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In Vitro Expression of the Recombinant hFSH Gene using Retrovirus Vector System  

Min, Gyeong-Heon (Department of Physiology, Catholic University of Daegu School of Medicine)
Kwon, Mo-Sun (Department of Physiology, Catholic University of Daegu School of Medicine)
Kim, Teoan (Department of Physiology, Catholic University of Daegu School of Medicine)
Koo, Bon-Chul (Department of Physiology, Catholic University of Daegu School of Medicine)
Publication Information
Reproductive and Developmental Biology / v.35, no.1, 2011 , pp. 115-121 More about this Journal
Abstract
hFSH is a glycoprotein secreted from anterior pituitary and consists of ${\alpha}$ and ${\beta}$ subunits. Because of its major biological functions including sperm formation in the male and for follicular growth, FSH is used to cure woman's sterility. In this study we tried to produce recombinant hFSH in vitro using a retrovirus expression vector. Two major components of the vector we constructed are: ( i ) a DNA fragment containing ${\alpha}$ and ${\beta}$ genes fused by a DNA sequence coding carboxyl terminal peptide (CTP) of human chorionic gonadotropin, (ii) a DNA fragment corresponding woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Evaluation of expression profile of the recombinant FSH using reverse transcription PCR and enzyme-linked immunosorbent assay (ELISA). Among three cell lines tested, HeLa cells were the best for hFSH expression (5,395 mIU/ml), then followed by chicken embryonic fibroblast (CEF) cells and Chinese hamster ovary (CHO) cells in the order of hFSH production. In addition to the amount, the FSH produced from HeLa cells was highest in terms of biological activity which was determined by measuring cAMP.
Keywords
hFSH; Retrovirus vector; CTP; WPRE; Biological activity;
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1 Sugahara T, Sato A, Kudo M, Ben-Menahem D, Pixley MR, Hsueh AJ, Boime I (1996): Expression of biologically active fusion genes encoding the common ${\alpha}$ subunit and the follicle-stimulating hormone ${\beta}$ subunit. J BioI Chem 271:10445-10448.   DOI
2 Walsh G (2006): Biopharmaceutical benchmarks. Net Biotechonol 24:769-776.
3 Wurm FM (2004): Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22:1393-1398.   DOI   ScienceOn
4 Zufferey R, Donello JE, Trono D, Hope TJ (1999): Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes by retroviral vectors. J Virol 73:2886-2892.
5 Montesino R, Garcia R, Quintero O, Cremata JA (1998): Variation in N-linked oligosaccharide structures on heterologous proteins secreted by the methylotrophic yeast Pichia pastoris. Protein Expr Purif 14:197-207.   DOI   ScienceOn
6 Moyle WR, Myers RV, Wang Y, Han Y, Lin W, Kelley GL, Ehrlich PH, Rao SN, Bernard MP (1998): Functional homodimeric glycoprotein hormones: implications for hormone action and evolution. Chem BioI 5:241-254.   DOI   ScienceOn
7 Nishikawa M, Huang L (2001): Nonviral vectors in the new millennium: delivery barriers in gene transfer. Hum Gene Ther 12:861-870.   DOI   ScienceOn
8 Out HJ, Mannaerts BMJL, Driessen SGAJ, Bennink HJTC (1996): Recombinant follicle stimulating hormone (rFSH; Puregon) in assisted reproduction: more oocytes, more pregnancies. Results from five comparative studies. Hum Reprod Update 2:162-71.   DOI   ScienceOn
9 Out HJ., Bennink HJ, de Laat WN (1999): What are the clinical benefits of recombinant gonadotrphins? the development of recombinant FSH (Puregon): a scientific business. Hum Reprod 14:2189-2190.   DOI
10 Padmanbhan V, Chappel SC, Beitins IZ (1987): An improved in vitro bioassay for follicle-stimulating hormone (FSH): suitable for measurement of FSH in unextracted human serum. Endocrinol 121:1089-1098.   DOI   ScienceOn
11 Palomares LA, Estrada MS, Ramirez OT (2004): Production of recombinant protein: Challenges and solutions. Methods Mol BioI 267:15-52.
12 Pavlou AK, Reichert JM (2004): Recombinant protein therapeutics-success rates, market trends and values to 2010. Nature Biotechnol 22:1513-1519.   DOI   ScienceOn
13 Kirby SL (1999): The therapeutic potential of erythropoietin receptor transgenes. Cytokines Cell Mol Ther 5:97-104.
14 Klein J, Lobel L, Pollak S, Lustbader B, Ogden RT, Sauer MV, Lustbader JW (2003): Development and characterization of a long-acting recombinant hFSH agonist. Hum Reprod 18:50-56.   DOI   ScienceOn
15 Lapolt PS, Nishimori K, Fares FA, Perlas E, Boime I, Hsueh AJW (1992): Enhanced stimulation of follicle maturation and ovulatory potential by long acting follicle-stimulating hormone agonist with extended carboxylterminal peptide. Endocrinol 131:2514-2520.   DOI   ScienceOn
16 Kay MA, Glorioso JC, Naldini L (2001): Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. Nature Med 7:33-40.   DOI   ScienceOn
17 Fares FA, Suganuma N, Nishimori K, LaPolt PS, Hsueh AJ, Boime I (1992): Design of a long-acting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin ${\beta}$ subunit to the foIIitropin ${\beta}$ subunit. Proc Natl Acad Sci USA 89:4304-4308.   DOI   ScienceOn
18 Jennings CJ, Moreland K, Peterson CM (1996): In vitro fertilization. Drugs 52:313-343.   DOI   ScienceOn
19 Jones HW Jr, Jones GS, Andrews MC, Acosta A, Bundren C, Garcia J, Sandow B, Veeck L, Wilkes C, Witmyer J, Wortham JE, Wright G (1982): The program for in vitro fertilization at Norfolk. Fertil Steril 38:14-21.
20 Kim MO, Kim SH, Shin MJ, Lee DB, Kim TW, Kim KS, Ha JH, Lee S, Park YB, Kim SJ, Ryoo ZY (2006): Human erythropoietin induces lung failure and erythrocytosis in transgenic mice. Mol Cells 23:17-22.
21 Kim NS, Chang KH, Chung BS, Kim SH, Kim JH, Lee GM (2003): Characterization of humanized anti-body produced by apoptosis-resistant CHO cells under sodium butyrate-induced condition. J Microbiol Biotechnol 13:926-936.
22 Kim T (2002): Retrovirus-mediated gene transfer. In Transgenic Animal Technology. Edited by Pinkert CA. 2nd ed. Academic Press, San Diego, CA, pp 173-193.
23 Speroff L, Glass RH, Kase NG (1989): Clinical Gynecologic Endocrinology and Infertility, pp 583-609. 3rd Ed. Williams & Wikins, Baltimore, USA.
24 Baenziger JU, Green ED (1988): Pituitary glycoprotein hormone oligosaccharides: Structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin. Biochim Biophys Acta 947:287-306.   DOI   ScienceOn
25 Bagatti GC, Datola CA, Gostoli G, Mascia M, Polletta P, Roncalli G, Simoni P (2001): Characterization and comparison of recombinant human follicle-stimulating hormones. J Clin Res 4:91-104.
26 Prevost RR (1998): Recombinant follicle-stimulating hormone: New biotechnology for infertility. Pharmacotherapy 18:1001-1010.
27 Reichert Jr, LE, Dattatreyamurty B (1989): The follicle-stimulating hormone (FSH) receptor in testis: interaction with FSH, mechanism of signal transduction, and properties of the purified receptor. BioI Reprod 40:13-26.   DOI   ScienceOn
28 Schaison G, Young J, Pholsena M, Nahoul K, Couzinet B (1993): Failure of combined follicle-stimulating hormone-testosterone administration to initiate and/or maintain spermatogenesis in men with hypogonadotropic hypogonadism. J Clin Endocrinol Metab 77:1545-1549.   DOI   ScienceOn
29 Liu ML, Winther BL, Kay MA (1996): Pseudotransduction of hepatocytes by using concentrated pseudotyped vesicular stomatitis virus G glycoprotein (VSVG)-Moloney murine leukemia virus-derived retrovirus vectors: comparison of VSV-G and amphotropic vectors for hepatic gene transfer. J Viral 70:2497-2502.
30 Link N, Aubel C, Kelm JM, Marty RR, Greber D, Djonov V, Bourhis J, Weber W, Fussenegger M (2006): Therapeutic protein transduction of mammalian cells and mice by nucleic acid-free lentiviral nanoparticles. Nucleic Acids Res 34:e16.   DOI   ScienceOn
31 Loumaye E, Campbell R, Salat-Baroux J (1995): Human follicle-stimulating hormone produced by recombinant DNA technology: a review for clinicians. Hum Reprod Update 1:188-199.   DOI   ScienceOn
32 Chung JY, Ahn HK, Lim SW, Sung YH, Koh YW, Park SK, Lee GM (2003): Development of recombinant Chinese hamster ovary cell lines producing human thrombopoietin or its analog. J Microbiol Biotechnol 13:759-766.
33 Chang GD, Chen CJ, Lin CY, Chen HC, Chen H (2003): Improvement of glycosylation in insect cells with mammalian glycosyltransferases. J Biotechnol 102:61-71.   DOI   ScienceOn
34 Bergh C, Howles CM, Borg K, Hamberger L, Josefsson B, Nilsson L, Wikland M (1997): Recombinant human follicle stimulating hormone (r-hFSH; Gonal-F) versus highly purified urinary FSH (Metrodin HP): results of a randomized comparative study in women undergoing assisted reproductive techniques. Hum Reprod 12:2133-2139.   DOI   ScienceOn
35 Braun P, LaBaer J (2003): High throughput protein production for functional proteomics. Trends biotechnol 21:383-388.   DOI   ScienceOn
36 Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK (1993): Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: Concentration to very high titer and effcient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci USA 90:8033-8037.   DOI   ScienceOn