitale, J. , Hehir, K. , and Gordon, K. (1992). Cloning of the goat β-casein-encoding gene and expression in transgenic mice Gene 121, 255–62.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Shani, M. , Barash, I. , Nathan, M. , Ricca, G. , Searfoss, G. H. , Dekel, I. , Faerman, A. , Givol, D. , and Hurwitz, D. R. (1992). Expression of human serum albumin in the milk of transgenic mice Transgenic Res. 1, 195–208.
| PubMed |
Soulier, S. , Vilotte, J. L. , Stinnakre, M. G. , and Mercier, J. C. (1992). Expression analysis of ruminant α-lactalbumin in transgenic mice: developmental regulation and general location of important cis-regulatory elements FEBS Lett. 297, 13–18.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Suzuki, T. , Shin, B. C. , Fujikura, K. , Matsuzaki, T. , and Takata, K. (1998). Direct gene transfer into rat liver cells by in vivo electroporation FEBS Lett. 425, 436–40.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Vihinen, T. , Maatta, A. , Jaakkola, P. , Auvinen, P. , and Jalkanen, M. (1996). Functional characterization of mouse syndecan-1 promoter J. Biol. Chem. 271, 12 532–41.
| Crossref | GoogleScholarGoogle Scholar |
Wall, R. J. , Kerr, D. E. , and Bondioli, K. R. (1997). Transgenic daily cattle: genetic engineering on a large scale J. Dairy Sci. 80, 2213–24.
| PubMed |
Wechselberger, C. , Ebert, A. D. , Bianco, C. , Khan, N. I. , Sun, Y. , Wallace-Jones, B. , and Salomon, D. S. (2001). Cripto-1 enhances migration and branching morphogenesis of mouse mammary epithelial cells Exp. Cell Res. 266, 95–105.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Whitelaw, C. B. , Archibald, A. L. , Harris, S. , McClenaghan, M. , Simons, J. P. , and Clark, A. J. (1991). Targeting expression to the mammary gland: intronic sequences can enhance the efficiency of gene expression in transgenic mice Transgenic Res. 1, 3–13.
| PubMed |
Winklehner-Jennewein, P. , Geymayer, S. , Lechner, J. , Welte, T. , Hansson, L. , Geley, S. , and Doppler, W. (1998). A distal enhancer region in the human β-casein gene mediates the response to prolactin and glucocorticoid hormones Gene 217, 127–39.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Yoshimura, M. , and Oka, T. (1990). Transfection of β-casein chimeric gene and hormonal induction of its expression in primary murine mammary epithelial cells Proc. Natl Acad. Sci. USA 87, 3670–4.
| PubMed |
Yuh, I. S. , Jang, B. B. , and Hong, B. J. (1998). Effects of adenosine and epidermal growth factor on the growth of mouse mammary epithelial cells Cell Biol. Int. 22, 131–5.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Zhang, G. , Gurtu, V. , and Kain, S. R. (1996). An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells Biochem. Biophys. Res. Commun. 227, 707–11.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Zhang, K. , Lu, D. , Xue, J. , Huang, Y. , and Huang, S. (1998). Construction of mammary gland-specific expression vectors for human clotting factor IX and its secretory expression in goat milk Chin. J. Biotechnol. 13, 271–6.
Ziomek, C. A. (1998). Commercialization of proteins produced in the mammary gland Theriogenology 49, 139–44.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Zolotukhin, S. , Potter, M. , Hauswirth, W. , Guy, J. , and Muzyczka, N. (1996). A ‘humanized’ green fluorescent protein cDNA adapted for high-level expression in mammalian cells J. Virol. 70, 4646–54.
| PubMed |
