Diphtheria toxin receptor-mediated conditional and targeted cell ablation in transgenic mice.

Specific cell ablation is a useful method for analyzing the in vivo function of cells. We have developed a simple and sensitive method for conditional cell ablation in transgenic mice, called "toxin receptor-mediated cell knockout." We expressed the diphtheria toxin (DT) receptor in transgenic mice using a hepatocyte-specific promoter and found that injection of DT caused fulminant hepatitis. Three independently established transgenic lines demonstrated a good correlation between the sensitivity of hepatocytes to DT and the expression level of the DT receptors. Moreover, the degree of hepatocyte damage was easily controlled over a wide range of doses of injected DT without any obvious abnormalities in other cells or tissues. This system is useful for generating mouse models of disease and for studying the recovery or regeneration of tissues from cell damage or loss. As DT is a potent inhibitor of protein synthesis in both growing and non-growing cells, the method is applicable to a wide range of cells and tissues in mice or in other DT-insensitive animals.

Immunohistochemical characterization of transgenic mice highly expressing human lysosomal alpha-galactosidase.

Human lysosomal alpha-galactosidase predominantly hydrolyzes ceramide trihexoside. A transgenic mouse line, C57BL/6CrSIc-TgN(GLA) 1951 Rin, highly expressing human alpha-galactosidase, has been established and investigated biochemically and immunohistochemically in order to clarify the distribution of the expressed enzyme proteins and to evaluate it as a donor model of organ transplantation therapy for Fabry disease caused by a genetic defect of alpha-galactosidase. In these transgenic mice, about five copies of the transgene were integrated, and alpha-galactosidase activity was expressed in liver, kidney, heart, spleen, small intestine, submaxillary gland, skeletal muscle, cerebrum, cerebellum, bone marrow cells and serum. The enzyme activity was about 22 to 11,080-fold higher than that in non-transgenic mice. In liver, heart and kidney tissues, which are important organs for transplantation studies, sufficient amounts of alpha-galactosidase mRNAs were transcribed, and the expressed enzymes, with molecular weights of 54-60 kDa, are abundant in the liver (enzyme activity: 53,965 nmol h-1 mg-1 protein) and heart (39,906 nmol h-1 mg-1 protein), followed by in the kidney tissue (9177 nmol h-1 mg-1 protein), respectively. An immunohistochemical microscopic study clearly demonstrated the distribution of the expressed enzyme proteins in kidney and liver tissues. Highly expressed alpha-galactosidase was detected in glomerular cells, tubular cells and hepatocytes. These transgenic mice will be useful as a donor model for experimental organ transplantation, and also it will enable recurrent biopsies and long-term observation. The organ transplantation data on mice will provide us with important information.

Factores socioeducativos asociados al no uso de métodos anticonceptivos en universitarias de cuatro países de Latinoamérica / Socio-educational factors associated with the non-use of contraceptive methods in university students from four Latin American countries

INTRODUCCIÓN: El cuidado anticonceptivo es importante una vez que se inicia la vida sexual, pero esto no ha sido medido en distintas realidades de Latinoamérica. OBJETIVO: Determinar los factores socio-educativos asociados al no uso de métodos anticonceptivos en universitarias de cuatro países de Latinoamérica. METODOLOGÍA: Estudio transversal analítico, se encuestó a estudiantes mujeres que ya habían iniciado su vida sexual, se le preguntó por el uso de condón (preservativo), método del ritmo, anticoncepción oral y anticoncepción oral de emergencia. Estas fueron descritas y asociadas a variables socio-educativas. RESULTADOS: El 7% (47) no usaba ninguno de los 4 métodos anticonceptivos; al realizar el análisis multivariado, no hubo diferencias estadísticamente significativas según el país, el año de estudios o si eran católicas/cristianas (todos los valores p>0,05), en cambio, las de universidades particulares tuvieron un mayor porcentaje de ausencia de uso de los 4 métodos anticonceptivos (RPa: 2,52; IC95%: 1,24-5,14; valor p=0,010). Según el uso de alguno de los 4 métodos, el país donde se encuestó tuvo muchas diferencias entre el uso de uno u otro método; el año de la carrera no estuvo asociado al no uso de alguno de los cuatro métodos; las que fueron católicas o cristianas usaron menos la anticoncepción oral (p<0,001) y las que estudiaban en universidades particulares usaron más el método del ritmo (p<0,05). CONCLUSIONES: Un porcentaje importante no usó ninguno de los cuatro métodos anticonceptivos más comunes, estando esto asociado al tipo de universidad.

INTRODUCTION: The care of contraception is important once you start the sex lives, but this hasn't been measured in different realities of Latin-America. OBJECTIVE: To determine the socio-educational factors associated with non-use of contraceptive methods in universities in four Latin American countries. METHODOLOGY: Cross-sectional study. Surveyed women students, who have started their sexual lives. They were asked about the use of condoms, rhythm method, birth control pills and next day pill. These're described and associated to variables socio-educational. RESULTS: 7% (47) did not use any of the 4 contraceptive methods; when performing the multivariate analysis, there were no statistically significant differences by country, the year of study or if they were Catholic/Christian (all values p>0.05), on the other hand, those of particular universities had a higher percentage of non- take care of yourself with one of the 4 methods (RPa: 2,52; IC95%: 1,24-5,14; value p=0,010). According to the use of one of the 4 methods, the country where it was surveyed had many differences between the use of one or the other method; the year of the degree was not associated with the non-use of any of the four methods; those who were Catholic or Christian used less oral contraception (p <0.001) and those who studied at private universities used the rhythm method more (p <0.05). CONCLUSIONS: A significant percentage did not use any of the four most common contraceptive methods, this being associated with the type of university.

Non-invasive visualization of sperm mitochondria behavior in transgenic mice with introduced green fluorescent protein (GFP).

Using high sensitive polymerase chain reaction (PCR), we previously demonstrated that selective elimination of sperm mitochondrial DNA occurred during early embryogenesis in mouse. To analyze the process morphologically in more detail, a non-invasive, real-time observation of sperm mitochondria was used. Transgenic mice that express green fluorescent protein (GFP) exclusively in mitochondria (mtGFP-tg mice) were generated. The fluorescence in mtGFP-tg mice was strong and stable enough to carry out repeated observations under confocal laser scanning microscopy. In these mtGFP-tg mice it was revealed that the sperm mitochondria were selectively eliminated from egg cytoplasm during the two-cell stage of early embryogenesis. Therefore, mtGFP-tg mice should contribute to studies on sequential or repeated analysis of mitochondria.

Generation and characterization of transgenic mice expressing a human mutant alpha-galactosidase with an R301Q substitution causing a variant form of Fabry disease.

Transgenic mice expressing a human mutant alpha-galactosidase with an R301Q substitution, which was found in a patient with a variant form of Fabry disease, were established. The mice transcribed a sufficient amount of alpha-galactosidase mRNA, but the steady-state levels of the enzyme protein were decreased in liver, kidney and heart, only residual activity being detected in these tissues. The mice will be useful for the clarification of the defective regulation of the structurally altered enzyme protein expressed by the mutant gene at the organ or individual level as well as for the evaluation of drugs that stabilize and/or activate the mutant alpha-galactosidase.

Deficiency of a STE20/PAK family kinase LOK leads to the acceleration of LFA-1 clustering and cell adhesion of activated lymphocytes.

Lymphocyte-oriented kinase (LOK) is a member of the STE20/p21-activated kinase (PAK) family and expressed predominantly in lymphoid organs. Generation of LOK-deficient mice revealed that the leukocyte-function-associated antigen (LFA-1)/intercellular adhesion molecules (ICAM)-mediated aggregation of mitogen-stimulated T cells was greatly enhanced in the absence of LOK. Though levels of total LFA-1 and ICAMs as well as the active form of LFA-1 on T cell blasts were comparable in the presence and absence of LOK, clustering of active LFA-1 detected by binding of soluble ICAM-1 was accelerated in the absence of LOK. These results suggest that LOK is potentially involved in the regulation of LFA-1-mediated lymphocyte adhesion.

A sialidase-susceptible ganglioside, IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer, is a major disialoganglioside in WHT/Ht mouse thymoma and thymocytes.

The disialogangliosides of WHT/Ht mouse thymomas, which were obtained by subcutaneous transplantation of a thymoma that developed spontaneously in a WHT/Ht mouse, were purified and characterized. From the results of sugar-composition analysis, a permethylation study, enzymatic hydrolysis followed by TLC-immunostaining, negative-ion fast atom bombardment mass spectrometry (FAB/MS), and 1H-NMR spectroscopy, the structure of one of the five purified disialogangliosides was determined to be IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer. The other 4 disialogangliosides were tentatively characterized on the basis of sialidase treatment followed by TLC-immunostaining with cholera toxin B subunit and anti-Gg4Cer antibody to be IV alpha(NeuAc alpha-NeuGc)-Gg4Cer, IV alpha(NeuGc alpha-NeuAc)-Gg4Cer, IV alpha NeuAc,II3 alpha NeuAc-Gg4Cer, and IV alpha NeuGc,II3 alpha NeuGc-Gg4Cer. In addition, another component exhibiting one spot on TLC was a mixture of IV alpha NeuGc,II3 alpha NeuAc-Gg4Cer and IV alpha NeuAc,II3 alpha NeuGc-Gg4Cer. Then the occurrence of these gangliosides in WHT/Ht mouse thymocytes was examined. As one of two major disialogangliosides, the thymocytes contained IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer, which was characterized with a mass spectrum and mass chromatograms obtained by micro high-performance liquid chromatography-FAB/MS. The other major disialoganglioside was tentatively characterized to be II3 alpha-(NeuGc alpha-NeuGc)-Gg4Cer by sialidase treatment followed by TLC-immunostaining. A sialidase-susceptible monosialoganglioside, IV3 alpha NeuGc-Gg4Cer [GM1b(NeuGc)], had been reported to be characteristic of mouse immune tissues [Nakamura, K. et al. (1988) J. Biochem, 103, 201-208]. Taken together, the results suggest that the pathway from Gg4Cer to IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer through GM1b(NeuGc) is quite active in mouse immune tissues.

Effect of H-2 complex on the growth of embryo-derived teratomas in mice.

Seven-day-old embryos of several H-2 congenic strains were transplanted under the kidney capsules of syngeneic adult recipients to determine the genetic factors(s) governing the in vivo growth of embryo-derived teratomas. A.TH(H-2t2) and A.TL(H-2t1) strains showed significantly greater tumor weights than A.BY(H-2b) and A.SW(H-2s) strains. The A(H-2a) strain was intermediate in tumor size. A comparison of the genic constitution of the H-2 complex in each congenic strain suggested that the H-2D locus and/or its distal regions affected the growth of embryo-derived teratomas. The teratoma induced in the B10.A(H-2a) strain was smaller than that in the A(H-2a) strain, indicating that the genetic background of the A strain is favorable for teratoma growth. Histological observations demonstrated that the existence of embryonal carcinoma cells was necessary for the growth of teratomas. A radiation-sensitive immunological factor in the recipient probably plays a role in stimulating teratoma growth.

Drastic up-regulation of Fcepsilonri on mast cells is induced by IgE binding through stabilization and accumulation of Fcepsilonri on the cell surface.

It has been shown that IgE binding to FcepsilonRI on mast cells results in increased FcepsilonRI expression, which in turn enhances IgE-dependent chemical mediator release from mast cells. Therefore, prevention of the IgE-mediated FcepsilonRI up-regulation would be a promising strategy for management of allergic disorders. However, the mechanism of IgE-mediated FcepsilonRI up-regulation has not been fully elucidated. In this study, we analyzed kinetics of FcepsilonRI on peritoneal mast cells and bone marrow-derived mast cells. In the presence of brefeldin A, which prevented transport of new FcepsilonRI molecules to the cell surface, levels of IgE-free FcepsilonRI on mast cells decreased drastically during culture, whereas those of IgE-bound FcepsilonRI were stable. In contrast, levels of FcgammaRIII on the same cells were stable even in the absence of its ligand, indicating that FcepsilonRI alpha-chain, but not beta- and gamma-chains, was responsible for the instability of IgE-free FcepsilonRI. As far as we analyzed, there was no evidence to support the idea that IgE binding to FcepsilonRI facilitated synthesis and/or transport of FcepsilonRI to the cell surface. Therefore, the stabilization and accumulation of FcepsilonRI on the cell surface through IgE binding appears to be the major mechanism of IgE-mediated FcepsilonRI up-regulation.

Cell type-specific expression of the genes for the protein kinase C family: down regulation of mRNAs for PKC alpha and nPKC epsilon upon in vitro differentiation of a mouse neuroblastoma cell line neuro 2a.

By the use of cloned cDNAs for protein kinase C isozymes alpha, beta I, beta II, gamma, and those for novel protein kinase C, epsilon and zeta, the expression of the corresponding mRNA species was examined in various mouse tissues, human lymphoid cell lines, and mouse cell lines of neuronal origin. In adult brain, mRNAs for all the isozymes of PKC family are expressed. However, the expression of these mRNA species in brain is low at birth. A similar pattern of expression was also observed for beta I/beta II mRNAs in spleen. These expression patterns are in clear contrast to that for beta I/beta II mRNAs in thymus where the mRNAs are expressed at birth and the levels of expression decrease with age. Human lymphoid cell lines express large amounts of PKC beta mRNAs in addition to PKC alpha. Further, nPKC epsilon mRNA is expressed in some of these cell lines. On the other hand, all the mouse cell lines of neuronal origin tested express nPKC epsilon and zeta in addition to PKC alpha. In a mouse neuroblast cell line, Neuro 2a, down modulation of mRNAs for both PKC alpha and nPKC epsilon was observed in association with in vitro differentiation.

Transgenic mice susceptible to poliovirus.

Poliovirus-sensitive transgenic mice were produced by introducing the human gene encoding cellular receptors for poliovirus into the mouse genome. Expression of the receptor mRNAs in tissues of the transgenic mice was analyzed by using RNA blot hybridization and the polymerase chain reaction. The human gene is expressed in many tissues of the transgenic mice just as in tissues of humans. The transgenic mice are susceptible to all three poliovirus serotypes, and the mice inoculated with poliovirus show clinical symptoms similar to those observed in humans and monkeys. Rabbit antipoliovirus serum detects the antigens mainly in motor neurons in the anterior horn of the spinal cord and in nerve cells in the medulla oblongata and pons of the paralyzed transgenic mice. Therefore, cell types sensitive to poliovirus in the central nervous system of the transgenic mice appear to be identical to those of humans and monkeys. Furthermore, many more doses of oral poliovirus vaccine strains than of the virulent strains are required to cause paralysis in the transgenic mice. This may reflect the observation that the virulent strain multiplies more efficiently in the central nervous system than the attenuated strain. Thus, the transgenic mice may become an excellent new animal model to study molecular mechanisms of pathogenesis of poliovirus and to assess oral poliovirus vaccines.

Elimination of paternal mitochondrial DNA in intraspecific crosses during early mouse embryogenesis.

To examine whether mtDNA is uni- or biparentally transmitted in mice, we developed an assay that can detect sperm mtDNA in a single mouse embryo. In intraspecific hybrids of Mus musculus, paternal mtDNA was detected only through the early pronucleus stage, and its disappearance co-incided with loss of membrane potential in sperm-derived mitochondria. By contrast, in interspecific hybrids between M. musculus and Mus spretus, paternal mtDNA was detected throughout development from pronucleus stage to neonates. We propose that oocyte cytoplasm has a species-specific mechanism that recognizes and eliminates sperm mitochondria and mtDNA. This mechanism must recognize nuclearly encoded proteins in the sperm midpiece, and not the mtDNA or the proteins it encodes, because sperm mitochondria from the congenic strain B6.mtspr, which carries M. spretus mtDNA on background of M. musculus (B6) nuclear genes, were eliminated early by B6 oocytes as in intraspecific crosses. We conclude that cytoplasmic genomes are transmitted uniparentally in intraspecific crosses in mammals as in Chlamydomonas and that leakage of parental mtDNA is limited to interspecific crosses, which rarely occur in nature.

Poliovirus-sensitive transgenic mice as a new animal model.

Transgenic mice susceptible to poliovirus infection were produced by introducing a human gene encoding cellular receptors for poliovirus into the mouse genome. Expression of receptor mRNAs in tissues of the transgenic mice was analysed using Northern blot hybridization. The results indicate that the human gene is expressed in many tissues of the transgenic mice just as in human tissues, and that the amount of the receptor mRNAs varies from tissue to tissue. The transgenic mice inoculated with poliovirus by any of the routes tested in this study show clinical symptoms similar to those in humans and monkeys, although the sensitivity of the mice depended on the inoculation route. In any route, the virulent Mahoney strain of type 1 poliovirus is much more virulent than the attenuated Sabin 1 strain in the transgenic mice. These observations suggest that the transgenic mice become an excellent new animal model for studying molecular mechanisms of pathogenesis of poliovirus and for assessing oral poliovirus vaccines.

Establishment of antigen-specific IgE transgenic mice to study pathological and immunobiological roles of IgE in vivo.

We have established transgenic mice that carry the genes coding for heavy and light chains of TNP-specific IgE. They produced high titers of TNP-specific IgE (20-40 microg/ml in serum) and their mast cells were heavily loaded with IgE. The level of FcepsilonRI expression on their mast cells was 6-8 times higher than that in non-transgenic littermates. The expression of low-affinity IgE receptor FcepsilonRII (CD23) on splenic B cells was also 6-8 times higher in the transgenic mice. Consistent with this, substantial amounts of IgE were detected on B cells in the transgenic mice. When challenged with i.v. administration of the corresponding antigen, the transgenic mice exhibited systemic anaphylactic symptoms such as a drastic drop of body temperature and extravasation of administered dye. Biphasic (immediate and delayed) ear swelling response was also elicited in a TNP-specific manner by epicutaneous antigen challenge without any prior sensitization. Thus, IgE produced in the transgenic mice was found to be biologically active to induce both local and systemic allergic reactions in vivo upon the challenge of the corresponding antigen. Taken together, the antigen-specific IgE transgenic mice established for the first time in this study appear to provide an attractive model system to study the pathological roles of IgE in acute and chronic phases of allergic inflammation as well as their immunobiological roles in vivo. They may also be useful to develop novel therapeutic strategies for atopic disorders.

Characterization of three different transgenic mouse lines that carry human poliovirus receptor gene--influence of the transgene expression on pathogenesis.

Three transgenic mouse lines, ICR-PVRTg1, ICR-PVRTg5, and ICR-PVRTg21, which are susceptible to poliovirus, have been established by introducing the human gene for poliovirus receptor (PVR) into the genome of mouse strain ICR. Genetic characterizations of the PVR gene were carried out on these mouse lines to define the approximate copy number, insertion site, and expression of the transgene in the central nervous system (CNS). The transgene was integrated in the chromosome 4, 12, and 13 of ICR-PVRTg1, ICR-PVRTg5 and ICR-PVRTg21 mice, respectively, and was stably transmitted to progeny mice. ICR-PVRTg1 appeared to have the most abundant copy numbers of the transgene and showed the highest level of PVR mRNA and membrane associated PVR protein in the CNS among the three mouse lines. Those in ICR-PVRTg21 and ICR-PVRTg5 were at intermediate and lowest levels, respectively. In the CNS, PVR mRNA was detected at high levels only in neurons of the spinal cord and brain stem where poliovirus can replicate, suggesting that the PVR mRNA expression confers cell specificity to poliovirus in the CNS. ICR-PVRTg1 and ICR-PVRTg5 showed the highest and the lowest sensitivity to poliovirus, respectively, whereas ICR-PVRTg21 was in-between. These results may suggest that poliovirus sensitivity of the mice is attributed to relative levels of PVR expression.

Regulation of mouse kidney tubular epithelial cell-specific expression of core 2 GlcNAc transferase.

A mouse gene, Gsl5, controls the expression of Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Galbeta1-3)Gb4Cer and its precursor glycolipids in the kidney by regulating transcription of beta-1,6-GlcNAc transferase. Here we report that Gsl5 controls the expression of the core 2 structure [GlcNAcbeta1-6(Galbeta1-3)GalNAcalpha1-Ser/Thr] of glycoproteins as well as the glycolipid, GlcNAcbeta1-6(Galbeta1-3)GalNAcbeta1-3Galalpha1-4Galbeta1-4Glcbeta1-ceramide. Immunohistochemical studies using an anti-(core 2-Lex) monoclonal antibody demonstrated that lysosome-like vesicles of proximal tubule cells were clearly stained in a Gsl5 wild type mouse, but not in a Gsl5 mutant strain of mice. Western blotting of microsomal fractions of kidney tissue with the same antibody confirmed the histological findings. In situ hybridization with an antisense probe to the kidney-specific mRNA demonstrated that the mRNA is localized at proximal tubule-cells in the cortex adjacent to the medulla, but not detected in glomeruli nor in collecting duct cells in the medulla. The results obtained by immunohistological staining and in situ hybridyzation are compatible and lead to the conclusion that the kidney specific mRNA is expressed in a proximal tubular cell specific manner and produces core 2 GlcNAc transferase responsible for the production of glycoproteins localized at vesicles in the proximal tubular cells. Glycosylation regulated by Gsl5 gene may modify functions of membrane glycoproteins in proximal tubular cells.

Differential requirement of the cytoplasmic subregions of gamma c chain in T cell development and function.

The common cytokine receptor gamma chain (gammac), a shared component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, is critical for the development and function of lymphocytes. The cytoplasmic domain of gammac consists of 85 aa, in which the carboxyl-terminal 48 aa are essential for its interaction with and activation of the Janus kinase, Jak3. Evidence has been provided that Jak3-independent signals might be transmitted via the residual membrane-proximal region; however, its role in vivo remains totally unknown. In the present study, we expressed mutant forms of gammac, which lack either most of the cytoplasmic domain or only the membrane-distal Jak3-binding region, on a gammac null background. We demonstrate that, unlike gammac or Jak3 null mice, expression of the latter, but not the former mutant, restores T lymphopoiesis in vivo, accompanied by strong expression of Bcl-2. On the other hand, the in vitro functions of the restored T cells still remained impaired. These results not only reveal the hitherto unknown role of the gammac membrane-proximal region, but also suggest the differential requirement of the cytoplasmic subregions of gammac in T cell development and function.

Transgenic mice carrying the human poliovirus receptor: new animal models for study of poliovirus neurovirulence.

Recombinant viruses between the virulent Mahoney and attenuated Sabin 1 strains of poliovirus type 1 were subjected to neurovirulence tests using a transgenic (Tg) mouse line, ICR-PVRTg1, that carried the human poliovirus receptor gene. The Tg mice were inoculated intracerebrally with these recombinant viruses and observed for clinical signs, histopathological lesions, and viral antigens as parameters of neurovirulence of the viruses. These parameters observed in the Tg mice were different for different inoculated viruses. Dose-dependent incidences of paralysis and of death were observed in the Tg mice inoculated with any viruses used. This indicates that values of 50% lethal dose are useful to score a wide range of neurovirulence of poliovirus. The neurovirulence of individual viruses estimated by the Tg mouse model had a strong correlation with those estimated by monkey model. Consequently, the mouse tests identified the neurovirulence determinants on the genome of poliovirus that had been identified by monkey tests. In addition, the mouse tests revealed new neurovirulence determinants, that is, different nucleotides between the two strains at positions 189 and 21 and/or 935 in the 5'-proximal 1,122 nucleotides. The Tg mice used in this study may be suitable for replacing monkeys for investigating poliovirus neurovirulence.

Efficient conditional transgene expression in hepatitis C virus cDNA transgenic mice mediated by the Cre/loxP system.

Conditional gene expression has greatly facilitated the examination of the functions of particular gene products. Using the Cre/loxP system, we developed efficient conditional transgene activation of hepatitis C virus (HCV) cDNA (nucleotides 294-3435) in transgenic mice. Efficient recombination was observed in transgenic mouse liver upon intravenous administration of adenovirus that expresses Cre DNA recombinase. After transgene activation, most hepatocytes were stained with anti-core polyclonal antibody, and 21-, 37-, and 64-kDa proteins were detected by Western blot analysis in liver lysates using anti-core, E1, and E2 monoclonal antibodies, respectively. Serum core protein was detected in transgenic mice 7 days after transgene activation with concurrent increases in serum alanine aminotransferase levels. Subsequently, an anti-core antibody response was detected 14 days after infection. Furthermore, a CD4 and CD8 positive cell depletion assay normalized both the serum alanine aminotransferase increases and pathological changes in the liver. These results suggest that HCV proteins are not directly cytopathic and that the host immune response plays a pivotal role in HCV infection. Thus, this HCV cDNA transgenic mouse provides a powerful tool with which to investigate the immune responses and pathogenesis of HCV infection.

Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9.

Caspases are essential components of the mammalian cell death machinery. Here we test the hypothesis that Caspase 9 (Casp9) is a critical upstream activator of caspases through gene targeting in mice. The majority of Casp9 knockout mice die perinatally with a markedly enlarged and malformed cerebrum caused by reduced apoptosis during brain development. Casp9 deletion prevents activation of Casp3 in embryonic brains in vivo, and Casp9-deficient thymocytes show resistance to a subset of apoptotic stimuli, including absence of Casp3-like cleavage and delayed DNA fragmentation. Moreover, the cytochrome c-mediated cleavage of Casp3 is absent in the cytosolic extracts of Casp9-deficient cells but is restored after addition of in vitro-translated Casp9. Together, these results indicate that Casp9 is a critical upstream activator of the caspase cascade in vivo.