Generation of transgenic fibroblasts producing doxycycline‑inducible human interferon‑α or erythropoietin for a bovine mammary bioreactor

Kang,Hee  Young; Hong,Eui‑Ju; Hwang,Kyu‑Chan; Kim,Nam‑Hyung; Hwang,Woo‑Suk; Jeung,Eui‑Bae

doi:10.3892/mmr.2015.3483

Generation of transgenic fibroblasts producing doxycycline‑inducible human interferon‑α or erythropoietin for a bovine mammary bioreactor

Authors:
- Hee Young Kang
- Eui‑Ju Hong
- Kyu‑Chan Hwang
- Nam‑Hyung Kim
- Woo‑Suk Hwang
- Eui‑Bae Jeung
View Affiliations

Affiliations: Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361‑763, Republic of Korea, Department of Research and Development, Sooam Biotech Research Foundation, Seoul 137‑851, Republic of Korea , Department of Animal Sciences, Chungbuk National University, Cheongju, Chungbuk 361‑763, Republic of Korea
Published online on: March 13, 2015 https://doi.org/10.3892/mmr.2015.3483
Pages: 1137-1144

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Interferon α (IFN‑α) is a cytokine, produced predominantly in immune cells in response to pathogens, which interferes with viral replication in host cells. Another cytokine hormone, erythropoietin (EPO), is synthesized in interstitial fibroblasts of the kidney and acts as a stimulator for the production of red blood cells. Importantly, the two cytokines have been used in the treatment of certain hematological malignancies, including renal anemia. In the production of recombinant proteins, a transgenic expression system in bovine species is an efficient strategy for pharmaceutical production. In the present study, recombinant constructs capable of producing recombinant human IFN‑α and EPO proteins were established and were generated containing the mammary gland‑specific αS1‑casein promoter region (between ‑175 and +796 nt), as this promoter was revealed to have the highest level of activity in a previous promoter study. In order to minimize developmental toxicity by constitutive exogenous expression, a doxycycline (dox)‑inducible system was introduced to the IFN‑α/EPO‑expressing constructs. Therefore, a unitary tetracycline (tet)‑on the IFN‑α/EPO vector was established, which combined a tet‑on activator cassette controlled by the αS1‑casein promoter, with a responder cassette encoding the IFN‑α/EPO gene, controlled by the tetracycline response element (TRE) promoter. In these systems, the tet‑controlled transactivator is affected by mammary gland‑specific αS1‑casein promoter, and binding of the transcriptional activator to the TRE results in transcription of the downstream IFN‑α/EPO genes in the presence of dox. To assess this, the unitary tet‑on IFN‑α/EPO vector was introduced into a bovine mammary gland cell line (MAC‑T), and the cells were then treated with 0.1‑1 µg/ml dox. A marked increase was observed in the expression levels of IFN‑α/EPO. In addition, bovine transgenic fibroblasts containing a mammary gland‑specific and dox‑inducible IFN‑α/EPO construct were generated. These transgenic fibroblasts may provide a source for somatic cell nuclear transfer for the generation of transgenic cattle producing recombinant human IFN‑α/EPO protein during lactation.

View Figures

View References

1	Jonasch E and Haluska FG: Interferon in oncological practice: review of interferon biology, clinical applications and toxicities. Oncologist. 6:34–55. 2001. View Article : Google Scholar
2	Belardelli F: Role of interferons and other cytokines in the regulation of the immune response. APMIS. 103:161–179. 1995. View Article : Google Scholar : PubMed/NCBI
3	Biron CA, Nguyen KB, Pien GC, Cousens LP and Salazar-Mather TP: Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu Rev Immunol. 17:189–220. 1999. View Article : Google Scholar : PubMed/NCBI
4	Bogdan C: The function of type I interferons in antimicrobial immunity. Curr Opin Immunol. 12:419–424. 2000. View Article : Google Scholar : PubMed/NCBI
5	Jelkmann W: Erythropoietin after a century of research: younger than ever. Eur J Haematol. 78:183–205. 2007. View Article : Google Scholar : PubMed/NCBI
6	Neumann E: Regulation of erythropoiesis. Acta Med Austriaca Supp. 6:360–363. 1979.In German.
7	Argenti M: Hematosis and erythropoiesis in guinea pigs exposed to low oxygen pressure. Riv Med Aeronaut. 14:283–313. 1951.In Undetermined Language. PubMed/NCBI
8	Jelkmann W: Regulation of erythropoietin production. J Physiol. 589:1251–1258. 2011. View Article : Google Scholar :
9	Macdougall IC and Ashenden M: Current and upcoming erythropoiesis-stimulating agents, iron products and other novel anemia medications. Adv Chronic Kidney Dis. 16:117–130. 2009. View Article : Google Scholar : PubMed/NCBI
10	Yang X and Carter MG: Transgenic animal bioreactors: a new line of defense against chemical weapons? Proc Natl Acad Sci USA. 104:13859–13860. 2007. View Article : Google Scholar : PubMed/NCBI
11	Baneyx F: Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol. 10:411–421. 1999. View Article : Google Scholar : PubMed/NCBI
12	Ebert KM, Selgrath JP, DiTullio P, et al: Transgenic production of a variant of human tissue-type plasminogen activator in goat milk: generation of transgenic goats and analysis of expression. Biotechnology (NY). 9:835–838. 1991. View Article : Google Scholar
13	Krimpenfort P, Rademakers A, Eyestone W, et al: Generation of transgenic dairy cattle using ‘in vitro’ embryo production. Biotechnology (NY). 9:844–847. 1991. View Article : Google Scholar
14	Buhler TA, Bruyere T, Went DF, Stranzinger G and Burki K: Rabbit beta-casein promoter directs secretion of human interleukin-2 into the milk of transgenic rabbits. Biotechnology (NY). 8:140–143. 1990. View Article : Google Scholar
15	Cerdan MG, Young JI, Zino E, et al: Accurate spatial and temporal transgene expression driven by a 3.8-kilobase promoter of the bovine beta-casein gene in the lactating mouse mammary gland. Mol Reprod Dev. 49:236–245. 1998. View Article : Google Scholar : PubMed/NCBI
16	Ebert KM, DiTullio P, Barry CA, et al: Induction of human tissue plasminogen activator in the mammary gland of transgenic goats. Biotechnology (NY). 12:699–702. 1994. View Article : Google Scholar
17	Gordon K, Lee E, Vitale JA, Smith AE, Westphal H and Hennighausen L: Production of human tissue plasminogen activator in transgenic mouse milk. 1987. Biotechnology. 24:425–428. 1992.PubMed/NCBI
18	Ikonen T, Ojala M and Ruottinen O: Associations between milk protein polymorphism and first lactation milk production traits in Finnish Ayrshire Cows. J Dairy Sci. 82:1026–1033. 1999. View Article : Google Scholar : PubMed/NCBI
19	Buser AC, Gass-Handel EK, Wyszomierski SL, et al: Progesterone receptor repression of prolactin/signal transducer and activator of transcription 5-mediated transcription of the beta-casein gene in mammary epithelial cells. Mol Endocrinol. 21:106–125. 2007. View Article : Google Scholar
20	Doppler W, Windegger M, Soratroi C, et al: Expression level-dependent contribution of glucocorticoid receptor domains for functional interaction with STAT5. Mol Cell Biol. 21:3266–3279. 2001. View Article : Google Scholar : PubMed/NCBI
21	Raught B, Liao WS and Rosen JM: Developmentally and hormonally regulated CCAAT/enhancer-binding protein isoforms influence beta-casein gene expression. Mol Endocrinol. 9:1223–1232. 1995.PubMed/NCBI
22	Malewski T and Zwierzchowski L: Computer-aided analysis of potential transcription-factor binding sites in the rabbit beta-casein gene promoter. Biosystems. 36:109–119. 1995. View Article : Google Scholar : PubMed/NCBI
23	Rosen JM, Rodgers JR, Couch CH, et al: Multihormonal regulation of milk protein gene expression. Ann NY Acad Sci. 478:63–76. 1986. View Article : Google Scholar : PubMed/NCBI
24	Rosen JM, Wyszomierski SL and Hadsell D: Regulation of milk protein gene expression. Annu Rev Nutr. 19:407–436. 1999. View Article : Google Scholar : PubMed/NCBI
25	Hodges VM, Rainey S, Lappin TR and Maxwell AP: Pathophysiology of anemia and erythrocytosis. Crit Rev Oncol Hematol. 64:139–158. 2007. View Article : Google Scholar : PubMed/NCBI
26	Gossen M and Bujard H: Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA. 89:5547–5551. 1992. View Article : Google Scholar : PubMed/NCBI
27	Zhou X, Vink M, Klaver B, Berkhout B and Das AT: Optimization of the Tet-On system for regulated gene expression through viral evolution. Gene Ther. 13:1382–1390. 2006. View Article : Google Scholar : PubMed/NCBI
28	Gossen M, Freundlieb S, Bender G, Müller G, Hillen W and Bujard H: Transcriptional activation by tetracyclines in mammalian cells. Science. 268:1766–1769. 1995. View Article : Google Scholar : PubMed/NCBI
29	Jung EM, An BS, Kim YK, et al: Establishment of transgenic fibroblasts for producing recombinant human interferon-α and erythropoietin in bovine milk. Mol Med Rep. 7:406–412. 2013.
30	Wang CJ, Xiao CW, You TG, et al: Interferon-alpha enhances antitumor activities of oncolytic adenovirus-mediated IL-24 expression in hepatocellular carcinoma. Mol Cancer. 11:312012. View Article : Google Scholar
31	Biron CA: Interferons alpha and beta as immune regulators - a new look. Immunity. 14:661–664. 2001. View Article : Google Scholar : PubMed/NCBI
32	Gallucci S, Lolkema M and Matzinger P: Natural adjuvants: endogenous activators of dendritic cells. Nat Med. 5:1249–1255. 1999. View Article : Google Scholar : PubMed/NCBI
33	Son YD, Jeong YT, Park SY and Kim JH: Enhanced sialylation of recombinant human erythropoietin in Chinese hamster ovary cells by combinatorial engineering of selected genes. Glycobiology. 21:1019–1028. 2011. View Article : Google Scholar : PubMed/NCBI
34	Tuite MF, Dobson MJ, Roberts NA, et al: Regulated high efficiency expression of human interferon-alpha in Saccharomyces cerevisiae. EMBO J. 1:603–608. 1982.PubMed/NCBI
35	Wall RJ, Kerr DE and Bondioli KR: Transgenic dairy cattle: genetic engineering on a large scale. J Dairy Sci. 80:2213–2224. 1997. View Article : Google Scholar : PubMed/NCBI
36	Zuelke KA: Transgenic modification of cows milk for value-added processing. Reprod Fertil Dev. 10:671–676. 1998. View Article : Google Scholar
37	Huynh HT, Robitaille G and Turner JD: Establishment of bovine mammary epithelial cells (MAC-T): an in vitro model for bovine lactation. Exp Cell Res. 197:191–199. 1991. View Article : Google Scholar : PubMed/NCBI
38	Berry SD, Weber Nielsen MS, Sejrsen K, Pearson RE, Boyle PL and Akers RM: Use of an immortalized bovine mammary epithelial cell line (MAC-T) to measure the mitogenic activity of extracts from heifer mammary tissue: effects of nutrition and ovariectomy. Domest Anim Endocrinol. 25:245–253. 2003. View Article : Google Scholar : PubMed/NCBI
39	Baguisi A, Behboodi E, Melican DT, et al: Production of goats by somatic cell nuclear transfer. Nat Biotechnol. 17:456–461. 1999. View Article : Google Scholar : PubMed/NCBI
40	Kubota C, Yamakuchi H, Todoroki J, et al: Six cloned calves produced from adult fibroblast cells after long-term culture. Proc Natl Acad Sci USA. 97:990–995. 2000. View Article : Google Scholar : PubMed/NCBI