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Knock-in of Enhanced Green Fluorescent Protein or/and Human Fibroblast Growth Factor 2 Gene into β-Casein Gene Locus in the Porcine Fibroblasts to Produce Therapeutic Protein

  • Lee, Sang Mi (Department of Animal Science, College of Agriculture and Life Science, Chonnam National University) ;
  • Kim, Ji Woo (Department of Animal Science, College of Agriculture and Life Science, Chonnam National University) ;
  • Jeong, Young-Hee (Department of Animal Science, College of Agriculture and Life Science, Chonnam National University) ;
  • Kim, Se Eun (Department of Animal Science, College of Agriculture and Life Science, Chonnam National University) ;
  • Kim, Yeong Ji (Department of Animal Science, College of Agriculture and Life Science, Chonnam National University) ;
  • Moon, Seung Ju (Department of Animal Science, College of Agriculture and Life Science, Chonnam National University) ;
  • Lee, Ji-Hye (Department of Animal Science and Biotechnology, College of Agriculture and Life Sciences, Chungnam National University) ;
  • Kim, Keun-Jung (Department of Animal Science and Biotechnology, College of Agriculture and Life Sciences, Chungnam National University) ;
  • Kim, Min-Kyu (Department of Animal Science and Biotechnology, College of Agriculture and Life Sciences, Chungnam National University) ;
  • Kang, Man-Jong (Department of Animal Science, College of Agriculture and Life Science, Chonnam National University)
  • Received : 2014.03.27
  • Accepted : 2014.06.24
  • Published : 2014.11.01

Abstract

Transgenic animals have become important tools for the production of therapeutic proteins in the domestic animal. Production efficiencies of transgenic animals by conventional methods as microinjection and retrovirus vector methods are low, and the foreign gene expression levels are also low because of their random integration in the host genome. In this study, we investigated the homologous recombination on the porcine ${\beta}$-casein gene locus using a knock-in vector for the ${\beta}$-casein gene locus. We developed the knock-in vector on the porcine ${\beta}$-casein gene locus and isolated knock-in fibroblast for nuclear transfer. The knock-in vector consisted of the neomycin resistance gene (neo) as a positive selectable marker gene, diphtheria toxin-A gene as negative selection marker, and 5' arm and 3' arm from the porcine ${\beta}$-casein gene. The secretion of enhanced green fluorescent protein (EGFP) was more easily detected in the cell culture media than it was by western blot analysis of cell extract of the HC11 mouse mammary epithelial cells transfected with EGFP knock-in vector. These results indicated that a knock-in system using ${\beta}$-casein gene induced high expression of transgene by the gene regulatory sequence of endogenous ${\beta}$-casein gene. These fibroblasts may be used to produce transgenic pigs for the production of therapeutic proteins via the mammary glands.

Acknowledgement

Supported by : Rural Development Administration

References

  1. Sullivan, P. M., H. Mezdour, Y. Aratani, C. Knouff, J. Najib, R. L. Reddicki, S. H. Quarfordt, and N. Maeda. 1997. Targeted replacement of the mouse apolipoprotein E gene with the common human APOE3 allele enhances diet-induced hypercholesterolemia and atherosclerosis. J. Biol. Chem. 272:17972-17980. https://doi.org/10.1074/jbc.272.29.17972
  2. Sullivan, P. M., H. Mezdour, S. H. Quarfordt, and N Maeda.1998. Type III hyperlipoproteinemia and spontaneous atherosclerosis in mice resulting from gene replacement of mouse apoe with human APOE 2. J. Clin. Invest. 102:130-135. https://doi.org/10.1172/JCI2673
  3. Wang, B. and J. Zhou. 2003. Specific genetic modifications of domestic animals by gene targeting and animal cloning. Reprod. Biol. Endocrinol. 1:103. https://doi.org/10.1186/1477-7827-1-103
  4. Wilmut, I., A. E. Schnieke, J. McWhir, A. J. Kind, and K. H. S. Campbell. 1997. Viable offspring derived from fetal and adult mammalian cells. Nature 385:810-813. https://doi.org/10.1038/385810a0
  5. Wolf, E., W. Schernthaner, V. Zakhartchenko, K. Prelle, M. Stojkovic, and G. Brem. 2000. Transgenic technology in farm animals - progress and perspectives. Exp. Physiol. 85:615-625. https://doi.org/10.1111/j.1469-445X.2000.02110.x
  6. Yanagawa, Y., T. Kobayashi, M. Ohnishi, T. Kobayashi, S. Tamura, T. Tsuzuki, M. Sanbo, T. Yagi, F. Tashiro, and J. I. Miyazaki. 1999. Enrichment and efficient screening of ES cells containing a targeted mutation : The use of DT-A gene with the polyadenylation signal as a negative selection maker. Transgenic Res. 8:215-221. https://doi.org/10.1023/A:1008914020843
  7. Ahn, K. S., Y. J. Kim, M. Kim, B. H. Lee, S. Y. Heo, M. J. Kang, Y. K. Kang, J. W. Lee, K. K. Lee, J. H. Kim, W. G. Nho, S. S. Hwang, J. S. Woo, J. K. Park, S. B. Park, and H. Shim. 2011. Resurrection of an alpha-1,3-galactosyltransferase genetargeted miniature pig by recloning using postmortem ear skin fibroblast. Theriogenology 75:933-939. https://doi.org/10.1016/j.theriogenology.2010.11.001
  8. Chan, A. W. S. 1999. Transgenic animals: Current and alternative strategies. Cloning. 1:25-46. https://doi.org/10.1089/15204559950020076
  9. Clark, A. J. 1998. The mammary gland as a bioreactor: expression, processing, and production of recombinant proteins. J. Mammary Gland Biol. Neoplasia 3:337-350. https://doi.org/10.1023/A:1018723712996
  10. Clark, A. J., S. Burl, C. Denning, and P. Dickinson. 2000. Gene targeting in livestock: A preview. Transgenic Res. 9:263-275. https://doi.org/10.1023/A:1008974616402
  11. Denning, C. and H. Priddle. 2003. New frontiers in gene targeting and cloning: success, application and challenges in domestic animals and human embryonic stem cells. Reproduction 126:1-11. https://doi.org/10.1530/rep.0.1260001
  12. Hammer, R. E., V. G. Pursel, C. E. Rexroad, R. J. Wall, D. J. Bolt, K. M. Ebert, R. D. Palmiter, and R. L. Brinster. 1985. Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315:680-683. https://doi.org/10.1038/315680a0
  13. Doppler, W., B. Groner, and R. K. Ball. 1989. Prolactin and glucocorticoid hormones synergistically induce expression of transfected rat $\beta$-casein gene promoter constructs in a mammary epithelial cell line. Proc. Nat. Acad. Sci. USA. 86:104-108. https://doi.org/10.1073/pnas.86.1.104
  14. Gordon, J. W. and F. H. Ruddle. 1981. Integration and stable germ line transmission of genes injected into mouse pronuclei. Science 214:1244-1246. https://doi.org/10.1126/science.6272397
  15. Hamanaka, H., Y. Katoh-Fukui, K. Suzuki, M. Kobayashi, R. Suzuki, Y. Motegi, Y. Nakahara, A. Takeshita, M. Kawai, K. Ishiguro, M. Yokoyama, and S. C. Fujita. 2000. Altered cholesterol metabolism in human apolipoprotein $E_4$ knock-in mice. Hum. Mol. Genet. 9:353-361. https://doi.org/10.1093/hmg/9.3.353
  16. Houdebine, L. M. 2000. Transgenic animal bioreactors. Transgenic Res. 9:305-320. https://doi.org/10.1023/A:1008934912555
  17. Houdebine, L. M. 2009. Production of pharmaceutical proteins by transgenic animals. Comp. Immunol. Microbiol. Infect. Dis. 32:107-121. https://doi.org/10.1016/j.cimid.2007.11.005
  18. Kim, J. W., H. M. Kim, S. M. Lee, and M. J. Kang. 2012. Porcine knock-in fibroblasts expressing hDAF on $\alpha$-1,3-galactosyltransferase (GGTA1) gene locus. Asian Australas. J. Anim. Sci. 25:1473-1480. https://doi.org/10.5713/ajas.2012.12146
  19. Kumar, S., A. R. Clarke, M. L. Hooper, D. S. Horne, H. A. J. Law, J. Leaver, A. Springbett, E. Stevenson, and P. Simons. 1994. Milk composition and lactation of $\beta$-casein-deficient mice. Proc. Natl. Acad. Sci. USA. 91:6138-6142. https://doi.org/10.1073/pnas.91.13.6138
  20. Lee, S. M., H. M. Kim, S. J. Moon, and M. J. Kang. 2012. Cloning and molecular characterization of porcine $\beta$-casein gene (CNS2). Asian Australas. J. Anim. Sci. 25:421-427. https://doi.org/10.5713/ajas.2011.11240
  21. Piedrahita, J. A. 2000. Targeted modification of the domestic animal genome. Theriogenology 53:105-116. https://doi.org/10.1016/S0093-691X(99)00244-7
  22. Li, L., W. Shen, Q. Y. Pan, L. J. Min, Y. J. Sun, Y. W. Fang, J. X. Deng, and Q. J. Pan. 2006. Nuclear transfer of goat somatic cells transgenic for human lactoferrin. Yi. Chuan. 28:1513-1519. https://doi.org/10.1360/yc-006-1513
  23. McCreath, K. J., J. Howcroft, K. H. Campbell, A. Colman, A. E. Schnieke, and A. J. Kind. 2000. Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature 405:1066-1069. https://doi.org/10.1038/35016604
  24. Palmiter, R. D., P. L. Brinster, R. E. Hammer, M. E. Trumbauer, M. G. Rosenfeld, N. C. Birnberg, and R. M. Evans. 1982. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 300:611-615. https://doi.org/10.1038/300611a0
  25. Robl, J. M., Z. Wang, P. Kasinathan, and Y Kuroiwa. 2007. Transgenic animal production and animal biotechnology. Theriogenology 67:127-133. https://doi.org/10.1016/j.theriogenology.2006.09.034
  26. Schnieke, A. E., A. J. Kind, W. A. Ritchie, K. Mycock, A. R. Scott, M. Ritchie, I. Wilmut, A. Colman, and K. H. Campbell. 1997. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 278:2130-2133. https://doi.org/10.1126/science.278.5346.2130
  27. Shen, W., Z. T. Yang, L. Y. Tian, X. J. Wu, H. Chen, P. T. Huang, and J. X. Deng. 2004. The ht-PAm cDNA knock-in the goat beta-casein gene locus. Sheng. Wu. Gong. Cheng. Xue. Bao. 20:361-365.
  28. Shen, W., L. J. Min, L. Li, Q. J. Pan, X. J. Wu, Y. R. Zhou, and J. X. Deng. 2005. High-efficient gene targeting of goat mammary epithelium cell by the multi-selection mechanism. Yi. Chuan. Xue. Bao. 32:366-371.
  29. Shen, W., G. Lan, X. Yang, L. Li, L. Min, Z. Yang, L. Tian, X. Wu, Y. Sun, H. Chen, J. Tan, J. Deng, and Q. Pan. 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