Steatosis induced by the accumulation of apolipoprotein A-I and elevated ROS levels in H-ras12V transgenic mice contributes to hepatic lesions

Hepatic steatosis is considered to have an important impact on liver tumorigenesis, despite a lack of clear experimental evidence. Histopathological analysis of H-ras12V transgenic mice showed liver lesions on a steatosis background had significantly higher incidence than on a non-steatosis background. Further investigation showed that apolipoprotein A-I was elevated and accumulated around fatty vacuoles. This elevated level of apolipoprotein A-I was coupled with an elevated level of H-ras12V protein and ROS. In conclusion, our results suggest that the expression of H-ras12V oncogene leads to elevated levels of ROS and apolipoprotein A-I that contribute to steatosis. The steatosis, in turn, promotes the development of hepatic lesions induced by H-ras12V oncogene.     

Constitutive expression of p21H-ras(Val12) in pyramidal neurons results in reorganization of mouse neocortical afferents

The effect of constitutive expression of p21H-ras(Val12) in pyramidal neurons upon the establishment of afferent input has been investigated in the primary somatosensory cortex of transgenic mice. In these animals, relevant transgene expression is confined to cortical pyramidal neurons and starts postnatally at a period when neuronal morphogenesis has been largely completed. We have shown recently that overexpression of p21H-ras(Val12) in these cells results in considerable enlargement of their size and consequently in expansion of the cortex. In the present study we demonstrate that the density of terminals representing intra- or interhemispheric afferents within cortical layers II/III, however, is only slightly decreased. The density of thalamocortical boutons within layer IV is even higher and the number of afferent contacts to transgenic pyramidal neurons is significantly increased compared to the wild-type. The number of catecholaminergic and cholinergic terminals is augmented proportionally to cortical size or even overproportionally, respectively. Along intercortical and striatal fibers arising from p21H-ras(Val12)-expressing pyramidal neurons, frequency of varicosities is significantly increased, but remains unchanged on cortical cholinergic and catecholaminergic axons originating from "nontransgenic" neurons. Additionally, a higher number of multiple synaptic bodies are found in transgenic mice, suggesting subtle effects on synaptic plasticity. It is concluded that the enlargement of pyramidal neurons due to transgenic expression of p21H-ras(Val12) is paralleled by significant changes in the quantity and pattern of afferent connections. Moreover, expression of p21H-ras(Val12) in pyramidal cells induces an enhanced establishment of efferent boutons.     

A promoter that drives gene expression preferentially in male transgenic rats

Gender-preferential gene expression is a widespread phenomenon in humans. It is important to study how gender differences influence the pathogenesis of various diseases and response to specific drugs. The aim of this study is to determine if the mouse albumin enhancer/promoter may serve as the promoter to introduce gender-preferential gene expression in transgenic animals. We created four independent transgenic rat lines in which the human C-reactive protein transgene was under the control of mouse albumin enhancer/promoter. Quantitative real time RT-PCR analysis showed that transgene expression in the liver of male rats was significantly higher than transgene expression in the female rats (P < 0.05).There was a 5.3-fold (male/female) difference in line-519, and a 12.2-fold (male/female) difference in line-488. Enzyme-linked immunosorbent assay showed that the serum of male transgenic rats had a 13- to 679-fold difference at the protein level on transgene production compared with female transgenic rats. The male-to-female difference in gene expression was 10- to 17-fold in the liver of transgenic rats. Orchiectomy dramatically reduced protein production from the transgene in the liver. Testosterone administration into female rats did not increase the transgene expression, but estrogen administration into the male rats reduced transgene expression. This study provides a valuable tool for investigating the pathological roles of genes that are expressed in a gender-preferential manner in human disease.     

Influence of a nonfragile FHIT transgene on murine tumor susceptibility

FHIT, at a constitutively active chromosome fragile site, is often a target of chromosomal aberrations and deletion in a large fraction of human tumors. Inactivation of murine Fhit allelessignificantly increases susceptibility of mice to spontaneous and carcinogen-induced tumorigenesis. In this study, transgenic mice, carrying a human FHIT cDNA under control of the endogenous promoter, were produced to determine the effect of Fhit expression, from a nonfragile cDNA transgene outside the fragile region, on carcinogen-induced tumor susceptibility of wildtype and Fhit heterozygous mice. Mice received sufficient oral doses of N-nitrosomethybenzylamine (NMBA) to cause forestomach tumors in >80% of nontransgenic control mice. Although the level of expression of the FHIT transgene in the recombinant mouse strains was much lower than the level of endogenous Fhit expression, the tumor burden in NMBA-treated male transgenic mice was significantly reduced, while female transgenic mice were not protected. To determine if the difference in protection could be due to differences in epigenetic changes at the transgene loci in male versus female mice, we examined expression, hypermethylation and induced re-expression of FHIT transgenes in male and female mice or cells derived from them. The transgene was methylated in male and female mice and in cell lines established from male and female transgenic kidneys, the FHIT locus was both hypermethylated and deacetylated. It is likely that the FHIT transgene is more tightly silenced in female transgenic mice, leading to a lack of protection from tumor induction.     

Tissue-specific and hormonally regulated expression of a rat alpha 2u globulin gene in transgenic mice.

To investigate the tissue-specific and hormonal regulation of the rat alpha 2u globulin gene family, we introduced one cloned member of the gene family into the mouse germ line and studied its expression in the resulting transgenic mice. Alpha 2u globulingene 207 was microinjected on a 7-kilobase DNA fragment, and four transgenic lines were analyzed. The transgene was expressed at very high levels, specifically in the liver and the preputial gland of adult male mice. The expression in male liver was first detected at puberty, and no expression was detected in female transgenic mice. This pattern of expression is similar to the expression of endogenous alpha 2u globulin genes in the rat but differs from the expression of the homologous mouse major urinary protein (MUP) gene family in that MUPs are synthesized in female liver and not in the male preputial gland. We conclude that these differences between rat alpha 2u globulin and mouse MUP gene expression are due to evolutionary differences in cis-acting regulatory elements. The expression of the alpha 2u globulin transgene in the liver was abolished by castration and fully restored after testosterone replacement. The expression could also be induced in the livers of female mice by treatment with either testosterone or dexamethasone, following ovariectomy and adrenalectomy. Therefore, the cis-acting elements responsible for regulation by these two hormones, as well as those responsible for tissue-specific expression, are closely linked to the alpha 2u globulin gene.     

Transgenic mice to study T-cell receptor gene regulation and repertoire formation

Transgenic mice have been obtained with genes coding for an alpha beta T-cell receptor that recognizes the male-specific antigen H-Y in association with the Db class I major histocompatibility complex molecule. Most if not all of the T-cells express the beta chain encoded by the transgene and show allelic exclusion of endogenous beta genes. In contrast, the expression of the alpha transgene does not completely block rearrangement and formation of functional endogenous alpha genes. In H-2b transgenic female mice the transgenic T-cell receptor is functionally expressed on at least 30% of CD8+ peripheral T-lymphocytes as indicated by their ability to lyse male target cells. Also in transgenic H-2b male mice a large proportion of peripheral T-cells appear to express the transgenic receptor. However, these cells do not react with male target cells because they show only low level or no expression of CD8 cell interaction molecules. Tolerance is established in the male transgenic thymus through deletion of CD4+CD8+ immature thymocytes.     

Interplay between oncogenic K-Ras and wild-type H-Ras in Caco2 cell transformation

Mutation of RAS genes is one of the most common oncogenic alterations in cancer and acquisition of activating RAS mutations has been demonstrated to cause progression of colorectal adenoma to cancer. The aim of this study was to identify changes in the proteome of the intermediate-stage colorectal cancer cell line Caco2, induced by ectopic expression of two distinct RAS proteins, KRAS(V12) and HRAS(V12), in their mutated, constitutively active form. Using 2D-gel electrophoresis, followed by LC-MS/MS we identified almost 200 differentially expressed proteins in pair-wise comparisons of Caco2 vs Caco2-KRAS(V12) and Caco2 vs Caco2-HRAS(V12). Although many of the affected proteins were unique for each pair, there were also substantial similarities. Interestingly, transformation by the mutant KRAS(V12) gene resulted in elevated expression levels and activity of endogenous H-ras protein. Silencing the latter with a specific RNAi reversed several proteomic changes observed in KRAS(V12)-transformed cells, suggesting that oncogenic K-ras partly exerts its effects through endogenous H-ras activation. Alterations in the expression of cytoskeletal and cell adhesion proteins, caused by HRAS siRNA treatment, correlated with a reduction in the invasive properties of Caco2-KRAS(V12) cells. Our data suggest a novel interplay between K-ras and H-ras, with possible implications for colorectal carcinogenesis.     

原发性肝癌H-ras12V小鼠 肠道微生态的初步研究

目的探索原发性肝癌小鼠(H-ras12V小鼠)肠道微生态的特异性。方法设立7月龄的H-ras12V小鼠为实验组,C57BL/6J常态小鼠为对照组。小鼠在标准饮食下饲养2个月。于实验第8周无菌收集小鼠粪便,进行V3-V4高可变区16SrDNA基因组测序,分析两组小鼠肠道菌群多样性和组成的差异。结果肝癌小鼠成瘤率为100%。α多样性:稀释曲线趋于平缓,样品测序数据量较合理。β多样性:通过Anosim分析确定两组组间差异显著大于组内差异(R=0.2857,P=0.0037)。菌群结构:门水平优势菌群有拟杆菌门、厚壁菌门、变形菌门等,属水平优势菌群有Barnesiella、Alloprevotella、拟杆菌属等。显著性差异物种:通过Kruskal-Wallis test分析,发现拟杆菌属等7个属差异有统计学意义(P0.05)。结论原发性肝癌H-ras12V小鼠肠道微生态多样性和菌群结构有显著差异,其中拟杆菌属及其特殊结构鞘脂与肝硬化及肿瘤关系密切,可能与肝癌存在联系。     

Transgenerational transmission of bovine satellite DNA in transgenic mice

Genetical, cytogenetical and molecular analysis was made for 5 generations of mice transgenic for bovine satellite DNA (Sat). In all cases transgenic mice were generated by crosses of transgenic males and females with normal (CBA x C57B1) mice. No abnormalities in the founder development were noticed. A normal (near 50 %) ratio of transgenic and nontransgenic offsprings was observed in blastocysts. However, profound differences occurred in the rate of transgene bearing offsprings, depending on the sex of grandparents rather than of parents. The grandfather Sat transmission resulted in the appearance of 0-52.4 % transgenic grandchildren, whereas the grandmother transmission ended in the theoretically expected rate. This means that stabilization of transsatellite took place upon the female germ line transmission (a positive grandmother effect). It is essential that in hemizygous transsatellite mice Sat integration led to the occurrence of mammary tumors, inflammation of uterine horns, and infringement of mother care of transgenic females. Simultaneous FISH and G-banding showed Sat to be localized in the internal region of chromosome 12 near Pax 9 and Brms 11 genes. Commonly, these genes are implicated in tumorigenesis as their expression decreases. Thus, a kind of silencing effect of these genes' expression may be supposed.     

Mice expressing mutant myosin heavy chains are a model for familial hypertrophic cardiomyopathy.

BACKGROUND: Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease characterized by ventricular hypertrophy, myocellular disarray, arrhythmias, and sudden death. Mutations in several contractile proteins, including cardiac myosin heavy chains, have been described in families with this disease, leading to the hypothesis that HCM is a disease of the sarcomere. MATERIALS AND METHODS: A mutation in the myosin heavy chain (Myh) predicted to interfere strongly with myosin's binding to actin was designed and used to create an animal model for HCM. Five independent lines of transgenic mice were produced with cardiac-specific expression of the mutant Myh. RESULTS: Although the mutant Myh represents a small proportion (1-12%) of the heart's myosin, the mice exhibit the cardiac histopathology seen in HCM patients. Histopathology is absent from the atria and primarily restricted to the left ventricle. The line exhibiting the highest level of mutant Myh expression demonstrates ventricular hypertrophy by 12 weeks of age, but the further course of the disease is strongly affected by the sex of the animal. Hypertrophy increases with age in female animals while the hearts of male show severe dilation by 8 months of age, in the absence of increased mass. CONCLUSIONS: The low levels of the transgene protein in the presence of the phenotypic features of HCM suggest that the mutant protein acts as a dominant negative. In addition, the distinct phenotypes developed by aging male or female transgenic mice suggest that extragenic factors strongly influence the development of the disease phenotype.     

Boar proacrosin expressed in spermatids of transgenic mice does not reach the acrosome and disrupts spermatogenesis

Transgenic mice that express boar proacrosin were produced to examine mechanisms for targeting hydrolytic enzymes to the acrosome. A 2.3 kb transgene was constructed by ligating the cDNA for boar preproacrosin with the mouse protamine 2 promoter region. Six founder mice that incorporated the transgene were identified by polymerase chain reaction and Southern blot analysis. Northern blots indicated that the two male founders (Ac.2 and Ac.5) and male progeny from three female founders (Ac.3, Ac.4, Ac.6) expressed the transgene mRNA in testis, but not in somatic tissues. In these transgenic animals boar proacrosin was detected by immunohistochemistry in condensing spermatids, but was not localized in the acrosome. This acrosomal targeting defect of the transgene product may result from its delayed expression during the later steps of haploid differentiation. Furthermore, both male founders and all Ac.4 and Ac.6 males were infertile, as determined by multipe matings for at least 2 months. Ac.3 males were either infertile or rarely transmitted the transgene to their offspring The infertile males mated, produced copulatory plugs, and had seminal vesicle weights and testosterone levels within the normal range. However, they produced significantly fewer spermatozoa and had lower testis weights than controls. Although the mitotic and meiotic phases of spermatogenesis appeared normal by histological criteria, condensing spermatids were missing from most tubules, and multinucleated cells were present in the lumen of seminiferous tubules and in the epididymis. We hypothesize that boar proacrosin which fails to reach the acrosome is activated in these transgenic mice, and that its proteolytic activity disrupts spermatogenesis during spermatid formation. © 1996 Wiley-Liss, Inc.     

The human growth hormone transgene: expression in hemizygous and homozygous mice

Female transgenic mice carrying the mouse metallothionein-I/human growth hormone (hGH) fusion gene are sterile. Transmission of the transgene has been limited to the male germ line, resulting in the production of hemizygous (He) progeny containing only a single (paternal) copy of the gene. Using ovary transfer, we have developed procedures for producing homozygous (Ho) TG mice, viz., male TG mice were mated with control (non-TG) females carrying ovaries donated by female TG mice. In both He and Ho TG animals, serum levels of hGH were higher (1.5-fold) in males than in females, tended to decrease with age of the animal, and were increased (about 5-fold) by zinc induction. However, in comparison to He animals of the same sex, the Ho TG mice attained a greater body weight and had more than 2-fold higher levels of liver hGH-mRNA and serum hGH, both under basal conditions and in response to zinc induction. That is, the expression of the transgene was qualitatively similar in He and Ho TG mice, but the level of transgene activity was greater in the Ho animals. We interpret this to indicate that both copies (maternal and paternal) of the transgene were active and expressed additively (or cooperatively) in the Ho TG animal.     

Haploid male germ cells show no susceptibility to transformation by simian virus 40 large tumour antigen in transgenic mice.

Cell-type specific tumorigenesis can be induced in transgenic mice by the directed expression of simian virus 40 (SV 40) large tumour antigen (TAg). In an attempt to determine the susceptibility of haploid male germ cells to neoplastic transformation by this oncogene, transgenic mice were generated that harboured a chimeric gene composed of the SV40 T antigen genes fused to the 2.3-kb 5' flanking sequences of the rat proacrosin gene. It was previously shown that this regulatory sequence is able specifically to direct the expression of CAT reporter gene in male germ cells with the onset of translation in early haploid male germ cells. The transgene showed regulated expression in male germ cells. Although T antigen immunostaining was detected specifically in spermatids, no testicular pathology was observed. This indicates that spermatids show no susceptibility to transformation by oncogene TAg. However, in about 10% of animals of two independent transgenic lines, we could find non-testicular tumours in abdomen with a sarcoma-like structure in advanced age which showed SV40 TAg expression.     

Germ cell specific promoter drives ectopic transgene expression during embryogenesis

In this study, we used the male germ cell-specific phosphoglycerate kinase 2 (Pgk2) promoter to generate Pgk2Cre transgenic mice to allow investigation of genes critically involved in meiosis. The Pgk2 promoter had been used previously to target transgene expression to spermatocytes and spermatids in several laboratories including ours. In several Cre targeting experiments using other promoters, ectopic Cre expression had been observed, but the timing and extent of this expression was not analyzed. We demonstrate that in adult mice the Pgk2Cre transgene is expressed specifically in spermatocytes and spermatids, as expected. However, in offspring from matings of Pgk2Cre mice and an H19loxP indicator strain, we discovered that recombination events had occurred in several, but not all, tissues to varying extents. The lacZ-loxP transgenic indicator strain was next used to uncover ectopic Cre expression even in single cells, which indicated that the Pgk2Cre transgene is expressed between days 11 and 15 during embryogenesis in several tissues and organs. Using an RT PCR assay we were unable to detect endogenous Pgk2 mRNA during embryogenesis or in adult tissues other than testis. In conclusion, the Pgk2 promoter is a valid choice for targeting gene expression to meiotic male germ cells, since transient ectopic expression is unlikely to have a discernable effect in most studies, but it may be inappropriate for utilization with Cre recombinase.     

The human ubiquitin C promoter drives selective expression in principal neurons in the brain of a transgenic mouse line

The specificity of promoters used to drive the expression of proteins of interest is a crucial determinant of transgenesis. Numerous strategies have been developed to restrict expression on a certain cell population. On the other hand it has also remained challenging to obtain ubiquitous expression of transgenes which is needed for example to generate recombination reporter mice or to induce expression by recombination mediated excision of STOP-cassettes. We have generated transgenic mice with the expression of nuclear β-galactosidase driven by the human ubiquitin C promoter thought to mediate ubiquitous expression. However, in the brains of these transgenic mice the expression of the transgene was strikingly limited to principal neurons, while no expression was detected in interneurons or glial cells. These results indicate that the human ubiquitin C promoter might be useful to selectively target projections neurons of the brain.     

The Murine Stem Cell Virus Promoter Drives Correlated Transgene Expression in the Leukocytes and Cerebellar Purkinje Cells of Transgenic Mice

The murine stem cell virus (MSCV) promoter exhibits activity in mouse hematopoietic cells and embryonic stem cells. We generated transgenic mice that expressed enhanced green fluorescent protein (GFP) under the control of the MSCV promoter. We obtained 12 transgenic founder mice through 2 independent experiments and found that the bodies of 9 of the founder neonates emitted different levels of GFP fluorescence. Flow cytometric analysis of circulating leukocytes revealed that the frequency of GFP-labeled leukocytes among white blood cells ranged from 1.6% to 47.5% across the 12 transgenic mice. The bodies of 9 founder transgenic mice showed various levels of GFP expression. GFP fluorescence was consistently observed in the cerebellum, with faint or almost no fluorescence in other brain regions. In the cerebellum, 10 founders exhibited GFP expression in Purkinje cells at frequencies of 3% to 76%. Of these, 4 mice showed Purkinje cell-specific expression, while 4 and 2 mice expressed GFP in the Bergmann glia and endothelial cells, respectively. The intensity of the GFP fluorescence in the body was relative to the proportion of GFP-positive leukocytes. Moreover, the frequency of the GFP-expressing leukocytes was significantly correlated with the frequency of GFP-expressing Purkinje cells. These results suggest that the MSCV promoter is useful for preferentially expressing a transgene in Purkinje cells. In addition, the proportion of transduced leukocytes in the peripheral circulation reflects the expression level of the transgene in Purkinje cells, which can be used as a way to monitor transgene expression properties in the cerebellum without invasive techniques.