Skip to main content

Advertisement

SpringerLink
Log in

RNA Interference in Pigs: Comparison of RNAi Test Systems and Expression Vectors

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

We have examined the use of RNA interference as a means of downregulating gene expression and provide the first comparison of shRNA and artificial miRNA constructs for transgenic livestock. Several in vitro assays were performed to identify the most effective RNAi constructs. shRNA and miRNA constructs achieved significant downregulation of two porcine target genes: the milk whey protein beta-lactoglobulin and the tumour suppressor p53. Results of different assays were, however, sometimes at variance, indicating that no one assay can be relied upon to predict the effectiveness of an RNAi construct. Our findings are that screening of RNAi constructs is most informative if carried out in primary cells that express the target gene and are competent for somatic cell nuclear transfer. Importantly, the use of miRNA constructs makes tissue specific gene knockdown in large animals a realistic possibility.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. McCreath, K. J., Howcroft, J., Campbell, K. H. S., Colman, A., Schnieke, A. E., & Kind, A. J. (2000). Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature, 405, 1066–1069.

    Article  CAS  Google Scholar 

  2. Denning, C., Dickinson, P., Burl, S., Wylie, D., Fletcher, J., & Clark, A. J. (2001). Gene targeting in primary fetal fibroblasts from sheep and pig. Cloning and Stem Cells, 3, 221–231.

    Article  CAS  Google Scholar 

  3. Kuroiwa, Y., Kasinathan, P., Matsushita, H., Sathiyaselan, J., Sullivan, E. J., Kakitani, M., et al. (2004). Sequential targeting of the genes encoding immunoglobulin-mu and prion protein in cattle. Nature Genetics, 36, 775–780.

    Article  CAS  Google Scholar 

  4. Yu, G., Chen, J., Yu, H., Liu, S., Chen, J., Xu, X., et al. (2006). Functional disruption of the prion protein gene in cloned goats. Journal of General Virology, 87, 1019–1027.

    Article  CAS  Google Scholar 

  5. Dai, Y., Vaught, T. D., Boone, J., Chen, S. H., Phelps, C. J., Ball, S., et al. (2002). Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs. Nature Biotechnology, 20, 251–255.

    Article  CAS  Google Scholar 

  6. Lai, L., Kolber-Simonds, D., Park, K. W., Cheong, H. T., Greenstein, J. L., Im, G. S., et al. (2002). Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science, 295, 1089–1092.

    Article  CAS  Google Scholar 

  7. Rogers, C. S., Hao, Y., Rokhlina, T., Samuel, M., Stoltz, D. A., Li, Y., et al. (2008). Production of CFTR-null and CFTR-DeltaF508 heterozygous pigs by adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer. Journal of Clinical Investigation, 118, 1571–1577.

    Article  CAS  Google Scholar 

  8. Kunath, T., Gish, G., Lickert, H., Jones, N., Pawson, T., & Rossant, J. (2003). Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype. Nature Biotechnology, 21, 559–561.

    Article  CAS  Google Scholar 

  9. Dieckhoff, B., Petersen, B., Kues, W. A., Kurth, R., Niemann, H., & Denner, J. (2008). Knockdown of porcine endogenous retrovirus (PERV) expression by PERV-specific shRNA in transgenic pigs. Xenotransplantation, 15, 36–45.

    Article  Google Scholar 

  10. Ramsoondar, J., Vaught, T., Ball, S., Mendicino, M., Monahan, J., Jobst, P., et al. (2009). Production of transgenic pigs that express porcine endogenous retrovirus small interfering RNAs. Xenotransplantation, 16, 164–180.

    Article  Google Scholar 

  11. Wal, J. M. (1998). Cow’s milk allergens. Allergy, 53, 1013–1022.

    Article  CAS  Google Scholar 

  12. Kagan, R. S. (2003). Food allergy: An overview. Environmetal Health Perspectives, 111, 223–225.

    Article  Google Scholar 

  13. Thomson, A. J., Marques, M. M., & McWhir, J. (2003). Gene targeting in livestock. Reproduction Supplement, 61, 495–508.

    CAS  Google Scholar 

  14. Donehower, L. A., Harvey, M., Slagle, B. L., McArthur, M. J., Montgomery, C. A., Jr, Butel, J. S., et al. (1992). Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature, 356, 215–221.

    Article  CAS  Google Scholar 

  15. Jacks, T., Remington, L., Williams, B. O., Schmitt, E. M., Halachmi, S., Bronson, R. T., et al. (1994). Tumor spectrum analysis in p53-mutant mice. Current Biology, 4, 1–7.

    Article  CAS  Google Scholar 

  16. Hemann, M. T., Fridman, J. S., Zilfou, J. T., Hernando, E., Paddison, P. J., Cordon-Cardo, C., et al. (2003). An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo. Nature Genetics, 33, 396–400.

    Article  CAS  Google Scholar 

  17. Dickins, R. A., Hemann, M. T., Zilfou, J. T., Simpson, D. R., Ibarra, I., Hannon, G. J., et al. (2005). Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nature Genetics, 37, 1289–1295.

    CAS  Google Scholar 

  18. Swindle, M. M., & Smith, A. C. (2000). Information resources on swine in biomedical research. United States Department of Agriculture Animal Welfare Information Center Resource Series No 11

  19. Hasuwa, H., Kaseda, K., Einarsdottier, T., & Okabe, M. (2002). Small interfering RNA and gene silencing in transgenic mice and rats. FEBS Letters, 532, 227–230.

    Article  CAS  Google Scholar 

  20. Paddison, P. J., Caudy, A. A., Bernstein, E., Hannon, G. J., & Conklin, D. S. (2002). Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes and Development, 16, 948–958.

    Article  CAS  Google Scholar 

  21. Yu, J. Y., DeRuiter, S. L., & Turner, D. L. (2002). RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proceedings of the National Academy of Sciences USA, 99, 6047–6052.

    Article  CAS  Google Scholar 

  22. Miyagishi, M., & Taira, K. (2002). U6 promoter-driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nature Biotechnology, 20, 497–500.

    Article  CAS  Google Scholar 

  23. Paul, C. P., Good, P. D., Winer, I., & Engelke, D. R. (2002). Effective expression of small interfering RNA in human cells. Nature Biotechnology, 20, 505–508.

    Article  CAS  Google Scholar 

  24. Brummelkamp, T. R., Bernards, R., & Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science, 296, 550–553.

    Article  CAS  Google Scholar 

  25. Hitz, C., Wurst, W., & Kühn, R. (2007). Conditional brain-specific knockdown of MAPK using Cre/loxP regulated RNA interference. Nucleic Acids Research, 35, e90.

    Article  Google Scholar 

  26. Kotnik, K., Popova, E., Todiras, M., Mori, M. A., Alenina, N., Seibler, J., et al. (2009). Inducible transgenic rat model for diabetes mellitus based on shRNA-mediated gene knockdown. PLoS One, 4, e5124.

    Article  Google Scholar 

  27. Rao, M. K., Pham, J., Imam, J. S., MacLean, J. A., Murali, D., Furuta, Y., et al. (2006). Tissue-specific RNAi reveals that WT1 expression in nurse cells controls germ cell survival and spermatogenesis. Genes and Development, 20, 147–152.

    Article  CAS  Google Scholar 

  28. Kissler, S., Stern, P., Takahashi, K., Hunter, K., Peterson, L. B., & Wicker, L. S. (2006). In vivo RNA interference demonstrates a role for Nramp1 in modifying susceptibility to type 1 diabetes. Nature Genetics, 38, 479–483.

    Article  CAS  Google Scholar 

  29. Nagy, P., Arndt-Jovin, D. J., & Jovin, T. M. (2003). Small interfering RNAs suppress the expression of endogenous and GFP-fused epidermal growth factor receptor (erbB1) and induce apoptosis in erbB1-overexpressing cells. Experimental Cell Research, 285, 39–49.

    Article  CAS  Google Scholar 

  30. Dieckhoff, B., Karlas, A., Hofmann, A., Kues, W. A., Petersen, B., Pfeifer, A., et al. (2007). Inhibition of porcine endogenous retroviruses (PERVs) in primary porcine cells by RNA interference using lentiviral vectors. Archives of Virology, 152, 629–634.

    Article  CAS  Google Scholar 

  31. Polyak, K., Waldman, T., He, T. C., Kinzler, K. W., & Vogelstein, B. (1996). Genetic determinants of p53-induced apoptosis and growth arrest. Genes and Development, 10, 1945–1952.

    Article  CAS  Google Scholar 

  32. Hublarova, P., Greplova, K., Holcakova, J., Vojtesek, B., & Hrstka, R. (2010). Switching p53-dependent growth arrest to apoptosis via the inhibition of DNA damage-activated kinases. Cellular and Molecular Biology Letters, 15, 473–484.

    Article  CAS  Google Scholar 

  33. Kumar, R., Conklin, D. S., & Mittal, V. (2003). High-throughput selection of effective RNAi probes for gene silencing. Genome Research, 13, 2333–2340.

    Article  CAS  Google Scholar 

  34. Garraway, S. M., Xu, Q., & Inturrisi, C. E. (2007). Design and evaluation of small interfering RNAs that target expression of the N-methyl-d-aspartate receptor NR1 subunit gene in the spinal cord dorsal horn. Journal of Pharmacology and Experimental Therapeutics, 322, 982–988.

    Article  CAS  Google Scholar 

  35. Wang, S., Lv, X., Zhang, K., Lin, T., Liu, X., Yuan, J., et al. (2009). Knockdown of the prion gene expression by RNA interference in bovine fibroblast cells. Molecular Biology Reports, Online First October 11.

  36. Zhou, H., Xia, X. G., & Xu, Z. (2005). An RNA polymerase II construct synthesizes short-hairpin RNA with a quantitative indicator and mediates highly efficient RNAi. Nucleic Acids Research, 33, e62.

    Article  Google Scholar 

  37. Idogawa, M., Sasaki, Y., Suzuki, H., Mita, H., Imai, K., Shinomura, Y., et al. (2009). A single recombinant adenovirus expressing p53 and p21-targeting artificial microRNAs efficiently induces apoptosis in human cancer cells. Clinical Cancer Research, 15, 3725–3732.

    Article  CAS  Google Scholar 

  38. Wu, S. C. (2009). RNA interference technology to improve recombinant protein production in Chinese hamster ovary cells. Biotechnology Advances, 27, 417–422.

    Article  CAS  Google Scholar 

  39. Santoro, R., Lienemann, P., & Fussenegger, M. (2009). Epigenetic engineering of ribosomal RNA genes enhances protein production. PLoS One, 4, e6653.

    Article  Google Scholar 

  40. Bohula, E. A., Salisbury, A. J., Sohail, M., Playford, M. P., Riedemann, J., Southern, E. M., et al. (2003). The efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript. Journal of Biological Chemistry, 278, 15991–15997.

    Article  CAS  Google Scholar 

  41. Vickers, T. A., Koo, S., Bennett, C. F., Crooke, S. T., Dean, N. M., & Baker, B. F. (2003). Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis. Journal of Biological Chemistry, 278, 7108–7118.

    Article  CAS  Google Scholar 

  42. Kretschmer-Kazemi Far, R., & Sczakiel, G. (2003). The activity of siRNA in mammalian cells is related to structural target accessibility: A comparison with antisense oligonucleotides. Nucleic Acids Research, 31, 4417–4424.

    Article  CAS  Google Scholar 

  43. Overhoff, M., Alken, M., Far, R. K., Lemaitre, M., Lebleu, B., Sczakiel, G., et al. (2005). Local RNA target structure influences siRNA efficacy: A systematic global analysis. Journal of Molecular Biology, 348, 871–881.

    Article  CAS  Google Scholar 

  44. Sun, G., & Rossi, J. J. (2009). Problems associated with reporter assays in RNAi studies. RNA Biology, 6, 406–411.

    Article  CAS  Google Scholar 

  45. Grimm, D., Streetz, K. L., Jopling, C. L., Storm, T. A., Pandey, K., Davis, C. R., et al. (2006). Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature, 441, 537–541.

    Article  CAS  Google Scholar 

  46. Stewart, C. K., Li, J., & Golovan, S. P. (2008). Adverse effects induced by short hairpin RNA expression in porcine fetal fibroblasts. Biochemical and Biophysical Research Communications, 370, 113–117.

    Article  CAS  Google Scholar 

  47. Giering, J. C., Grimm, D., Storm, T. A., & Kay, M. A. (2008). Expression of shRNA from a tissue-specific pol II promoter is an effective and safe RNAi therapeutic. Molecular Therapy, 16, 1630–1636.

    Article  CAS  Google Scholar 

  48. McBride, J. L., Boudreau, R. L., Harper, S. Q., Staber, P. D., Monteys, A. M., Martins, I., et al. (2008). Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: Implications for the therapeutic development of RNAi. Proceedings of the National Academy of Sciences USA, 105, 5868–5873.

    Article  CAS  Google Scholar 

  49. Yamamoto, M., Maehara, Y., Oda, S., Ichiyoshi, Y., Kusumoto, T., & Sugimachi, K. (1999). The p53 tumor suppressor gene in anticancer agent-induced apoptosis and chemosensitivity of human gastrointestinal cancer cell lines. Cancer Chemotherapy and Pharmacology, 43, 43–49.

    Article  CAS  Google Scholar 

  50. Dunkern, T. R., Wedemeyer, I., Baumgärtner, M., Fritz, G., & Kaina, B. (2003). Resistance of p53 knockout cells to doxorubicin is related to reduced formation of DNA strand breaks rather than impaired apoptotic signaling. DNA Repair, 2, 49–60.

    Article  CAS  Google Scholar 

  51. Stern, P., Astrof, S., Erkeland, S. J., Schustak, J., Sharp, P. A., & Hynes, R. O. (2008). A system for Cre-regulated RNA interference in vivo. Proceedings of the National Academy of Sciences USA, 105, 13895–13900.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported financially by grant SCHN 971/2-2 from the Deutsche Forschungsgemeinschaft. The authors would like to Eckhard Wolf, Mayuko Kurome and Barbara Kessler for the nuclear transfer work, Peggy Müller and Margret Bahnweg for excellent technical assistance, and Davide Cavanna and Bernadette Antoni for plasmid construction.

Author information

Authors and Affiliations

  1. Chair of Livestock Biotechnology, TU Muenchen, Liesel-Beckmann-Str. 1, 85354, Freising, Germany

    Claudia Merkl, Simon Leuchs, Anja Saalfrank, Alexander Kind & Angelika Schnieke

Authors
  1. Claudia Merkl
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Simon Leuchs
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anja Saalfrank
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alexander Kind
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Angelika Schnieke
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Angelika Schnieke.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 75 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Merkl, C., Leuchs, S., Saalfrank, A. et al. RNA Interference in Pigs: Comparison of RNAi Test Systems and Expression Vectors. Mol Biotechnol 48, 38–48 (2011). https://doi.org/10.1007/s12033-010-9346-6

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-010-9346-6

Keywords

Search

Navigation