Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

In vivo RNA interference demonstrates a role for Nramp1 in modifying susceptibility to type 1 diabetes

Nature Genetics volume 38, pages 479–483 (2006)Cite this article

Abstract

Type 1 diabetes is an autoimmune disease influenced by multiple genetic loci. Although more than 20 insulin-dependent diabetes (Idd) loci have been implicated in the nonobese diabetic (NOD) mouse model1,2, few causal gene variants have been identified3,4. Here we show that RNA interference5,6 (RNAi) can be used to probe candidate genes in this disease model. Slc11a1 encodes a phagosomal ion transporter, Nramp1, that affects resistance to intracellular pathogens and influences antigen presentation7,8,9. This gene is the strongest candidate among the 42 genes in the Idd5.2 region; a naturally occurring mutation in the protective Idd5.2 haplotype results in loss of function of the Nramp1 protein10. Using lentiviral transgenesis11, we generated NOD mice in which Slc11a1 is silenced by RNAi. Silencing reduced the frequency of type 1 diabetes, mimicking the protective Idd5.2 region. Our results demonstrate a role for Slc11a1 in modifying susceptibility to type 1 diabetes and illustrate that RNAi can be used to study causal genes in a mammalian model organism.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Dose effect of the protective B10-derived Idd5.2 region.
Figure 2: Design of a lentiviral vector for Nramp1 knockdown.
Figure 3: Generation of Nramp1-knockdown Idd5.1 congenic NOD mice.
Figure 4: Nramp1 silencing affects S. enterica infection and diabetes frequency.

Similar content being viewed by others

References

  1. Makino, S. et al. Breeding of a non-obese, diabetic strain of mice. Jikken Dobutsu 29, 1–13 (1980).

    CAS  PubMed  Google Scholar 

  2. Todd, J.A. & Wicker, L.S. Genetic protection from the inflammatory disease type 1 diabetes in humans and animal models. Immunity 15, 387–395 (2001).

    Article  CAS  Google Scholar 

  3. Ueda, H. et al. Association of the T-cell regulatory gene CTLA-4 with susceptibility to autoimmune disease. Nature 423, 506–511 (2003).

    Article  CAS  Google Scholar 

  4. Vijayakrishnan, L. et al. An autoimmune disease-associated CTLA-4 splice variant lacking the B7 binding domain signals negatively in T cells. Immunity 20, 563–575 (2004).

    Article  CAS  Google Scholar 

  5. Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998).

    Article  CAS  Google Scholar 

  6. Elbashir, S.M. et al. Duplexes of 21-nucleotides RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

    Article  CAS  Google Scholar 

  7. Vidal, S.M., Malo, D., Vogan, K., Skamene, E. & Gros, P. Natural resistance to infection with intracellular parasites: isolation of a candidate for Bcg. Cell 73, 469–485 (1993).

    Article  CAS  Google Scholar 

  8. Vidal, S. et al. The Ity/Lsh/Bcg locus: natural resistance to infection with intracellular pathogens is abrogated by disruption of the Nramp1 gene. J. Exp. Med. 182, 655–666 (1995).

    Article  CAS  Google Scholar 

  9. Wojciechowski, W., DeSanctis, J., Skamene, E. & Radzioch, D. Attenuation of MHC class II expression in macrophages infected with Mycobacterium bovis bacillus Calmette-Guerin involves class II transactivator and depends on the Nramp1 gene. J. Immunol. 163, 2688–2696 (1999).

    CAS  PubMed  Google Scholar 

  10. Wicker, L.S. et al. Fine mapping, gene content, comparative sequencing, and expression analyses support Ctla-4 and Nramp-1 as candidates for Idd5.1 and Idd5.2 in the nonobese diabetic mouse. J. Immunol. 173, 164–173 (2004).

    Article  CAS  Google Scholar 

  11. Lois, C., Hong, E.J., Pease, S., Brown, E.J. & Baltimore, D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295, 868–872 (2002).

    Article  CAS  Google Scholar 

  12. Kanagawa, O., Xu, G., Tevaarwerk, A. & Vaupel, B.A. Protection of nonobese diabetic mice from diabetes by gene(s) closely linked to IFN-γ receptor loci. J. Immunol. 164, 3919–3923 (2000).

    Article  CAS  Google Scholar 

  13. Rubinson, D.A. et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat. Genet. 33, 401–406 (2003).

    Article  CAS  Google Scholar 

  14. Tiscornia, G., Singer, O., Ikawa, M. & Verma, I.M. A general method for gene knock-down in mice by using lentiviral vectors expressing small interfering RNA. Proc. Natl. Acad. Sci. USA 100, 1844–1848 (2003).

    Article  CAS  Google Scholar 

  15. Lu, W., Yamamoto, V., Ortega, B. & Baltimore, D. Mammalian Ryk is a Wnt coreceptor required for stimulation of neurite outgrowth. Cell 119, 97–108 (2004).

    Article  CAS  Google Scholar 

  16. Kwaks, T.H. et al. Identification of anti-repressor elements that confer high and stable protein production in mammalian cells. Nat. Biotechnol. 21, 553–558 (2003).

    Article  CAS  Google Scholar 

  17. Klehr, D., Maass, K. & Bode, J. Scaffold-attached regions from the human interferon beta domain can be used to enhance the stable expression of genes under the control of various promoters. Biochemistry 30, 1264–1270 (1991).

    Article  CAS  Google Scholar 

  18. Schwarz, D.S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003).

    Article  CAS  Google Scholar 

  19. Khvorova, A., Reynolds, A. & Jayasena, S.D. Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216 (2003).

    Article  CAS  Google Scholar 

  20. Reynolds, A. et al. Rational siRNA design for RNA interference. Nat. Biotechnol. 22, 326–330 (2004).

    Article  CAS  Google Scholar 

  21. Nishino, M. et al. Functional polymorphism in Z-DNA-forming motif of promoter of SLC11A1 gene and type 1 diabetes in Japanese subjects: Association study and meta-analysis. Metabolism 54, 628–633 (2005).

    Article  CAS  Google Scholar 

  22. Takahashi, K. et al. Promoter polymorphism of SLC11A1 (formerly NRAMP1) confers susceptibility to autoimmune type 1 diabetes mellitus in Japanese. Tissue Antigens 63, 231–236 (2004).

    Article  CAS  Google Scholar 

  23. Sanjeevi, C.B. et al. Polymorphism at NRAMP1 and D2S1471 loci associated with juvenile rheumatoid arthritis. Arthritis Rheum. 43, 1397–1404 (2000).

    Article  CAS  Google Scholar 

  24. Shaw, M.A. et al. Linkage of rheumatoid arthritis to the candidate gene NRAMP1 on 2q35. J. Med. Genet. 33, 672–677 (1996).

    Article  CAS  Google Scholar 

  25. Steinman, L. & Zamvil, S.S. Virtues and pitfalls of EAE for the development of therapies for multiple sclerosis. Trends Immunol. 26, 565–571 (2005).

    Article  CAS  Google Scholar 

  26. Qiu, S., Adema, C.M. & Lane, T. A computational study of off-target effects of RNA interference. Nucleic Acids Res. 33, 1834–1847 (2005).

    Article  CAS  Google Scholar 

  27. Jackson, A.L. & Linsley, P.S. Noise amidst the silence: off-target effects of siRNAs? Trends Genet 20, 521–524 (2004).

    Article  CAS  Google Scholar 

  28. Searle, S. & Blackwell, J.M. Evidence for a functional repeat polymorphism in the promoter of the human NRAMP1 gene that correlates with autoimmune versus infectious disease susceptibility. J. Med. Genet. 36, 295–299 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Hill, N.J. et al. NOD Idd5 locus controls insulitis and diabetes and overlaps the orthologous CTLA-4/IDDM12 and NRAMP1 loci in humans. Diabetes 49, 1744–1747 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank J. Blackwell for the gift of Slc11a1 cDNA; J. Bode for the gift of the SAR DNA; J. Todd for advice and support; P. Sandy, C. Dillon, B. Haines and A. Ventura for technical discussions and A. Ayerdi, M. Serrano and T. Chavarria for technical assistance. This work was supported in part by the Cambridge-MIT Institute. L.S.W. and K.H. are supported by grants from the Juvenile Diabetes Research Foundation (JDRF) and the Wellcome Trust. The availability of NOD congenic mice through the Taconic Farms Emerging Models Program has been supported by grants from the Merck Genome Research Institute, the US National Institute of Allergy and Infectious Disease and the JDRF.

Author information

Author notes

  1. Stephan Kissler and Patrick Stern: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA

    Stephan Kissler & Patrick Stern

  2. Laboratory of Developmental Immunology, Massachusetts General Hospital for Children, Harvard Medical School, Boston, 02114, Massachusetts, USA

    Kazue Takahashi

  3. Department of Medical Genetics, Diabetes and Inflammation Laboratory, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, CB2 2XY, UK

    Kara Hunter & Linda S Wicker

  4. Department of Pharmacology, Merck Research Laboratories, Rahway, 07065, New Jersey, USA

    Laurence B Peterson

Authors
  1. Stephan Kissler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Patrick Stern
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kazue Takahashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kara Hunter
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Laurence B Peterson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Linda S Wicker
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Stephan Kissler.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Dose effect of protective alleles at Idd5.2 in the absence of protective alleles at Idd5.1. (PDF 340 kb)

Supplementary Fig. 2

Variegated expression in pLL3.7 transgenic mice. (PDF 241 kb)

Supplementary Fig. 3

Lentiviral construct expression in pLB-915 founder. (PDF 300 kb)

Supplementary Fig. 4

Reduced expression caused by interference between lentiviral constructs. (PDF 346 kb)

Supplementary Fig. 5

Nramp1 silencing protects from experimental autoimmune encephalomyelitis (EAE). (PDF 356 kb)

Supplementary Table 1

Primer sequences used for cloning and target sequences used for shRNA design. (PDF 10 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kissler, S., Stern, P., Takahashi, K. et al. In vivo RNA interference demonstrates a role for Nramp1 in modifying susceptibility to type 1 diabetes. Nat Genet 38, 479–483 (2006). https://doi.org/10.1038/ng1766

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng1766

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing