Interference of EGFP RNA in Human NT-2/D1 Cell Lines Using Human U6 Promoter-based siRNA PCR Products

  • Kwak, Young-Don (Burnett College of Biomedical Sciences, Biomolecular Science Center, University of Central Florida) ;
  • Sugaya, Kiminobu (Burnett College of Biomedical Sciences, Biomolecular Science Center, University of Central Florida)
  • Published : 2006.06.30

Abstract

RNA interference (RNAi), a process of sequence-specific gene suppression, has been known as a natural gene regulatory mechanism in a wide range of lower organisms. Recently, we have reported that a transfection of human U6 promoter (hU6) driven hairpin small-interference RNA (siRNA) plasmid specifically knocks down the target gene by post-transcriptional gene silencing in mammalian cells. Here we report that transfection of polymerase chain reaction (PCR) products, containing human U6 promoter with hairpin siRNA, knocks down the target gene expression in human teratocarcinoma NT-2/D1 cells. Moreover, we showed 3' end termination sequence, 5 Ts, is not critical elements for knocking down in PCR-based siRNA system. Therefore, the PCR-based siRNA system is a promising tool not only for the screening but also to temporally regulate gene expression in the human progenitor cells.

References

  1. Akashi, H., M. Miyagishi, and K. Taira (2001) Suppression of gene expression by RNA interference in cultured plant cells. Antisense Nucleic Acid Drug Dev. 11: 359-367 https://doi.org/10.1089/108729001753411326
  2. Kamath, R. S., M. Martinez-Campos, P. Zipperlen, A. G. Fraser, and J. Ahringer (2001) Effectiveness of specific RNA-mediated interference through ingested doublestranded RNA in Caenorhabditis elegans. Genome Biol. 2: RESEARCH0002
  3. Kennerdell, J. R. and R. W. Carthew (2000) Heritable gene silencing in Drosophila using double-stranded RNA. Nat. Biotechnol. 18: 896-898 https://doi.org/10.1038/78531
  4. Sharp, P. A. (2001) RNA interference-2001. Genes Dev. 15: 485-490 https://doi.org/10.1101/gad.880001
  5. Hannon, G. J. (2002) RNA interference. Nature 418: 244-251 https://doi.org/10.1038/418244a
  6. Zamore, P. D., T. Tuschl, P. A. Sharp, and D. P. Bartel (2000) RNAi: double-stranded RNA directs the ATPdependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101: 25-33 https://doi.org/10.1016/S0092-8674(00)80620-0
  7. Bernstein, E., A. A. Caudy, S. M. Hammond, and G. J. Hannon (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409: 363-366 https://doi.org/10.1038/35053110
  8. Hammond, S. M., S. Boettcher, A. A. Caudy, R. Kobayashi, and G. J. Hannon (2001) Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293: 1146-1150 https://doi.org/10.1126/science.1064023
  9. Stark, G. R., I. M. Kerr, B. R. Williams, R. H. Silverman, and R. D. Schreiber (1998) How cells respond to interferons. Annu. Rev. Biochem. 67: 227-264 https://doi.org/10.1146/annurev.biochem.67.1.227
  10. Caplen, N. J., S. Parrish, F. Imani, A. Fire, and R. A. Morgan (2001) Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA 98: 9742-9747
  11. Elbashir, S. M., J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, and T. Tuschl (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494-498 https://doi.org/10.1038/35078107
  12. Donze, O. and D. Picard (2002) RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Nucleic Acids Res. 30: e46 https://doi.org/10.1093/nar/30.10.e46
  13. Kwak, Y. D., H. Koike, and K. Sugaya (2003) RNA interference with small hairpin RNAs transcribed from a human U6 promoter-driven DNA vector. J. Pharmacol. Sci. 93: 214-217 https://doi.org/10.1254/jphs.93.214
  14. Brummelkamp, T. R., R. Bernards, and R. Agami (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296: 550-553 https://doi.org/10.1126/science.1068999
  15. Paule, M. R. and R. J. White (2000) Survey and summary: transcription by RNA polymerases I and III. Nucleic Acids Res. 28: 1283-1298 https://doi.org/10.1093/nar/28.6.1283
  16. Barton, G. M. and R. Medzhitov (2002) Retroviral delivery of small interfering RNA into primary cells. Proc. Natl. Acad. Sci. USA 99: 14943-14945
  17. Wiznerowicz, M. and D. Trono (2003) Conditional suppression of cellular genes: lentiviral vector-mediated druginducible RNA interference. J. Virol. 77: 8957-8961 https://doi.org/10.1128/JVI.77.16.8957-8951.2003
  18. Lee, V. M. and P. W. Andrews (1986) Differentiation of NTERA 2 clonal human embryonal caicinoma cells into neurons involves the induction of all three neurofilament proteins. J. Neurosci. 6: 514-521
  19. Guillemain, I., S. Gaboyard, G. Fontes, M. Saunier, A. Privat, and G. Patey (2000) Differential expression of Bcl- 2-related proteins in differentiating NT2 cells. Neuroreport 11: 1421-1425 https://doi.org/10.1097/00001756-200005150-00014
  20. Ferreira, S., M. J. Dupire, A. Delacourte, J. Najib, and M. L. Caillet-Boudin (2000) Synthesis and regulation od apolipoprotein E during the differentiation of human neuronal precursor NT2/D1 cells into postmitotic neurons. Exp. Neurol. 116: 415-421
  21. Younkin, D. P., C. M. Tang, M. Hardy, U. R. Reddy, Q. Y. Shi, S. J. Pleasure, V. M. Lee, and D. Pleasure (1993) Inducible expression of neuronal glutamate receptor channels in the NT2 human cell line. Proc. Natl. Acad. Sci. USA 90: 2174-2178
  22. Castanotto, D., H. Li, and J. J. Rossi (2002) Functional siRNA expression from transfected PCR products. RNA 8:1454-1460 https://doi.org/10.1017/S1355838202021362
  23. Scherer, L. J., Y. Yildiz, J. Kim, L. Cagnon, B. Heale, and J. J. Rossi (2004) Rapid assessment of anti-HIV siRNA efficacy using PCR-derived Pol III shRNA cassettes. Mol. Ther. 10: 597-603 https://doi.org/10.1016/j.ymthe.2004.05.003