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Expression of Human Serum Albumin in Milk of Transgenic Mice Using Goat β-casein/Human Serum Albumin Fusion Gene

  • Wu, H.T. (National Laboratory Animal Center, National Applied Research Laboratories) ;
  • Chou, C.K. (National Laboratory Animal Center, National Applied Research Laboratories) ;
  • Huang, M.C. (Department of Animal Science, National Chung Hsing University)
  • Received : 2003.08.13
  • Accepted : 2004.01.26
  • Published : 2004.06.01

Abstract

The gene encoding human serum albumin (HSA) was cloned from human liver cDNA library by PCR. The HSA cDNA in size of 2,176 bp, including 1,830 bp of open reading frame, was cloned into the plasmid carried with the 5'flanking sequence of goat $\beta$-casein gene (-4,044 to +2,025 bp) to get a tissue specific expression vector in mammary gland named pGB562/HSA (12.5 kb). A 9.6 kb DNA fragment in which the sequence is in order of goat $\beta$-casein gene regulatory sequence, HSA cDNA and SV40 polyadenylation signals was isolated from the pGB562/HSA by SacI and DraIII cutting, and used to microinject into the pronuclei of mouse fertilized eggs to produce transgenic mice. Three transgenic mice (2 female and 1 male) were identified by PCR and dot Southern blot analysis. The copy numbers of integrated transgene were more than 10 copies in line #21 and #26 as well as over 50 copies in line #31 of transgenic mice. HSA protein collected from the milk of lactating transgenic mice was confirmed by immuno-detection of Western and slot blot. The concentrations of HSA in the milk were from 0.05 to 0.4 mg/ml. An obvious antigen and antibody conjugate could be observed in immunohistochemical stain of mammary gland tissue from lactating day 11 of HSA transgenic mice. The transmission of transgene and its expression was recognized according to the results of RT-PCR and sequences analyses of their progeny.

Keywords

References

  1. Bischoff, R., E. Degryse, F. Perraud, W. Dalemans, D. Ali-Hadji, D. Thepot, E. Devinoy, L. M. Houdebine and A. Pavirani. 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. 305:265-268.
  2. Bleck, G. T. and R. D. Bremel. 1994. Variation in expression of a bovine alpha-lactalbumin transgene in milk of transgenic mice. J. Dairy Sci. 77:1897-1904.
  3. Cerdan, M. G., J. I. Young, E. Zino, T. L. Falzone, V. Otero, H. N. Torres and M. Rubinstein. 1998. 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.
  4. Ebert, K. M., P. DiTullio, C. A. Barry, J. E. Schindler, S. L. Ayres, T. E. Smith, L. J. Pellerin, H. M. Meade, J. Denman and B. Roberts. 1994. Induction of human tissue plasminogen activator in the mammary gland of transgenic goats. Bio/Technology 12:699-702.
  5. Faerman, A., I. Barash, R. Puzis, M. Nathan, D. Hurwitz and M. Shani. 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. 43:461-470.
  6. Gutierrez, A., H. M. Meade, P. DiTullio, D. Pollock, M. Harvey, R. Jimenez-Flores, G. B. Anderson, J. D. Murray and J. F. Medrano. 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. 5:271-279.
  7. Huang, M. C. 1988. Studies on microinjection of foreign gene into mammalian egg. 1. Evaluation of the efficiency of microinjection into mouse fertilized eggs. J. Agric. Forest 37:97-110.
  8. Huang, M. C. and C. S. Lin. 1996. Cloning and restriction mapping of goat $\beta$-casein gene. J. Agric. Forest. 45:83-94.
  9. Hogan, B. L. M., F. Constantini and E. Lacy. 1994. Manipulating the Mouse Embryo: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  10. Kang, Y. K., C. S. Lee, A. S. Chung and K. K. Lee. 1998. Prolactin inducible enhancer activity of the first intron of the bovine $\beta$-casein gene. Mol. Cell 8:259-265.
  11. Kim, S. J., Y. Y. Cho, K. W. Lee, D. Y. Yu, C. S. Lee, Y. M. Han and K. K. Lee. 1994. Expression of human lactoferrin in milk of transgenic mice by using bovine $\beta$-casein/human lactoferrin cDNA fusion gene. Mol. Cell 4:355–360.
  12. Liang, C. T., H. C. Wang and C. H. Liu. 2000. A modified immunohistochemical staining method employed in formalin or alcohol-fixed, paraffin-embedded porcine tissue. J. Chin. Soc. Vet. Sci. 26:213-222.
  13. Lin, C. S., Y. C. Wu, Y. L. Sun and M. C. Huang. 2002. Postnatal Expression of Growth/Differentiation Factor-8 (GDF-8) Gene in European and Asian Pigs. Asian-Aust. J. Anim. Sci. 15:1-6.
  14. Maga, E. A., G. B. Anderson and J. D. Murray. 1995. The effect of mammary gland expression of human lysozyme on the properties of milk from transgenic mice. J. Dairy Sci. 78:2645-2652.
  15. Nakao, K., N. Nakagata and M. Katsuki. 1997. Simple and efficient vitrification procedure for cryopreservation of mouse embryos. Exp. Anim. 46:231-234.
  16. National Research Council (NRC). 1996: Guide for the Care and Use of Laboratory Animals. Institute of Laboratory Animal Resources, Commission on Life Sciences. National Academy Press, Washington, DC.
  17. Peter, Jr. T. 1996. The albumin molecule: Its structure and chemical properties. In: All about albumin. Academic Press, San Diego. pp. 9-75.
  18. Petitclerc, D., J. Attal, M. C. Théron, M. Bearzotti, P. Bolifraud, G. Kann, M. G. Stinnakre, H. Pointu, C. Puissant and L. M. Houdebine. 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. 40:169-178.
  19. Provot, C., M. A. Persuy and J. C. Mercier. 1995. Complete sequence of the ovine $\beta$-casein-encoding gene and interspecies comparison. Gene 154:259-263.
  20. Rijnkels, M., P. M. Kooiman, P. J. A. Krimpenfort, H. A. De Boer and F. R. Pieper. 1995. Expression analysis of the individual bovine $\beta$-, $\alpha$s2- and $\kappa$-casein genes in transgenic mice. Biochem. J. 311:929-937.
  21. Roberts, B., P. DiTullio, J. Vitale, K. Hehir and K. Gordon. 1992. Cloning of the goat $\beta$-casein-encoding gene and expression in transgenic mice. Gene 121:255-262.
  22. Shani, M., I. Barash, M. Nathan, G. Ricca, G. H. Searfoss, I. Dekel, A. Faerman, D. Givol and D. R. Hurwitz. 1992. Expression of human serum albumin in the milk of transgenic mice. Trans. Res. 1:195-208.
  23. Wall, R. J., D. E. Kerr, K. R. Bondioli. 1997. Transgenic dairy cattle: genetic engineering on a large scale. J. Dairy Sci. 80:2213-2224.
  24. Wu, H. T., C. S. Lin and M. C. Huang. 2003. 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 goat $\beta$-casein gene. Reprod. Fert. Dev. 15:231-239.
  25. Wu, H. T., C. K. Chou, C. S. Lin and M. C. Huang. 2003. Effects of glucose concentration on in vitro fertilization in BALB/c Mice. Reprod. Dom. Anim. 38:470-474.