Determination of the Absolute Number of Transgene Copies in CMVFUT Transgenic Pigs

Daniel Lipiński, Joanna Zeyland 1 , Andrzej Pławski 2 ,  and Ryszard Słomski
  • 1 Department of Biochemistry and Biotechnology, University of Life Sciences, Wołyńska 35, 60-637 Poznań, Poland
  • 2 Institute of Human Genetics, Polish Academy of Sciences, Strzeszyńska 32, 60-479 Poznań, Poland

Determination of the Absolute Number of Transgene Copies in CMVFUT Transgenic Pigs

The aim of this research was to determine the number of transgene copies in the DNA of transgenic pigs. The copy number of the transgene was analysed in the transgenic animals with introduced pCMVFUT genetic construct containing a coding sequence of human H transferase under a control of CMV promoter. The copy number of the transgene that had integrated with the genome of the transgenic animals was analysed by qPCR with SYBR Green dye, which enabled nonspecific double-stranded DNA detection. CMVFT-2F and CMVFT-2R primers were used to amplify a 149 bp fragment of DNA. Forward primer had a sequence complementary to a promoter sequence and reverse primer to a coding sequence of H transferase. The copy number of the transgene in the examined samples was established by plotting the CT values obtained on a standard curve, which had been set by the usage of the CT values for the successive standard dilutions with known copy number (1.438-1.431 copies). As a standard we used pCMVFut genetic construct hydrolyzed with Not I restriction enzyme to a linear form. The real-time PCR results helped to establish the range of 3 - 4 as the number of the transgene copies that had integrated to the swine genome.

If the inline PDF is not rendering correctly, you can download the PDF file here.

  • Ballester M., Castelló A., Ibáñez E., Sánchez A., Folch J. M. (2004). Real-time quantitative PCR-based system for determining transgene copy number in transgenic animals. Biotechniques, 37: 610-613.

  • Costa C., Zhao L., Burton W. V., Bondioli K. R., Williams B. L., Hoagland T. A., Ditullio P. A., Ebert K. M., Fodor W. L. (1999). Expression of the human alpha 1,2-fucosyltransferase in transgenic pigs modifies the cell surface carbohydrate phenotype and confers resistance to human serum-mediated cytolysis. FASEB J., 13: 1762-1773.

  • Folger K. R., Thomas K., Capecchi M. R. (1985). Nonreciprocal exchanges of information between DNA duplexes coinjected into mammalian cell nuclei. Mol. Cell. Biol., 5: 59-69.

  • Folger K. R., Wong E. A., Wahl G., Capecchi M. R. (1982). Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules. Mol. Cell. Biol., 2: 372-1387.

  • Galili U., Shohet S. B., Kobrin E., Stults C. L., Macher B. A. (1988). Man, apes, and Old World monkeys differ from other mammals in the expression of alpha-galactosyl epitopes on nucleated cells. J. Biol. Chem., 263: 17755-17762.

  • Galili U., Swanson K. (1991). Gene sequences suggest inactivation of alpha-1,3-galactosyltransferase in catarrhines after the divergence of apes from monkeys. Proc. Nat. Acad. Sci. U. S. A., 88: 7401-7404.

  • Ingham D. J., Beer S., Money S., Hansen G. (2001). Quantitative real-time PCR assay for determining transgene copy number in transformed plants. Biotechniques, 31: 132-140.

  • Jura J., Słomski R., Smorąg Z., Gajda B., Wieczorek J., Lipiński D., Kalak R., Juzwa W., Zeyland J. (2004). Production of transgenic pigs suitable for xenotransplantation with the use of standard DNA microinjection. Ann. Anim. Sci., 4, 2: 321-327.

  • Keshet I., Lieman-Hurwitz J., Cedar H. (1986). DNA methylation affects the formation of active chromatin. Cell, 44: 535-543.

  • Lipiński D., Jura J., Zeyland J., Juzwa W., Mały E., Kalak R., Bochenek M., Pławski A., Szalata M., Smorąg Z., Słomski R. (2010). Production of transgenic pigs expressing human α1,2-fucosyltransferase to avoid humoral xenograft rejection. Med. Wet., 66: 316-322.

  • Milot E., Strouboulis J., Trimborn T., Wijgerde M., de Boer E., Langeveld A., Tan-Un K., Vergeer W., Yannoutsos N., Grosveld F., Fraser P. (1996). Heterochromatin effects in the frequency and duration of LCR-mediated gene transcription. Cell, 87: 105-114.

  • Muller K., Heller H., Doerfler W. (2001). Foreign DNA integration. J. Biol. Chem., 276: 14271-14278.

  • Sandrin M. S., McKenzie I. F. (1994). Gal alpha (1,3)Gal, the major xenoantigen(s) recognised in pigs by human natural antibodies. Immunol. Rev., 141: 169-190.

  • Sharma A., Okabe J., Birch P., McClellan S. B., Martin M. J., Platt J. L., Logan J. S. (1996). Reduction in the level of Gal(alpha1,3)Gal in transgenic mice and pigs by the expression of an alpha(1,2)fucosyltransferase. Proc. Nat. Acad. Sci. U. S. A., 93: 7190-7195.

  • Shitara H., Sato A., Hayashi J., Mizushima N., Yonekawa H., Taya C. (2004). Simple method of zygosity identification in transgenic mice by real-time quantitative PCR. Transgenic Res., 13: 191-194.

  • Tesson L., Heslan J. M., Ménoret S., Anegon I. (2002). Rapid and accurate determination of zygosity in transgenic animals by real-time quantitative PCR. Transgenic Res., 11: 43-48.


Journal + Issues