Development and Reproduction (한국발생생물학회지:발생과생식)

The Korean Society of Developmental Biology (한국발생생물학회)
권호 표지

Co-expression of IRES-mediated hG-CSF cDNA and hGH Gene under the Control of Goat beta-Casein Promoter

  • Oh, Keon-Bong (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Lee, Chul-Sang (Dept. of Biology, College of Natural Science, Kunsan National University)
  • Received : 2009.01.24
  • Accepted : 2010.03.02
  • Published : 2010.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

We developed a novel dicistronic system for the expression of target cDNA sequences in the milk of transgenic animals using goat beta-casein/hGH fusion construct, pGbc5.5hGH (Lee, 2006) and internal ribosome entry site (IRES) sequences of encephalomyocarditis virus (EMCV). Granulocyte colony-stimulating factor (hG-CSF) cDNA was linked to 3' untranslated region of hGH gene in the pGbc5.5hGH via EMCV IRES sequences. Transgenic mice were generated by microinjection and transgene expression was examined in the milk and mammary gland of transgenic mice at 10 days of lactation. Northern blot analysis showed that hGH gene and hG-CSF cDNA were transcribed as a single dicistronic mRNA. The hG-CSF and hGH proteins were independently translated from the dicistronic mRNA and secreted into the milk of transgenic mice. The highest concentration of hG-CSF and hGH in the milk of transgenic mice were $237{\mu}g/m{\ell}$ and $8,990{\mu}g/m{\ell}$, respectively. In contrast, another hG-CSF expression cassette, in which hG-CSF genomic sequences were inserted into a commercial milk-specific expression vector (pBC1), generated a lower level ($91{\mu}g/m{\ell}$) of hG-CSF expression in the milk of transgenic mice. These results demonstrated that the novel pGbc5.5hGH-based dicistronic construct could be useful for an efficient cDNA expression in the milk of transgenic animals.

Keywords

References

  1. Barash I, Nathan M, Kari R, Ilan N, Shani M, Hurwitz DR (1996) Elements within the beta-lactoglobulin gene inhibit expression of human serum albumin cDNA and minigenes in transfected cells but rescue their expression in the mammary gland of transgenic mice. Nucleic Acids Res 24:602-610. https://doi.org/10.1093/nar/24.4.602
  2. Bochkov YA, Palmenberg AC (2006) Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location. Biotechniques 41:283-284. https://doi.org/10.2144/000112243
  3. Borman AM, Bailly JL, Girard M, Kean KM (1995) Picornavirus internal ribosome entry segments: comparison of translation efficiency and the requirements for optimal internal initiation of translation in vitro. Nucleic Acids Res 23:3656-3663. https://doi.org/10.1093/nar/23.18.3656
  4. Borman AM, Le Mercier P, Girard M, Kean KM (1997) Comparison of picornaviral IRES-driven internal initiation of translation in cultured cells of different origins. Nucleic Acids Res 25:925-932. https://doi.org/10.1093/nar/25.5.925
  5. Clark AJ, Cowper A, Wallace R, Wright G, Simons JP (1992) Rescuing transgene expression by co-integration. Biotechnology (NY) 10:1450-1454. https://doi.org/10.1038/nbt1192-1450
  6. Clark AJ, Bissinger P, Bullock DW, Damak S, Wallace R, Whitelaw CB, Yull F (1994) Chromosomal position effects and the modulation of transgene expression. Reprod Fertil Dev 6:589-598. https://doi.org/10.1071/RD9940589
  7. Colman A (1996): Production of proteins in the milk of transgenic livestock: problems, solutions, and successes. Am J Clin Nutr 63:639S-645S. https://doi.org/10.1093/ajcn/63.4.639
  8. Devinoy E, Thepot D, Stinnakre MG, Fontaine ML, Grabowski H, Puissant C, Pavirani A, Houdebine LM, (1994) High level production of human growth hormone in the milk of transgenic mice: the upstream region of the rabbit whey acidic protein (WAP) gene targets transgene expression the mammary gland. Transgenic Res 3:79-84. https://doi.org/10.1007/BF01974085
  9. Drohan WN, Zhang DW, Paleyanda RK, Chang R, Wroble M, Velander W, Lubon H (1994) Inefficient processing of human protein C in the mouse mammary gland. Transgenic Res 3:355-364. https://doi.org/10.1007/BF01976767
  10. Dvorianchikov GA, Serova IA, Andreeva LE, Dias LP, Azevedo S, Serov OL (2005) Secretion of biologically active human granulocyte colony-stimulating factor (G-CSF) in milk of transgenic mice. Genetika 41: 1331-1337.
  11. Ebert KM, Selgrath JP, DiTullio P, Denman J, Smith TE, Memon MA ,Schindler JE, Monastersky GM, Vitale JA, Gordon K (1991) 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. https://doi.org/10.1038/nbt0991-835
  12. Ebert KM, DiTullio P, Barry CA, Schindler JE, Ayres SL, Smith TE, Pellerin LJ, Meade HM, Denman J, Roberts B (1994) Induction of human tissue plasminogen activator in the mammary gland of transgenic goats. Biotechnology (NY) 12:699-702. https://doi.org/10.1038/nbt0794-699
  13. Fujiwara Y, Miwa M, Takahashi R, Kodaira K, Hirabayashi M, Suzuki T, Ueda M (1999) High-level expressing YAC vector for transgenic animal bioreactors. Mol Reprod Dev 52:414-420. https://doi.org/10.1002/(SICI)1098-2795(199904)52:4<414::AID-MRD10>3.0.CO;2-S
  14. Hoff J (2000) Methods of blood collection in the mouse. Lab Animal 29:47-53.
  15. Holcik M, Sonenberg N, Korneluk RG (2000) Internal ribosome initiation of translation and the control of cell death. Trends Genet 16:469-473. https://doi.org/10.1016/S0168-9525(00)02106-5
  16. Huang YJ, Huang Y, Baldassarre H, Wang B, Lazaris A, Leduc M, Bilodeau AS, Bellemare A, Cote M, Herskovits P, Touati M, Turcotte C, Valeanu L, Lemee N, Wilgus H, Begin I, Bhatia B, Rao K, Neveu N, Brochu E, Pierson J, Hockley DK, Cerasoli DM, Lenz DE, Karatzas CN, Langermann S (2007) Recombinant human butyrylcholinesterase from milk of transgenic animals to protect against organophosphate poisoning. Proc Natl Acad Sci USA 104:13603-13608. https://doi.org/10.1073/pnas.0702756104
  17. Jang SK, Krausslich HG, Nicklin MJ, Duke GM, Palmenberg AC, Wimmer E (1988) A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol 62:2636-2643.
  18. Ko JH, Lee CS, Kim KH, Pang MG, Koo JS, Fang N, Koo DB, Oh KB, Youn WS, Zheng GD, Park JS, Kim SJ, Han YM, Choi IY, Lim J, Shin ST, Jin SW, Lee KK, Yoo OJ (2000) Production of biologically active human granulocyte colony stimulating factor in the milk of transgenic goat. Transgenic Res 9:215-222. https://doi.org/10.1023/A:1008972010351
  19. Krysiak R, Gdula-Dymek A, Bednarska-Czerwinska A, Okopien B (2007) Growth hormone therapy in children and adults. Pharmacol Rep 59:500-516.
  20. Lee CS, Kim K, Yu DY, Lee KK (1996) An efficient expression of human growth hormone (hGH) in the milk of transgenic mice using rat beta-casein/hGH fusion genes Appl Biochem Biotechnol 56:211-222. https://doi.org/10.1007/BF02786953
  21. Lee CS (2006) Goat $\beta$-casein promoter directs hGH expression at a high level and in a mammary-specific manner in transgenic mice. Korean J Genetics 28:131-137
  22. Mizuguchi H, Xu Z, Ishii-Watabe A, Uchida E, Hayakawa T (2000) IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. Mol Ther 1:376-382. https://doi.org/10.1006/mthe.2000.0050
  23. Murray RD, Shalet SM (2000) Growth hormone: current and future therapeutic applications. Expert Opin Pharmacother 1:975-990. https://doi.org/10.1517/14656566.1.5.975
  24. Ninomiya T, Hirabayashi M, Sagara J, Yuki A (1994) Functions of milk protein gene 5' flanking regions on human growth hormone gene. Mol Reprod Dev 37: 276-83. https://doi.org/10.1002/mrd.1080370306
  25. Oh KB, Lee CS (2009) Characterization of double transgenic mice harboring both goat $\beta$-casein/hGH and goat $\beta$- casein/hG-CSF hybrid genes. Dev Reprod 13:191-198.
  26. Shen W, Lan G, Yang X, Li L, Min L, Yang Z, Tian L, Wu X, Sun Y, Chen H, Tan J, Deng J, Pan Q (2007) Targeting the exogenous htPAm gene on goat somatic cell beta-casein locus for transgenic goat production. Mol Reprod Dev 74:428-434. https://doi.org/10.1002/mrd.20595
  27. Sonenberg N, Hinnebusch AG (2009) Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell 136:731-745. https://doi.org/10.1016/j.cell.2009.01.042
  28. van Berkel PH, Welling MM, Geerts M, van Veen HA, Ravensbergen B, Salaheddine M, Pauwels EK, Pieper F, Nuijens JH, Nibbering PH (2002) Large scale production of recombinant human lactoferrin in the milk of transgenic cows. Nat Biotechnol 20:484-487. https://doi.org/10.1038/nbt0502-484
  29. Wong ET, Ngoi SM, Lee CG (2002) Improved co-expression of multiple genes in vectors containing internal ribosome entry sites (IRESes) from human genes. Gene Ther 9:337-344. https://doi.org/10.1038/sj.gt.3301667
  30. Zhang J, Wang Y, Guangsan L, Yingyun W, Guofa H, Yu S, Miao D (2001) Co-expression of multiple genes constructs in transgenic mice. Biotechnology Letters 23:1249-1255. https://doi.org/10.1023/A:1010585328200