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Genetically Modified NOD Mouse Core Facility (HMS)

Location: Harvard Medical School, Pathology, NRB 1052G, 77 Avenue Louis Pasteur, Boston, MA 02115

Summary:

The Genetically Modified NOD Mouse Core provides Center investigators, as well as researchers elsewhere, with access to transgenic and mutant lines derived from the NOD mouse model: some will be generated within the Core; others are established lines of proven experimental value that are maintained in the Core.

The Core will construct transgenic mice in strains that have a high susceptibility to diabetes (in particular in the NOD line). This includes trangenesis by conventional pronuclear injection or by delivery of RNAi cassettes on lentiviral vectors.

The Core will also provide a panel of existing transgenic and mutant lines. These lines are chosen because of their established interest in allowing the dissection of immunological tolerance in Type 1 Diabetes, and in response to Center investigator needs.

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Organisms and Viruses

  • B6-H2g7 ( Mus musculus )

    Development: B6.H2g7 is a congenic line developed by H Kikutani. This strain carries the NOD-derived MHC class II g7, haplotype, Iocated on Chromosome 17, but has all other genes from the B6 strain. The line is currently at the >20th generation.

    B6-H2g7 mice are viable, fertile, and present with no particular phenotype. They show no insulitis or diabetes (Luhder F et al, Mathis D, 2000). When crossed with BDC2.5 transgenic line, BDC2.5/B6g7 mice develop typical diabetes in most features, but, in contrast to the disease in BDC2.5/N or regular NOD animals, there is a quick transition from initial islet infiltration to massive destruction of the insulin-producing beta cells (Gonzalez A et al, Mathis D, 1997).

  • BDC2.5/B6H2g7 ( Mus musculus )

    Development: The BDC2.5 TCR alpha and TCR beta sequences were co-injected into (B6xSJL)F2 eggs. The integration site for these BDC2.5Tcr transgenes was localized to chromosome 13 near the D13mit125 marker (Katz et al, Mathis, 1993). Transgenes have been transferred on B6.H2g7 background, a congenic line developed by H Kikutani. This strain carries the NOD-derived MHC class II g7 haplotype, Iocated on Chromosome 17, but has all other genes from the B6 strain (Gonzalez A et al, Mathis D, 1997). The BDC2.5/B6g7 line is currently at the >20th backcross generation.

    Mice carrying the BCD2.5 transgenes show a generalized and very extensive islet infiltration after a few weeks of age, and a rapid onset diabetes that affects 40-70% of mice by 8 weeks of age (Gonzalez A et al, Mathis D, 1997; Poirot L. et al, Mathis D., 2004).

  • BDC2.5/N ( Mus musculus )

    Development: BDC2.5/N mice carry both rearranged TCR alpha and beta genes from the diabetogenic H2-Ag7 restricted BDC2.5 CD4+ T cell clone BDC-2.5. The BDC2.5 TCRa and TCRb sequences were co-injected into (B6xSJL)F2 eggs. To achieve the natural expression of Tcra, the rearranged V alpha J alpha sequence from BDC2.5 was cloned into the cassette vector generated by Kouskoff et al (1996), which contains both the 5' upstream promoter and the 3' downstream enhancer regions of the Tcra gene. The integration site for these BDC2.5Tcr transgenes was localized to chromosome 13 near the D13mit125 marker (Katz et al , Mathis D. 1993). The line is currently at the >99 backcross generation to NOD/LtJ.

    On the NOD background, mice carrying the transgenes have a reduced incidence of diabetes relative to NOD/LtJ controls (12% incidence at age 30 weeks). BDC2.5/N animals show a generalized and very extensive islet infiltration after a few weeks of age, but most remain free of overt diabetes for long periods or develop forms of insulitis that does not cause beta cells destruction. When coupled with the homozygous Rag1 knock Out, mice develop diabetes extremely early (mean age of 25 days). (Katz et al Mathis D. 1993, Gonzalez et al, Benoist C., 2001, Mombaerts et al, Papaioannou VE., 1992).

  • BDC2.5/N Thy1.1 ( Mus musculus )

    Development: The BDC2.5 TCR alpha and beta sequences were co-injected into (B6xSJL)F2 eggs. The integration site for these BDC2.5 TCR transgenes was localized to chromosome 13 near the D13mit125 marker (Katz et al , Mathis D. 1993). BDC2.5/N Thy1.1 mice are created by crossing BDC2.5/N with NOD.Thy1a (NOD.NON-Thy1a/1LtJ). Thy1.1 is an allogenic marker from PL/J back-crossed onto NOD. (Fabien N et al, Thivolet C. 1995). The BDC2.5/N Thy1.1 line is currently at the N22 F6 backcross generation.

    On the NOD background, mice carrying the transgenes have a reduced incidence of diabetes relative to NOD/LtJ controls (12% incidence at age 30 weeks). BDC2.5/N animals show a generalized and very extensive islet infiltration after a few weeks of age, but most remain free of overt diabetes for long periods or develop forms of insulitis that does not cause beta cells destruction. The BDC2.5/N Thy1.1 congenic strain carries the T lymphocyte specific Thy1a (Thy1.1) allele. Donor T cells can be easily distinguished from recipient T cells by both flow cytometric and histological analysis.

  • beta2M0/N ( Mus musculus )

    Development: The beta2Mo mutant strain was generated by a targeted disruption of the beta2M gene into129-derived E14TG2a ES cell line (Koller BH et al, 1990). The NOD/LtJ strain was produced by backcrossing the mutation 10 times to NOD/LtJ inbred mice (Christianson et al, 1996). The line is currently at the >12th backcross generation on NOD/LtJ.

    The elimination of cell surface MHC class I expression blocks both insulitis and autoimmune diabetes in NOD/Lt mice (Katz J et al, 1993; Wicker et al, 1994; Wang et al, 1996).

  • CD40[0]/N ( Mus musculus )

    Development: The CD40 gene was disrupted by replacing exon 3 by the neomycin resistance gene (Kawabe et al, Kikutani H, 1994). Mice carrying the CD40 null mutation were transferred to the NOD background (Korganow AS et al, Mathis D, 1999). The line is currently at the >13th backcross to NOD/LtJ .

    Homozygous mutant mice show a deficiency in T cell activation. The mutation causes a significant reduction of CD23 expression on mature B cells and relatively decreased number of IgM bright and IgD dull B cells. The mutant mice mount IgM responses but no IgG, IgA, and IgE responses to thymus dependent antigens. However, IgG as well as IgM responses to thymus-independent antigens are normal (Kawabe et al, Kikutani H, 1994).

  • CTLA4[0]/N ( Mus musculus )

    Development: A neomycin expression cassette was inserted into exon 3 of Ctla4 (cloned from 129/Sv) and transfected into R1 (129X1/SvJ x 129S1/Sv)F1-Kitl+ embryonic stem cells. These ES cells were injected into C57BL/6 background. (Chambers CA et al, 1997). Luhder F et al subsequently backcrossed this mutation to NOD/LtJ. The line is currently at the 31th backcross (2005).

    CTLA-4 deficient mice are viable, fertile, and normal in size but develop a fatal lymphoproliferative disorder. All homozygous mice on various backgrounds, including C57BL/6, BALB/c, and 129SV die at 3-4 weeks of age due to massive polyclonal expansion of T-cells and massive lymphocyte infiltration into non-lymphoid organs, such as heart, liver, lung and pancreas. On NOD/LtJ background, CTL4 deficient mice die around 3 weeks of age of massive lymphocyte proliferation and infiltration into multiple organs, or of hyperacute diabetes when crossed to a pancreas-reactive TCR transgene. (Luhder et al, 2000).

  • Ealpha16/N Tg ( Mus musculus )

    A cloned Ek alpha MHC Class II gene was introduced by microinjection into (C57BL/6 x SJL) F1 mouse oocytes. These strains, as NOD mice, are generally deficient in the MHC II E complex because of a large deletion in the promoter of he Ealpha gene. The transgene thus restore proper expression of the E alpha E beta complex, and immune reactivities linked to it (Le Meur et al, 1985).

    The line is currently at the >99th backcross generation in NOD/LtJ (Bohme J et al, 1990).

    Transgenic mice are viable and fertile. Ealpha 16 abundance in the spleen of all transgenic mice is equivalent to that in BALB/c and B10-BR controls. E alpha 16 expresses the I-E complex on all cells that normally display class II molecules. On the NOD genetic background, the transgene E alpha 16 confers almost complete protection from insulitis by preventing lymphocyte infiltration into the pancreatic islets of 10 week old mice.

  • Foxp3[DTR]/N ( Mus musculus )

    Development: Transgene-directed expression of the human Diphteria Toxin Receptor (DTR) can be used for lineage ablation studies, to determine the role of particular cell types. Mouse cells are resistant to DT for lack of a receptor, but targeted expression of DTR as a transgene renders DTR-expressing cells sensitive to DT treatment in vivo (Saito et al., 2001). Mice expressing the human DTR under the control of foxp3 transcriptional control elements were generated by BAC transgenesis. The BAC construct spanned from 150 kb upstream to 70kb downstream of the Foxp3 transcription start site. A DTR-eGFP cDNA with a stop codon was inserted between the first and second codon of the Foxp3 open reading frame. The recombinant Foxp3-DTR-eGFP BAC was injected into fertilized NOD oocytes, and offspring were genotyped by PCR. (Feuerer et al, 2009).

    Heterozygous NOD.Foxp3.DTR + transgenic mice show no obvious phenotype. The eGFP component can be used for identification of Treg cells, although the fluorescence intensity is lower than that of common reporter lines. DT administration into young adults results in 80%-90% depletion of Foxp3+ T cells in secondary lymphoid organs after a few days. Treg cell pools recover quickly after the end of DT treatment, and Treg cell numbers recovered to 60% of normal by three days after the last injection. (Feuerer et al, 2009) The pancreatic islet of the DT-injected mice showed a strong immune infiltrate compared to littermate control when DT is injected every other day in young adult for 9 days. Very mild infiltrate can be detected in lung and liver of Foxp3 DTR trangenic mice after such a regimen (Feuerer et al, 2009).

  • IFN-gamma[0]/N ( Mus musculus )

    Development: The targeting vector has a neomycin resistance gene inserted into exon 2, which introduces a terminaison codon after the first 30 amino acids of the mature IFN-gamma protein. The targeting vector was transferred into AB-1 embryonic stems. (Dalton et al,1993). The mutation was then transferred to the NOD background (Wang B et al, Mathis D, 1997). The line is currently at the 7th backcross in NOD/LTJ (2008).

    Deficient mice develop normally and are healthy in the absence of pathogens. NOD Mice homozygous for the IFN-gamma KO targeted mutation are viable and fertile. The genetic absence of IFN-gamma does not prevent either insulitis or diabetes in the NOD mice, but increases the time to onset. Splenocytes taken from IFN-gamma deficient 3 diabetic mice are fully capable of transferring diabetes to naive recipients (Hultgren et al,1996). In both NOD. IFN-gamma -/- and NOD. IFN-gamma - /+ mice, IL-12 administration generates a massive and destructive insulitis and increases the number of pancreatic CD4(+) cells (Trembleau et al, 2003). NOD IFN-gamma 0 homozygous mice do not display increased acinar cell apoptosis and abnormal salivary protein expression, typically observed in parental NOD mice prior to Sjogren's syndrome-like autoimmune exocrinopathy (Cha et al, 2004). When NOD. IFN-gamma -/- mice are infected with Coxsackievirus B4, Insulitis or diabetes development is delayed by several weeks compared to NOD mice. When mice are infected at 12 weeks of age, neither acceleration nor long-term protection is elicited in NOD IFN-gamma - /- mice (Serreze et al, 2005).

  • IFN-gammaR[0]/N ( Mus musculus )

    Development: The IFN-gammaR gene was disrupted by inserting the neomycin resistance gene into exon 5, which encodes an extra-cellular membrane proximal portion of the Receptor. (Huang S et al., 1993). Mice carrying the IFN-gamma null mutation were backcrossed with NOD mice with intercrosses to produce experimental animals (Wang et al, 1997). The line is at the >9th backcross in NOD/LtJ.

    Mice without the IFN-gammaR have no overt anomalies, and their immune system appear to develop normally. However, mutant mice have a defective immune resistance, despite normal cytotoxic and T helper cell responses. (Huang et al, 1993). NOD mice homozygous for the IFN-gammaR0 mutation show a marked inhibition of insulitis, both in the kinetics and penetrance, and no signs of diabetes, either spontaneously or after experimental provocation. (Wang B et al., 1997). However, extended backcrossing of this mouse line to the NOD mouse result in a segregation of the IFN-gamma R-deficient genotype from the diabetes-resistant phenotype. These results indicate that the protection of NOD mice from the development of diabetes is not directly linked to the defective IFN-gammaR gene but, rather, is influenced by the presence of a diabetes-resistant gene(s) closely linked to the IFN-gammaR loci derived from the 129 mouse strain (Kanagawa et al, 2000). Outcross studies of NOD mice with several different non autoimmune prone strains, including B6, have identified multiple polymorphic genetic loci conferring susceptibility or resistance to Type 1 diabetes (Wicker et al., 1995; Vyse TJ, Todd JA., 1998 ). Serreze DV et al show that pancreatic insulitis levels are not diminished in 9-wk-old NOD. IFN-gammaR B Tg females, and overt diabetes develops in the few that survive to an older age.

  • IL10[0]/N ( Mus musculus )

    Development: The first exon of IL10 was disrupted after the fourth codon, a neomycin resistance cassette, and an additional termination codon in the third exon, was used for homologous recombination in E14-1 ES cells (129P2/OlaHsd-derived) (Kuhn R et al, Muller W, 1993). The mutation was then transferred to the NOD/Lt background using a marker assisted protocol (Serreze DV et al, Rabinovitch A, 2001). NOD/Lt mice heterozygous for this IL100 allele and homozygous for diabetes susceptibility loci (Idd) were intercrossed to develop mice homozygous for IL10tm1Cgn and all Idd loci. The line is currently at the 11th backcross to NOD/LtJ (2005).

    Used to study gamma interferon.

    Mice homozygous for the IL10 targeted mutation are viable and fertile when housed under SPF conditions.

  • IL4[0]/N ( Mus musculus )

    Development: The targeting vector has a neomycin resistance gene inserted into exon 2, which introduces a terminaison codon after the first 30 amino acids of the mature IFN-gamma protein. The targeting vector was transferred into AB-1 embryonic stems. (Dalton et al, 1993). The mutation was then transferred.

    Deficient mice develop normally and are healthy in the absence of pathogens. NOD Mice homozygous for the IFN-gamma KO targeted mutation are viable and fertile. The genetic absence of IFN-gamma does not prevent either insulitis or diabetes in the NOD mice, but increases the time to onset. Splenocytes taken from IFN-gamma deficient 3 diabetic mice are fully capable of transferring diabetes to naive recipients (Hultgren B et al, 1996). In both NOD. IFN-gamma -/- and NOD. IFN-gamma - /+ mice, IL-12 administration generates a massive and destructive insulitisand increases the number of pancreatic CD4(+) cells. (Trembleau S et al, 2003). NOD IFN-gamma 0 homozygous mice do not display increased acinar cell apoptosis and abnormal salivary protein expression, typically observed in parental NOD mice prior to Sjogren's syndrome-like autoimmune exocrinopathy. (Cha S et al, 2004). When NOD IFN-gamma -/- mice are infected with Coxsackievirus B4, Insulitis or diabetes development is delayed by several weeks compared to NOD mice. When mice are infected at 12 weeks of age, neither acceleration nor long-term protection is elicited in NOD. IFN-gamma - /- mice. (Serreze DV et al, 2005).

  • MIP-GFP/N ( Mus musculus )

    Development: This transgenic allele expresses Enhanced Green Fluorescent Protein fused to a 2.1kb fragment of human Growth Hormone under the control of Mouse Insulin Promoter 1. The transgenic construct was injected directly into NOD/LtJ oocytes. (Hara et al, (2003). The line was backcrossed onto the NOD/Ltj background.

    Transgenic mice develop normally and behave similarly to controls with respect to glucose tolerance and pancreatic insulin content. Histology confirms transgenic mice have normal islet architecture with co-expression of insulin and GFP. (Hara et al, 2003).The enhanced GFP reporter allows the beta cells to be easily identified and purified for further studies.Studies completed at The Jackson Laboratory (Leiter EH et al 2007) indicate there is strong non-mosaic expression of green fluorescent protein in NOD/LtJ-Tg(Ins1-EGFP/GH1)14Hara/HaraJ islets. 100% of homozygous Ins1-EGFP transgenic males and females, identified by qPCR, become diabetic by 9 weeks of age. Pancreatic histopathology of homozygous mice shows beta cell loss without insulitis. Hemizygous Ins1-EGFP mice are viable and are used for breeding. A 30-week incidence study comparing NOD/LtJ controls with mice hemizygous for the Ins1-EGFP transgene shows severely depressed diabetes incidence in these hemizygotes, with males atypically at greater risk than females. Glucose tolerance in prediabetic, Ins1-EGFP transgenic hemizygous 8-week-old males is selectively impaired compared to wild type controls. Although these 8-week-old Ins1-EGFP transgenic and wild type males did not differ in plasma insulin content; a significant decline was noted in hemizygous Ins1-EGFP transgenic males compared to normoglycemic wildtype males when sampled at 30 wk. Adoptive transfer of highly diabetogenic CTL (AI4 TCRTg Rag) CD8+ T cells produced diabetes within 8 days post-injection into NOD/Lt females, but failed to produce any diabetes or even home to islets weeks after injection into hemizygous females. Pancreatic histopathology of 31 week old, non-diabetic, hemizygous Ins1-EGFP transgenic males and females indicate peri and intra islet fibrosis, peri-insulitis and depleted beta cell granulation in 70% of the animals, while only 30% of the mice have intra islet insulitis.

  • muMT[0]/N ( Mus musculus )

    Development: The membrane exons of the gene encoding the mu-chain constant region were disrupted by insertion of a neomycin resistant gene (Kitamura et al., 1991). The mutation was then transferred from the original chimeric stock with a mixed 129/Sv and C57 BL/6 genome onto the NOD/Lt background (Serreze DV et al., 1996). The line is currently at the 14th backcross to NOD/LtJ (2005).

    Heterozygous mice are normal and fertile. NOD.uMTdeficient mice have normal numbers of T cells but are free of overt Diabetes and insulitis resistant. The frequency of disease in the B lymphocyte intact segregants is equivalent to that of standard NOD mice (Serreze DV et al., 1996). Homozygous uMT knock out animals display a high incidence of lymphoma of both T- and B-cell origin compared with these mutations on other genetic backgrounds.

  • NOD.Nk1.1 ( Mus musculus )

    Development: The surface marker NK1.1 is a receptor of the NKR-P1 family normally expressed in the C57BL/6 (B6) strain but not in the NOD strain. The NK1.1 cell surface receptor has been bred onto NOD mice, which carry a reported 10 cM of Chr. 6 from C57BL/6. A homozygous line was initiated from the progeny of the intercross. (Carnaud et al, Herbelin A, 2001). The line is currently at the >50th backcross to NOD/LtJ (2005).

    NK cells are B6-like and can be differentiated from NOD-like NK cells. NOD.NK1.1 mice express the NK1.1 marker selectively on the surface of their NK and NKT cell subsets. In addition, the mice may manifest slightly reduced disease incidence.

  • RAG-1[0]/N ( Mus musculus )

    Development: A neomycin resistance cassette was inserted in the RAG-1 gene, resulting in a 1356 bp deletion in the 5' end of the coding sequence into 129-derived AB1 ES cell line. The C57BL/6J strain was generated by backcrossing mice carrying the Rag-10 mutation 10 times to C57BL/6J inbred mice. (Mombaerts P et al, 1992). Rag-1 deficient mice were transferred to the NOD/LtSz strain background. The line is currently at the 18th backcross to NOD.

    RAG-1 deficient mice are viable and fertile.They are unable to initiate V(D)J recombination in Ig and TCR genes and lack functional T and B lymphocytes. Although RAG-1 expression has been reported in the central nervous system of the mouse, no obvious neuroanatomical or behavioral abnormalities have been found in the RAG-1-deficient mice. On the NOD background, NOD.Rag10 mice are devoid of mature T or B cells. NOD/LtSz-Rag10 recipients of adoptively transferred spleen cells from diabetic NOD/Lt+/+ mice rapidly develop diabetes.

  • RIP-rtTA/N ( Mus musculus )

    Development: The transgene encodes the reverse tetracycline regulatable transactivator (rtTA)(Gossen M, Bujard H,1992; Gossen M, BujardH,1995) under the rat insulin promoter (Ins2 or RIP), and is expressed in the pancreatic beta cells. The RIP-rtTA transgenic construct was injected into (C57BL/6J x SJL)F1 oocytes and backcrossed onto the NOD background for 30 generations.

    Mice hemizygous for this transgenic insert are viable, fertile, normal in size, display normal NOD diabetes onset, and do not display any other gross physical or behavioral abnormalities. This strain provides a Tet-On tool to control the inducible expression of genes in the pancreatic beta cells during various stages of diabetes development. When NOD.RIP-rtTA mice are mated to a second transgenic strain carrying the target gene regulated by the tetO responsive elements, expression of the target gene is turned on by the tetracycline analog, doxycycline (dox). Dox can be administered orally in the food or water.

  • RIP-tTA/N Tg ( Mus musculus )

    Development: The tetracycline regulatable transactivator (tTA, (Gossen M, Bujard H,1992; Gossen M, Bujard ,1995) is placed under the control of the rat insulin promoter (Ins2or RIP), and is expressed in the pancreatic beta cells. The RIP-tTA transgenic construct was injected into (C57BL/6J x SJL)F1 oocytes. The resulting line was backcrossed onto the NOD background for 22 generations.

    This strain provides a Tet-Off tool that permits the inducible expression of genes in the pancreatic beta cells during various stages of diabetes development. Mice hemizygous for this transgenic insert are viable, fertile, normal in size, display normal NOD diabetes onset, but do not display any other gross physical or behavioral abnormalities. When mated to a second transgenic strain carrying the target gene regulated by the tetO responsive elements, expression of the target gene is turned off by the tetracycline analog, doxycycline (dox). Dox can be administered orally in the food or water.

  • Tcra[0]/N ( Mus musculus )

    Development: The Tcrao targeted mutation was generated in GK129 ES cells (derived from substrain129P2/OlaHsd) via homologous recombination. The neomycin resistance gene was inserted into the first constant region of the Tcra gene. (Philpott KL et al., 1992.) The Tcratm1Mjo allele on the C;129 mixed background was then transferred to the NOD background (Katz JD et al., 1995). The line is currently at the 12th backcross to NOD/LtJ (2004).

    NOD mice homozygous for the Tcra targeted mutation lack alpha beta T cells and are completely protected from diabetes development. Because of the complete elimination of alpha beta T cells, these mice are useful in adoptive transfer experiments or in crosses to TCRab transgenic lines. (Katz JD et al., 1995; Hoglund et al., 1999).

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Last updated: 2013-10-22T14:51:03.527-05:00

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