Allelic exclusion of a T cell receptor-beta minilocus.

A TCR-beta minilocus in germline configuration (beta M) has previously been shown to undergo rearrangement and expression in transgenic mice. To study allelic exclusion of TCR miniloci, several beta M transgenic mouse lines were generated and crossed with mice transgenic for a functionally rearranged TCR V beta 2 gene (beta R). PCR analysis of beta M beta R double transgenic mice revealed almost complete suppression of endogenous TCR V beta gene rearrangements, but blockage of minilocus V beta rearrangements was achieved with only one of five minilocus transgenic lines. This result cannot be explained by copy number or arrangement of the multiple miniloci integrated. It appears that the minilocus is not autonomously regulated which suggests that sequences flanking the integration sites influence accessibility of the minilocus for rearrangement and allelic exclusion. However, although productively rearranged genes were formed in double transgenic mice, surface expression of minilocus-encoded beta chains was not detected. This indicates that allelic exclusion may operate at a level after gene rearrangement.     

Cell-surface expression and alloantigenic function of a human nonclassical class I molecule (HLA-E) in transgenic mice

We have introduced the gene (E*01033) encoding the heavy chain of the human nonclassical MHC class I Ag, HLA-E, into the mouse genome. Two founder mice carry a 21-kb fragment, the others bear an 8-kb fragment. Each of the founder mice was mated to mice of an already established C57BL/10 transgenic line expressing human beta2-microglobulin (beta2m). Cell surface HLA-E was detected on lymph node cells by flow cytometry only in the presence of endogenous human beta2m. However, HLA-E-reactive mouse CTL (H-2-unrestricted) lysed efficiently the target cells originating from HLA-E transgenic mice without human beta2m, showing that the HLA-E protein can be transported to the cell surface in the absence of human beta2m, presumably by association with murine beta2m. Rejection of skin grafts from HLA-E transgenic mice demonstrates that HLA-E behaves as a transplantation Ag in mice. HLA-E transgenic spleen cells are effective in stimulating an allogeneic CTL response in normal and human classical class I (HLA-B27) transgenic mice. Furthermore, results from split-well analysis indicate that the majority of the primary in vivo-induced CTL recognizes HLA-E as an intact molecule (H-2-unrestricted recognition) and not as an HLA-E-derived peptide presented by a mouse MHC molecule, although a small fraction (ranging from 4 to 21%) of the primary in vivo-induced CTL is able to recognize HLA-E in an H-2-restricted manner. Based on these observations, we conclude that HLA-E exhibits alloantigenic properties that are indistinguishable from classical HLA class I molecules when expressed in transgenic mice.     

Expansion of myelopoietic precursors and inhibition of B-cell precursors in mice that express a T-cell receptor gamma (V gamma 1.1J gamma 4C gamma 4) transgene.

Knowledge of the genetic determinants that can affect renewal of multipotential stem cells and their commitment to specific cell lineages is essential to our understanding of multicellular development. However, despite the vast amount of accumulated knowledge in this area, genetic determinants that affect renewal and commitment of precursor cells are unknown. In this study, we demonstrate that three independently derived founder mouse strains, transgenic for the TcR V gamma 1.1J gamma 4C gamma 4 (TcR gamma 4) chain gene, differed significantly from normal mice in their development of T and B cells as well as myelopoietic precursor cells. Ontogenic programs consistent with an acceleration of T-cell development and a delayed appearance and suppressed levels of pre-B- and B-cell precursors were evident in these transgenic mice. In addition, TcR gamma 4 transgenic mice possessed a significantly elevated level of myelopoietic pluripotential precursors. 3H-thymidine cell suicide studies suggest that higher percentages of pluripotent precursors from the bone marrow of the TcR gamma 4 transgenic mice were in the S phase of the cell cycle. These modulations of the lymphoid and myelopoietic compartments, however, were not found in other T-cell receptor transgenic mice (e.g., TcR V gamma 1.2J gamma 2C gamma 2, TcR gamma 2; or V beta 8.1D beta J beta 2.4C beta 2, TcR beta) constructed with the same or similar cDNA expression vector. The results suggest that the expression of a specific T-cell receptor gamma chain gene, and/or an elevated level of particular subset of TcR gamma delta cells, may affect the proliferation and relative proportions of haemopoietic and lymphoid precursors.     

MHC-II-independent CD4+ T cells induce colitis in immunodeficient RAG-/- hosts

CD4(+) alpha beta T cells from either normal C57BL/6 (B6) or MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice engrafted into congenic immunodeficient RAG1(-/-) B6 hosts induced an aggressive inflammatory bowel disease (IBD). Furthermore, CD4(+) T cells from CD1d(-/-) knockout (KO) B6 donor mice but not those from MHC-I(-/-) (homozygous transgenic mice deficient for beta(2)-microglobulin) KO B6 mice induced a colitis in RAG(-/-) hosts. Abundant numbers of in vivo activated (CD69(high)CD44(high)CD28(high)) NK1(+) and NK1(-) CD4(+) T cells were isolated from the inflamed colonic lamina propria (cLP) of transplanted mice with IBD that produced large amounts of TNF-alpha and IFN-gamma but low amounts of IL-4 and IL-10. IBD-associated cLP Th1 CD4(+) T cell populations were polyclonal and MHC-II-restricted when derived from normal B6 donor mice, but oligoclonal and apparently MHC-I-restricted when derived from MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice. cLP CD4(+) T cell populations from homozygous transgenic mice deficient for beta(2)-microglobulin KO B6 donor mice engrafted into RAG(-/-) hosts were Th2 and MHC-II restricted. These data indicate that MHC-II-dependent as well as MHC-II-independent CD4(+) T cells can induce a severe and lethal IBD in congenic, immunodeficient hosts, but that the former need the latter to express its IBD-inducing potential.     

Analysis of the HLA-Cw3-specific cytotoxic T lymphocyte response of HLA-B7 X human beta 2m double transgenic mice

The cytolytic responses of either normal (non transgenic), HLA-B7 (single transgenic) or HLA-B7 x human beta 2 microglobulin (double transgenic) DBA/2 mice induced by transfected HLA-Cw3 P815 (H-2d) mouse mastocytoma cells were compared, to evaluate whether the expression of an HLA class I molecule in responder mice would favor the emergence of HLA-specific, H-2-unrestricted CTL. Only 8 of 300 HLA-Cw3-specific CTL clones tested could selectively lyse HLA-Cw3-transfected cells in an H-2-unrestricted manner, all having been isolated after hyperimmunization of double transgenic mice. These clones also lysed HLA-Cw3+ human cells. Unexpectedly, the lysis of the human but not that of the murine HLA-Cw3 cells was inhibited by Ly-2,3-specific mAb. Despite significant expression of HLA-B7 class I molecules on transgenic lymphoid cells, including thymic cells, limiting dilution analysis and comparative study of TCR-alpha and -beta gene rearrangements of the eight isolated clones (which suggested that they all derived from the same CTL precursor) indicated that the frequency of HLA-Cw3-specific H-2 unrestricted cytotoxic T lymphocytes remained low (even in HLA-B7 x human beta 2-microglobulin double transgenic mice). This suggests that coexpression of HLA class I H and L chain in transgenic mice is not the only requirement for significant positive selection of HLA class I-restricted cytotoxic mouse T lymphocytes.     

A direct test of the hypothesis that increased microtubule network density contributes to contractile dysfunction of the hypertrophied heart

Contractile dysfunction in pressure overload-hypertrophied myocardium has been attributed in part to the increased density of a stabilized cardiocyte microtubule network. The present study, the first to employ wild-type and mutant tubulin transgenes in a living animal, directly addresses this microtubule hypothesis by defining the contractile mechanics of the normal and hypertrophied left ventricle (LV) and its constituent cardiocytes from transgenic mice having cardiac-restricted replacement of native beta(4)-tubulin with beta(1)-tubulin mutants that had been selected for their effects on microtubule stability and thus microtubule network density. In each case, the replacement of cardiac beta(4)-tubulin with mutant hemagglutinin-tagged beta(1)-tubulin was well tolerated in vivo. When LVs in intact mice and cardiocytes from these same LVs were examined in terms of contractile mechanics, baseline function was reduced in mice with genetically hyperstabilized microtubules, and hypertrophy-related contractile dysfunction was exacerbated. However, in mice with genetically hypostabilized cardiac microtubules, hypertrophy-related contractile dysfunction was ameliorated. Thus, in direct support of the microtubule hypothesis, we show here that cardiocyte microtubule network density, as an isolated variable, is inversely related to contractile function in vivo and in vitro, and microtubule instability rescues most of the contractile dysfunction seen in pressure overload-hypertrophied myocardium.     

Establishment of La-tPA/G-CSF dual transgenic mice and expression in their mammary gland

Expression vectors of human granulocyte colony stimulating factor (G-CSG) and long acting tissue plasminogen activator (La-tPA) in mammary gland were constructed using promoters of mouse whey acid protein gene (WAP) and sheep β-lactoglobulin gene (BLG) with sizes of 2.6 and 5 kb respectively. Two kinds of transgenic mice of G-CSF and La-tPA were produced with microinjection. The expression of G-CSF and La-tPA was achieved in mammary glands of transgenic mice, respectively. In order to establish dual transgenic mice of La-tPA/G-CSF, transgenic mice carrying G-CSF and La-tPA gene characterized with specific expression in mammary gland were mated. La-tPA/G-CSF dual transgenic mice were screened out from the hybrid offspring by Once-PCR. The co-expression of La-tPA and G-CSF in mammary gland of the dual transgenic mice was confirmed by the milk assayed and Northern blot analysis. Some parameters about the dual transgenic mice indicated that there were fewer litters than that of normal mice. The ratio of du     

Hyperplasia and tumours in lung, breast and other tissues in mice carrying a RAR beta 4-like transgene.

Transgenic mice were generated which express a truncated nuclear retinoic acid receptor beta (RAR beta), closely resembling the natural isoform RAR beta 4, under the control of the MMTV promoter. The transgene was expressed in salivary gland, testis, lung and mammary tissue in two different lines. At approximately 11-14 months virtually all the transgenic mice showed hyperplasia of the lung alveolar epithelium with an excess of type II pneumocytes. Hyperplasia of the mammary alveoli and terminal ducts was also seen in some females. Salivary glands and some sebaceous glands were hyperplastic in most male transgenic mice, but only rarely in females or in non-transgenics. Primary benign and malignant tumours were more numerous in transgenic mice than in controls, with a total of 23 in 43 mice versus two in 33 non-transgenic animals. Treatment with dexamethasone to increase transgene expression resulted in exaggerated versions of the above phenotypes. Overexpression of RAR beta 4 therefore appears to predispose various tissues to hyperplasia and neoplasia, and this by contrast to the RAR beta 2 isoform, which has tumour suppressor activity. A survey of ratios of RAR beta 4:RAR beta 2 expression in human lung tumour cell lines showed an increase compared with normal lung tissue, suggesting that RAR beta 4 may play a similar role in human tumorigenesis.     

Overexpression of beta2-adrenergic receptors in mouse liver alters the expression of gluconeogenic and glycolytic enzymes

In the livers of humans and many other mammalian species, beta2-adrenergic receptors (beta2-ARs) play an important role in the modulation of glucose production by glycogenolysis and gluconeogenesis. In male mice and rats, however, the expression and physiological role of hepatic beta2-ARs are rapidly lost with development under normal physiological conditions. We previously described a line of transgenic mice, F28 (Andre C, Erraji L, Gaston J, Grimber G, Briand P, and Guillet JG. Eur J Biochem 241: 417-424, 1996), which carry the human beta2-AR gene under the control of its own promoter. In these mice, hepatic beta2-AR levels are shown to increase rapidly after birth and, as in humans, be maintained at an elevated level in adulthood. F28 mice display strongly enhanced adenylyl cyclase responses to beta-AR agonists in their livers and, compared with normal mice, have increased basal hepatic adenylyl cyclase activity. In this report we demonstrate that, under normal physiological conditions, this increased beta2-AR activity affects the expression of the gluconeogenic and glycolytic key enzymes phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and l-pyruvate kinase and considerably decreases hepatic glycogen levels. Furthermore, we show that the effects of beta-adrenergic ligands on liver glycogen observed in humans are reproduced in these mice: liver glycogen levels are strongly decreased by the beta2-AR agonist clenbuterol and increased by the beta-AR antagonist propranolol. These transgenic mice open new perspectives for studying in vivo the hepatic beta2-AR system physiopathology and for testing the effects of beta-AR ligands on liver metabolism.     

Sodium channel beta4 subunit: down-regulation and possible involvement in neuritic degeneration in Huntington's disease transgenic mice

Sodium channel beta4 is a very recently identified auxiliary subunit of the voltage-gated sodium channels. To find the primarily affected gene in Huntington's disease (HD) pathogenesis, we profiled HD transgenic mice using a high-density oligonucleotide array and identified beta4 as an expressed sequence tag (EST) that was significantly down-regulated in the striatum of HD model mice and patients. Reduction in beta4 started at a presymptomatic stage in HD mice, whereas other voltage-gated ion channel subunits were decreased later. In contrast, spinal cord neurons, which generate only negligible levels of expanded polyglutamine aggregates, maintained normal levels of beta4 expression even at the symptomatic stage. Overexpression of beta4 induced neurite outgrowth in Neuro2a cells, and caused a thickening of dendrites and increased density of dendritic spines in hippocampal primary neurons, indicating that beta4 modulates neurite outgrowth activities. These results suggest that down-regulation of beta4 may lead to abnormalities of sodium channel and neurite degeneration in the striatum of HD transgenic mice and patients with HD.     

T cell tolerance to Mlsa encoded antigens in T cell receptor V beta 8.1 chain transgenic mice.

To study T cell tolerance, transgenic mice were generated that expressed the Mlsa-reactive T cell receptor (TCR) beta chain V beta 8.1 (cDNA) under the control of the H-2Kb promoter/immunoglobulin heavy chain enhancer on approximately 90% of peripheral T cells. In transgenic mice bearing Mlsa, thymocytes expressing the TCR at a high density were deleted and the percentage of Thy 1.2+ lymph node cells was reduced. The CD4/CD8 ratio of mature T cells was reversed in Mlsa and Mlsb transgenic mice independent of the H-2. RNA analysis and immunofluorescence with TCR V beta-specific antibodies revealed that expression of endogenous TCR beta genes was suppressed. Both Mlsa and Mlsb TCR beta chain transgenic mice mounted a T-cell-dependent IgG response against viral antigens, whereas the capacity to generate alloreactive and virus-specific cytotoxic T cells was impaired in TCR beta chain transgenic Mlsa, but not in transgenic Mlsb mice.     

Mechanisms of accelerated immune-mediated diabetes resulting from islet beta cell expression of a Fas ligand transgene

Nonobese diabetic (NOD) mice transgenic for Fas ligand (FasL) on islet beta cells (HIPFasL mice) exhibit an accelerated diabetes distinct from the normal autoimmune diabetes of NOD mice. This study was undertaken to define the mechanism underlying accelerated diabetes development in HIPFasL mice. It was found that diabetes in HIPFasL mice is dependent on the NOD genetic background, as HIPFasL does not cause diabetes when crossed into other mice strains and is lymphocyte dependent, as it does not develop in HIPFasL(SCID) mice. Diabetes development in NOD(SCID) recipients of diabetic HIPFasL splenocytes is slower than when using splenocytes from diabetic NOD mice. Beta cells from HIPFasL mice are more susceptible to cytokine-induced apoptosis than wild-type NOD beta cells, and this can be blocked with anti-FasL Ab. HIPFasL islets are more rapidly destroyed than wild-type islets when transplanted into nondiabetic NOD mice. This confirms that FasL(+) islets do not obtain immune privilege, and instead NOD beta cells constitutively expressing FasL are more susceptible to apoptosis induced by Fas-FasL interaction. These findings are consistent with the accelerated diabetes of young HIPFasL mice being a different disease process from the autoimmune diabetes of wild-type NOD mice. The data support a mechanism by which cytokines produced by the insulitis lesion mediate up-regulation of beta cell Fas expression, resulting in suicide or fratricide of HIPFasL beta cells that overexpress FasL.     

Rescue of defective T cell development and function in Atm-/- mice by a functional TCR alpha beta transgene

The Atm-/- mice recapitulate most of the defects observed in ataxia-telangiectasia (A-T) patients, including a high incidence of lymphoid tumors and immune defects characterized by defective T cell differentiation, thymus hypoplasia, and defective T-dependent immune responses. To understand the basis of the T cell developmental defects in Atm-/- mice, a functional TCR alpha beta transgene was introduced into these mutant mice. Analysis of the Atm-/-TCR alpha beta+ mice indicated that the transgenic TCR alpha beta can rescue the defective T cell differentiation and partially rescue the thymus hypoplasia in Atm-/- mice, indicating that thymocyte positive selection is normal in the Atm-/- mice. In addition, cell cycle analysis of the thymocytes derived from Atm-/-TCR alpha beta+ and control mice suggested that Atm is involved in the thymocyte expansion. Finally, evaluation of the T-dependent immune responses in Atm-/-TCR alpha beta+ mice indicated that Atm is dispensable for normal T cell function. Therefore, the defective T-dependent immune responses in Atm-/- mice must be secondary to greatly reduced T cell numbers in these mutant mice.     

Interferon-regulated pathways that control hepatitis B virus replication in transgenic mice

We previously showed that the intrahepatic induction of cytokines such as alpha/beta interferon (IFN-alpha/beta) and gamma interferon (IFN-gamma) inhibits hepatitis B virus (HBV) replication noncytopathically in the livers of transgenic mice. The intracellular pathway(s) responsible for this effect is still poorly understood. To identify interferon (IFN)-inducible intracellular genes that could play a role in our system, we crossed HBV transgenic mice with mice deficient in IFN regulatory factor 1 (IRF-1), the double-stranded RNA-activated protein kinase (PKR), or RNase L (RNase L) (IRF-1(-/-), PKR(-/-), or RNase L(-/-) mice, respectively), three well-characterized IFN-inducible genes that mediate antiviral activity. We showed that unmanipulated IRF-1(-/-) or PKR(-/-) transgenic mice replicate HBV in the liver at slightly higher levels than the respective controls, suggesting that both IRF-1 and PKR individually appear to mediate signals that modulate HBV replication under basal conditions. These same animals were responsive to the antiviral effects of the IFN-alpha/beta inducer poly(I-C) or recombinant murine IFN-gamma, suggesting that under these conditions, either the IRF-1 or the PKR genes can mediate the antiviral activity of the IFNs or other IFN-inducible genes mediate the antiviral effects. Finally, RNase L(-/-) transgenic mice were undistinguishable from controls under basal conditions and after poly(I-C) or IFN-gamma administration, suggesting that RNase L does not modulate HBV replication in this model.     

Diabetes and tolerance in transgenic mice expressing class II MHC molecules in pancreatic beta cells

Insulin-dependent diabetes is caused by the loss of insulin-producing beta cells in pancreatic islets. It has been proposed that aberrant expression of Class II Major Histocompatibility Complex (MHC) molecules on beta cells stimulates an autoimmune attack against beta cell antigens. To test this hypothesis, we generated transgenic mice that express Class II MHC molecules (E alpha d/E beta b, or I-Eb) on beta cells. Diabetes was found in 100% of transgenic progeny from three expressing transgenic mouse lines, but without evidence for lymphocytic infiltrates. Furthermore, T lymphocytes appeared to be tolerant to the transgene I-Eb molecule, despite the absence of expression of I-Eb in the thymus or any other lymphoid tissue. The results suggest that novel expression of Class II MHC molecules on nonlymphoid cells is by itself insufficient to initiate autoimmune responses against tissue-specific antigens.     

The developmental regulation of the human zeta-globin gene in transgenic mice employing beta-galactosidase as a reporter gene.

We have investigated the developmental and tissue specific expression of the human embryonic zeta-globin gene in transgenic mice. A construct containing 550 bp of zeta-globin 5' flanking region, fused to a beta-galactosidase (lacZ) reporter gene and linked to the locus control region (LCR)-like alpha positive regulatory element (alpha PRE) was employed for the production of transgenic mice. Firstly, we compared the number of live born transgenic mice containing this construct to the number of live born transgenic mice containing the entire zeta-globin gene linked to the alpha PRE or the beta LCR. Data showed that 12% of mice generated from eggs injected with zeta-promoter/lacZ/alpha PRE DNA were transgenic compared to only 2% of mice generated from eggs injected with the entire zeta-globin gene linked to the alpha PRE or the beta LCR. The reduced number of live born transgenic mice containing the latter constructs suggests that death of transgenic embryos, possibly due to thalassaemia, may be occurring. X-gal staining of whole embryos containing the lacZ gene revealed that zeta-globin promoter activity was most pronounced at 8.5-9.5 days of development and was restricted to erythroid cells. By 15 days of development, no zeta-globin promoter activity was detected. These results suggest that the alpha PRE can direct high level expression from the zeta-globin promoter and that sequences required for the correct tissue and developmental specific expression of the human zeta-globin gene are present within 550 bp's of 5' flanking region. Sequences within the body of the zeta-globin gene or 3' of the cap site do not appear to be necessary for correct zeta-globin developmental regulation.     

Transgenic isolation of skeletal muscle and kidney defects in laminin beta2 mutant mice: implications for Pierson syndrome

Pierson syndrome is a recently defined disease usually lethal within the first postnatal months and caused by mutations in the gene encoding laminin beta2 (LAMB2). The hallmarks of Pierson syndrome are congenital nephrotic syndrome accompanied by ocular abnormalities, including microcoria (small pupils), with muscular and neurological developmental defects also present. Lamb2(-/-) mice are a model for Pierson syndrome; they exhibit defects in the kidney glomerular barrier, in the development and organization of the neuromuscular junction, and in the retina. Lamb2(-/-) mice fail to thrive and die very small at 3 weeks of age, but to what extent the kidney and neuromuscular defects each contribute to this severe phenotype has been obscure, though highly relevant to understanding Pierson syndrome. To investigate this, we generated transgenic mouse lines expressing rat laminin beta2 either in muscle or in glomerular epithelial cells (podocytes) and crossed them onto the Lamb2(-/-) background. Rat beta2 was confined in skeletal muscle to synapses and myotendinous junctions, and in kidney to the glomerular basement membrane. In transgenic Lamb2(-/-) mice, beta2 deposition in only glomeruli prevented proteinuria but did not ameliorate the severe phenotype. By contrast, beta2 expression in only muscle restored synaptic architecture and led to greatly improved health, but the mice died from kidney disease at 1 month. Rescue of both glomeruli and synapses was associated with normal weight gain, fertility and lifespan. We conclude that muscle defects in Lamb2(-/-) mice are responsible for the severe failure to thrive phenotype, and that renal replacement therapy alone will be an inadequate treatment for Pierson syndrome.     

Nuclear Magnetic Resonance Spectroscopy Study on Energy Metabolism, Intracellular pH, and Free Mg2+ Concentration in the Brain of Transgenic Mice Overexpressing Human Ornithine Decarboxylase Gene

We have generated a transgenic mouse line strikingly overexpressing the human ornithine decarboxylase (ODC) gene in their brain. Brain ODC activity was increased in the transgenic animals by a factor of 70 in comparison with their nontransgenic littermates. The content of brain putrescine, the product of ODC, was greater than 60 mumol/g of tissue in the transgenic mice, whereas in the normal animals it was below the level that could be detected by an HPLC method. The concentrations of the higher polyamines (spermidine and spermine) were not significantly different from control values. 31P nuclear magnetic resonance (31P NMR) spectroscopy analyses revealed a significantly reduced (40%) free Mg2+ concentration as calculated from the chemical shift differences of the nucleoside triphosphate alpha and beta peaks in the brains of the transgenic animals. The lower free Mg2+ concentration in the brains of ODC transgenic mice was not a consequence of altered intracellular pH or changes in cellular high-energy metabolites. 1H NMR showed no differences in brain choline/N-acetylaspartate and total creatine/N-acetylaspartate ratios between the two animal groups. These ODC transgenic animals may serve as models in vivo for studies on cerebral postischemic events and on epilepsy, as polyamines are supposed to be involved in these processes.