Asian-Australasian Journal of Animal Sciences

Asian Australasian Association of Animal Production Societies (아세아태평양축산학회)
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Construction of Mammalian Cell Expression Vector for pAcGFP-bFLIP(L) Fusion Protein and Its Expression in Follicular Granulosa Cells

  • Yang, Run Jun (Institute of Animal Sciences, Chinese Academy of Agricultural Sciences) ;
  • Li, Wu Feng (College of Life Science, Shanxi Agricultural University) ;
  • Li, Jun Ya (Institute of Animal Sciences, Chinese Academy of Agricultural Sciences) ;
  • Zhang, Lu Pei (Institute of Animal Sciences, Chinese Academy of Agricultural Sciences) ;
  • Gao, Xue (Institute of Animal Sciences, Chinese Academy of Agricultural Sciences) ;
  • Chen, Jin Bao (Institute of Animal Sciences, Chinese Academy of Agricultural Sciences) ;
  • Xu, Shang Zhong (Institute of Animal Sciences, Chinese Academy of Agricultural Sciences)
  • Received : 2009.03.21
  • Accepted : 2009.10.08
  • Published : 2010.03.01
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Abstract

FLICE inhibitory protein (FLIP) is one of the important anti-apoptotic proteins in the Fas/FasL apoptotic path which has death effect domains, mimicking the pro-domain of procaspase-8. To reveal the intracellular signal transduction molecules involved in the process of follicular development in the bovine ovary, we cloned the c-FLIP(L) gene in bovine ovary tissue with the reverse transcription polymerase chain reaction (RT-PCR), deleted the termination codon in its cDNA, and directionally cloned the amplified c-FLIP(L) gene into eukaryotic expression vector pAcGFP-Nl, including AcGFP, and successfully constructed the fusion protein recombinant plasmid. After identifying by restrictive enzyme BglII/EcoRI and sequencing, pAcGFP-bFLIP(L) was then transfected into follicular granulosa cells, mediated by Lipofectamine 2000, the expression of AcGFP observed and the transcription and expression of c-FLIP(L) detected by RT-PCR and Western blot. The results showed that the cattle c-FLIP(L) was successfully cloned; the pAcGFPbFLIP(L) fusion protein recombinant plasmid was successfuly constructed by introducing a BglII/EcoRI cloning site at the two ends of the c-FLIP(L) open reading frame and inserting a Kozak sequence before the start codon. AcGFP expression was detected as early as 24 h after transfection. The percentage of AcGFP positive cells reached about 65% after 24 h. A 1,483 bp transcription was amplified by RT-PCR, and a 83 kD target protein was detected by Western blot. Construction of the pAcGFP-bFLIP(L) recombinant plasmid should be helpful for further understanding the mechanism of regulation of c-FLIP(L) on bovine oocyte formation and development.

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References

  1. Carsten, S., S. Ingo, H. K. Peter and E. P. Marcus. 1999. The role of c-FLIP in modulation of CD95-induced apoptosis. J. Biol. Chem. 274:1541-1548 https://doi.org/10.1074/jbc.274.3.1541
  2. Ferguson, B. J., D. Esposito, J. Jovanović, A. Sankar, P. C. Driscoll and H. Mehmet. 2007. Biophysical and cell-based evidence for differential interactions between the death domains of CD95/Fas and FADD. J. Cell Death. Differ. 14:1717-1719 https://doi.org/10.1038/sj.cdd.4402191
  3. Giampietri, C., S. Petrungaro, F. G. Klinger, P. Coluccia, A. Paone, E. Vivarelli, A. Filippini, P. De Cesaris, M. De Felici and E. Ziparo. 2006. c-Flip expression and function in fetal mouse gonocytes. FASEB. J. 20:124-126
  4. Giampietri, C., S. Petrungaro, P. Coluccia, F. Antonangeli, A. Paone, F. Padula, P. De Cesaris, E. Ziparo and A. Filippini. 2006. c-Flip(L) is expressed in undifferentiated mouse male germ cells. FEBS. Lett. 580:6109-6114 https://doi.org/10.1016/j.febslet.2006.10.010
  5. Hengartner, M. 2000. The biochemistry of apoptosis. Nature 407:770-776 https://doi.org/10.1038/35037710
  6. Hideki, T., K. L. Chang, C. Y. Chan, O. Masayoshi, N. Masami, N. Yoshitake, O. Nobuo, K. Yoshinori, W. Rie, M. Rika, M. Kohji and M. Yoshiharu. 2003. In vitro and in vivo gene delivery by recombinant baculoviruses. J. Virol. 77:9799-9808 https://doi.org/10.1128/JVI.77.18.9799-9808.2003
  7. Jakobs, S., V. Subramaniam, A. Schönle, T. M. Jovin and S. W. Hell. 2000. EFGP and DsRed expressing cultures of Escherichia coli imaged by confocal, two-photon and fluorescence lifetime microscopy. FEBS. Lett. 479:131-135 https://doi.org/10.1016/S0014-5793(00)01896-2
  8. Magin, L. C., G. Kotzamanis, L. D'Aiuto, E. Wagner and C. Huxley. 2003. Retrofitting BACs with G418 resistance, luciferase, and oriP and EBNA-1- new vectors for in vitro and in vivo delivery. BMC. Biotechnol. 3:2-10 https://doi.org/10.1186/1472-6750-3-2
  9. Margalit, K. A., R. G. Cowan, R. M. Harman and S. M. Quirk. 2005. Apoptosis of bovine ovarian surface epithelial cells by Fas antigen/Fas ligand signaling. Reproduction 130:751-758 https://doi.org/10.1530/rep.1.00816
  10. Marilyn, K. 1980. Binding of wheat germ ribosomes to bisulfitemodified reovirus messenger RNA:Evidence for a scanning mechanism. J. Mol. Biol. 144:291-304 https://doi.org/10.1016/0022-2836(80)90092-3
  11. Moriyama, H. and S. Yonehara. 2007. Rapid up-regulation of c-FLIP expression by BCR signaling through the PI3K/Akt pathway inhibits simultaneously induced Fas-mediated apoptosis in murine Blymphocytes. Immunol. Lett. 109:36-46 https://doi.org/10.1016/j.imlet.2006.12.009
  12. Park, D., E. Shim, Y. Kim, Y. M. Kim, H. Lee, J. Choe, D. Kang, Y. S. Lee and D. Jeoung. 2008. C-FLIP promotes the motility of cancer cells by activating FAK and ERK, and increasing MMP-9 expression. Mol. Cells 25:184-195
  13. Skarzynski, D. J., M. Shibaya, Y. Tasaki, A. Korzekwa, S. Murakami, Woclawek, I. Potocka, M. Majewska and K. Okuda. 2007. Fas-mediated apoptosis is suppressed by calf serum in cultured bovine luteal cells. Reprod. Biol. 7:3-15
  14. Szperka, M. E., E. E. Connor, M. J. Paape, J. L.Williams and D. D. Bannerman. 2006. Sequencing, chromosomal mapping, and functional characterization of bovine FLICE-like inhibitory protein (FLIP). Cytogenet. Genome. Res. 112:90-97 https://doi.org/10.1159/000087518
  15. Zhang, S., H. M. Shen and C. N. Ong. 2005. Down-regulation of c-FLIP contributes to the sensitization effect of 3,3'-diindolylmethane on TRAIL-induced apoptosis in cancer cells. J. Mol, Cancer, Ther. 4:972-981
  16. Zhang, X., T. G. Jin, H. Yang, W. C. DeWolf, F. R. Khosravi and A. F. Olumi. 2004. Persistent c-FLIP(L) expression is necessary and sufficient to maintain resistance to tumor necrosis factorrelated apoptosis-inducing ligand-mediated apoptosis in prostate cancer. J. Cancer Res. 64:7086-7091 https://doi.org/10.1158/0008-5472.CAN-04-1498
  17. Zou, W., X. Liu, P. Yue, F. R. Khuri and S. Y. Sun. 2007. PPAR gamma ligands enhance TRAIL-induced apoptosis through DR5 upregulation and c-FLIP downregulation in human lung cancer cells. Cancer Biol. Ther. 6:99-106 https://doi.org/10.4161/cbt.6.1.3555