用地高辛标记探针检测转人肾素基因小鼠

采用地高辛标记探针,建立了一种快速检测和筛选转入肾素基因小鼠的方法。对实验中杂交液的选择、杂交液中探针的浓度,预杂交时间,抗体孵育前封闭时间和洗膜条件等影响因素进行了系统观察与分析,从而确定了以地高辛标记探针检测转入肾基因小鼠方法中的最佳实验条件。结果表明,该方法不仅安全,快速,而且灵敏度高,稳定性好。     

Skeletal myopathy in transgenic mice carrying human prototype c-Ha-ras gene

Skeletal myopathy was found in almost all-transgenic mice carrying the human prototype c-Ha-ras gene (rasH2 mouse). Microscopically, variation of the muscle fiber size, centrally placed nuclei, regenerating fibers, and interstitial fibrosis were evident; hyalinization and necrosis were sometimes observed in the skeletal muscle (femoralis and pectoralis) of the rasH2 mice. Inflammatory changes in the skeletal muscle or abnormality of adjacent peripheral nerve were not observed. The features were essentially similar to those of muscular dystrophy. Although the severity was relatively mild compared to 34-week-old rasH2 mice, the skeletal myopathy was also observed in younger male (10 weeks of age) rasH2 mice. In nontransgenic littermates, skeletal myopathy was not observed. The mRNA of human c-Ha-ras product was detected in femoral muscle from the rasH2 mice by RT-PCR. In conclusion, these data suggest that skeletal myopathy is occurring in almost all rasH2 mice. Integration of c-Ha-ras gene is thought to be crucial to pathogenesis of skeletal myopathy in the rasH2 mice. Further characterization of the muscular lesion and its pathogenesis are needed to explore the possibility of rasH2 mouse becoming a new model for muscular dystrophy.     

Mice transgenic for the human LCAT gene

The enzyme LCAT (Lecithin:cholesterol acyltransferase E.C. 2.3.1.43.) plays a central role in lipoprotein metabolism. It is secreted by the liver and catalyzes the transfer of a fatty acid from the 2-position of lecithin to the 3-beta-OH group of free cholesterol to produce cholesterol ester and lysolecithin.     

Lymphocyte infiltration into cerebellum in transgenic mice carrying human IL-2 gene

We created transgenic mice with an intact human genomic interleukin-2 gene (gIL-2) or a mouse metallothionein-I promoter-human IL-2 chimeric gene (MTgIL-2). Nine (2 gIL-2 and 7 MTgIL-2 transgenics) out of 12 transgenic mice which were obtained independently had motor ataxic symptoms. All transgenic offspring of the symptomatic founders showed the same symptoms as their transgenic parents. Morphological examination demonstrated perivascular lymphocyte accumulation in the cerebellar meninx which was followed by increased cell infiltration of neutrophils and monocytes in the destructive cerebellum of all transgenic mice. These findings suggest that the lymphocyte infiltration in the cerebellum is caused by the specific effect of the exogenously introduced human IL-2 gene.     

Expression of renin in large arteries outside the kidney revealed by human renin promoter/LacZ transgenic mouse

Renin plays a central role in controlling blood pressure as it catalyzes the first step in the production of angiotensin II. The aim of this study was to isolate fragments of the human renin (hREN) promoter able to direct tissue-specific and regulated expression of a LacZ reporter gene mimicking endogenous renin. We screened several hREN promoter/LacZ constructs for transgene expression in transient embryos at E15 when renin expression begins. We found that a 12-kb hREN promoter conferred high expression in the kidney at both embryonic and adult stages and that the transgene was expressed in the same cells as endogenous renin. We explored two pathophysiological models in which renin is stimulated and showed concomitant increases in beta-galactosidase and renin activities. In situ beta-galactosidase staining showed renin/transgene-expressing cells are recruited in the juxtaglomerular apparatus and in the afferent arterioles as well as in larger arteries outside the kidney. Using our model, renin expression in interlobular arteries was confirmed as being striped and, for the first time, expression of renin in larger arteries outside the kidney was shown. Therefore, this strain is a suitable model to investigate renin gene pathophysiological regulations in vivo.     

Strategies for expressing human antibody repertoires in transgenic mice

Repertoires of human antibodies can be created in transgenic mice carrying human immunoglobulin-gene loci in germline configuration. These ‘transloci’, introduced either as miniloci or as almost locus-sized regions, undergo rearrangement and hypermutation in mouse lymphoid tissue. Here, Marianne Brüggemann and Michael Neuberger review the use of such mice for raising antigen-specific human monoclonal antibodies, as well as their exploitation for studying regulatory aspects of antibody repertoire formation.     

Naso-maxillary deformity due to frontonasal expression of human transthyretin gene in transgenic mice

BACKGROUND: Retinoic acid, a metabolic product of retinol, is essential for craniofacial morphogenesis. Transthyretin (TTR) is a plasma protein delivering retinol to tissues. We produced several transgenic mouse lines using the human mutant TTR (hTTRMet30) gene to establish a mouse model of familial amyloidotic polyneuropathy. One of the lines showed an autosomal dominant inheritance of naso-maxillary deformity termed Nax. RESULTS: The Nax malformation was characterized by a hypoplastic developmental defect of the frontonasal region. Homozygous mice with higher transgene expressions showed more severe phenotypes, but a subline, in which the copy number and expression of the transgene was reduced, showed a normal phenotype, indicating that the hTTRMet30 expression caused the malformation. Nax mice began to express the hTTRMet30 gene in the nasal placode from embryonic day 10.5 (E10.5), which was 2 days earlier than in the other transgenic lines with a normal phenotype. Excessive cell death was observed in the nasal placode of the E10.5 Nax embryos. In addition, the forced expression of hTTRMet30 in the nasal placode of transgenic mice resulted in similar phenotypes. CONCLUSION: The expression of the hTTRMet30 gene in the nasal placode at E10.5 induced apoptotic cell death, leading to hypoplastic deformity in the frontonasal region.     

Functional expression of a human TCR{beta} gene in transgenic mice

A functionally rearranged TCRß (Tcrb) gene was isolatedfrom a cloned human T helper cell recognizing the CS.T3 epitopeof Plasmodium falciparum with HLA-DR2. Transgenic mice weregenerated by co-injection of the human gene together with themouse Tcrb enhancer. Analysis of transgenic mice shows thatthe functional Tcrb gene of xenogenic, i.e. human, origin exertsallelic exclusion of endogenous Tcrb genes. Cytofluorometricanalysis revealed expression of the human TCRß chainon virtually all thymocytes and peripheral T cells togetherwith endogenous TCRß chains and CD3 components. Nosurface expression of mouse TCRß chain or rearrangementof endogenous Tcr genes was detectable. Expression of the hybridreceptor causes a reduction in the number of thymocytes anda bias for CD4⁺CD8^– T cells in the thymus as comparedwith non-transgenic littermates. Peripheral transgenic T cellsmount a normal prollferative response against allogenelc targetsin mixed lymphocyte reactions. These results show that a hybridmouse/human TCR is able to pass positive and negative selectionin the thymus, and is functional in transgenic mice.     

Expression in brain sensory neurons of the transgene in transgenic mice carrying human tyrosine hydroxylase gene.

We have recently reported the production of transgenic (Tg) mice carrying the human tyrosine hydroxylase (TH) gene, and have described tissue-specific expression of the transgene in catecholaminergic (CAergic) neurons and adrenal glands. This paper describes the transgene expression in non-catecholaminergic (nCAergic) neurons in the brain of Tg mice by immunocytochemistry and in situ hybridization. In adult Tg mice, human TH was atypically expressed in the olfactory (typically, the anterior olfactory nucleus and pyriform cortex) and visual (typically, n. suprachiasmaticus and n. parabigeminalis) systems, in addition to typical CAergic neuron-rich nuclei in the brain. These results suggest the possibility that TH plays some novel roles in sensory systems.     

High-level, erythroid-specific expression of the human alpha-globin gene in transgenic mice and the production of human hemoglobin in murine erythrocytes

Using the dominant control region (DCR) sequences that flank the beta-globin gene locus, we have been able to achieve high-level expression of the human alpha-globin gene in transgenic mice. Expression in fetal liver and blood is copy number dependent and at levels comparable to that of the endogenous mouse alpha-globin genes. Transgenic fetuses with high-copy numbers of the transgene suffer severe anemia and die before birth. Using a construct with both the human alpha- and beta-globin genes and the beta-globin DCR, live mice with low-copy numbers were obtained. Both human globin genes are expressed at high levels in adult red cells to give human hemoglobin HbA in amounts equal to or greater than endogenous mouse hemoglobin. Expression of HbA in murine red cells is not accompanied by any increase in mean corpuscular volume (MCV) or mean corpuscular hemoglobin concentration (MCHC). However, these transgenic mice tend to have an increased number of reticulocytes in peripheral blood; consistent with some degree of hemolysis. Metabolic labeling experiments showed balanced mouse globin synthesis, but imbalanced human globin synthesis, with an alpha/beta biosynthetic ratio of approximately 0.6. Thus, these mice have mild anemia. These results are discussed with relation to the coordinate regulation of alpha- and beta-globin synthesis in erythroid tissues.     

Parent-specific expression of a human keratin 18/beta-galactosidase fusion gene in transgenic mice.

Insertion of a human keratin 18 (K18)-bacterial beta-galactosidase (LacZ) fusion gene into mice has led to a unique transgenic line in which expression of the transgene is subject to unusual germ line-specific, genomic imprinting effects. Fetal expression of the LacZ reporter gene depends on the gender of the transmitting parent, with appropriate expression in liver after maternal inheritance, and ectopic expression in retina and mesodermal tissues after paternal inheritance. This tissue-specific imprinting pattern is superimposed upon a basic expression pattern which is unaffected by parental inheritance. Insertion of the transgene has led to a recessive-lethal phenotype, with no parent-of-origin effects on viability, suggesting that the transgene has not inserted into an imprinted region of the genome. HpaII and HhaI methylation sensitive restriction sites within the bacterial LacZ reporter gene are completely methylated when activity of the maternally inherited transgene is detected in the fetal liver, and not methylated when the paternally inherited transgene is silent. Thus DNA methylation of LacZ is correlated with maternal inheritance and may be implicated in the genomic imprinting mechanism as others have suggested. However, in contrast to the commonly found correlation of expression and low DNA methylation, the LacZ gene was expressed in fetal liver when fully methylated. This result may imply the existence of negative regulatory activities that recognize the unmethylated LacZ gene.     

Augmentation of c-fos and c-jun expression in transgenic mice carrying the human T-cell leukemia virus type-Itax gene

To analyze the effect of human T-cell leukemia virus type I (HTLV-I) on cellular gene expression and its relation to tumorigenesis, two lines of transgenic mice carrying the long terminal repeat (LTR)-env-pX-LTR regions of the HTLV-I genome were produced. The transgene was expressed in many organs, including the brain, salivary gland, spleen, thymus, skin, muscle, and mammary gland. We found that the expression of the c-fos and c-jun genes, but not of thelyn and c-myc genes, was augmented 2- to 20-fold in histologically normal skin and muscle of these mice. The augmentation was tissue specific, suggesting the involvement of a cellular factor in the transgene action. In these mice, a three to seven times higher incidence of tumors was seen as compared with the control mice. These tumors included mesenchymal tumors, such as fibrosarcoma, neurofibroma, and lipoma, and adenocarcinomas of the mammary gland, salivary gland, and lung. The c-fos and c-jun genes were also activated in these tumors. The possible roles of elevated c-fos and c-jun gene expression in tumorigensis are discussed.The abbreviations used are ATL, adult T-cell leukemia; HTLV-I, human T-cell leukemia virus type I; IL-2, interleukin 2; IL-2R, interleukin 2 receptor; IL-6, interleukin 6; LTR, long terminal repeat.     

Lymphoid hyperplasia in transgenic mice over-expressing a secreted form of the human interleukin-1beta gene product

To evaluate the biological effects of over-expression of interleukin-1beta (IL-1beta) on the immune system we have generated transgenic mice, expressing the IL-1beta gene fused to a heterologous signal sequence under the control of the mouse immunoglobulin enhancer (Emu). A prominent hyperplasia and a disturbed microarchitecture of lymphoid tissues were observed in the transgenic mice. The CD4+ T cells in the hyperplastic lymphoid organs seemed to invade the majority of the lymphoid organs including B-cell restricted areas. Analysis of lymph node cells revealed an increased frequency of CD4+ CD44high CD62L- T cells and local secretion of IL-2 and IL-4, compatible with an elevated number of activated T cells. Furthermore, significant levels of human IL-1beta in sera and high concentrations of serum immunoglobulin G (IgG) were observed in the transgenic mice. The data suggest a role for IL-1beta in controlling lymphoid microarchitecture and, when over-expressed, breaking the threshold in T-helper-B-cell interaction.     

The diversity of antigen-specific monoclonal antibodies from transgenic mice bearing human immunoglobulin gene miniloci

An approach to the preparation of antigen-specific human monoclonal antibodies focuses on mice transgenic for human immunoglobulin gene miniloci; the V gene segments in these miniloci undergo productive rearrangement to yield mouse B cells expressing human immunoglobulin (Ig) chains. The general usefulness of this strategy hinges on whether it is feasible to obtain specific, high-affinity antibodies following immunization of such animals with a variety of antigens. To test this, we have investigated the antigen-specific responses in mice which carry human IgH miniloci (constaining just one or two V_H segments) instead of a functional mouse IgH locus. Although serum responses were relatively weak, monoclonal antibodies were readily obtained to all immunogens tested (a hapten, foreign proteins and human lymphoma cells). The affinities of two of the hapten-specific (anti-2-phenyl-oxazol-5-one) antibodies were 60 and 160 nM, values intermediate between what is typically obtained in the primary and secondary response of normal mice. Sequence analysis of the rearranged V genes revealed that junctional events made a major contribution to diversity with a considerable amount of apparently non-templated sequence at the V-D and D-J borders. Somatic hypermutation was also evident within the expressed V gene segments of many of the antigen-specific hybridomas. These findings augur well for the general usefulness of the transgenic approach for the isolation of high-affinity human antibodies to a wide range of antigens and suggests that the miniloci need not be particularly large.     

慢病毒载体法制备荧光素酶转基因小鼠

目的基于慢病毒介导的转基因方法制备荧光素酶（Luc）转基因小鼠。方法制备携带Luc基因的慢病毒,将其注入小鼠单细胞受精卵卵周隙以感染受精卵,然后将胚胎移植进假孕母鼠体内以获得仔鼠,应用小动物活体成像仪及PCR等在蛋白和DNA水平上筛选和鉴定Luc转基因小鼠。结果移植慢病毒隙感染后的成活胚胎63枚。将其移植至3只假孕母鼠,其中2只怀孕,共生仔鼠11只;利用小动物活体成像仪检测Luc表达,在蛋白水平证实11只F0代中,3只（命名为S1、S2、S3）表达Luc;DNA水平检测证实,3只Luc阳性小鼠的基因组中整合有外源转基因Luc。此外,Luc转基因首建鼠基因组中整合的Luc转基因可稳定遗传至下一代,并能正常表达。Luc转基因小鼠主要脏器如睾丸、肾脏、胃、肠、肺、脑、胸腺、肝脏和心脏等均可见Luc信号,但不同脏器间Luc强度有差异。结论成功制备Luc报告基因转基因小鼠。     

Expression of human full-length and minidystrophin in transgenic mdx mice: implications for gene therapy of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessivedisorder with a high spontaneous mutation rate and no effectivetreatment, hence development of genetic based therapies is animportant goal. We report that expression of a recombinant humanminidystrophin cDNA, compatible with current viral vectors,can significantly reduce the myopathic phenotype in transgenicmdx mice, even when expressed at only 20–30% of endogenousdystrophin levels at the sarcolemma. To the extent that dataobtained in mouse studies are applicable to DMD, the virtualelimination of morphological and biochemical abnormalities inthe mdx mouse supports the use of this cDNA in somatic genetherapy protocols for DMD.     

Anti-idiotype antibody induced cellular immunity in mice transgenic for human carcinoembryonic antigen

In the present study, we have analysed the detailed cellular immune mechanisms involved in tumour rejection in carcinoembryonic antigen (CEA) transgenic mice after immunization with dendritic cells (DC) pulsed with an anti-idiotype (Id) antibody, 3H1, which mimics CEA. 3H1-pulsed DC vaccinations resulted in induction of CEA specific cytotoxic T lymphocyte (CTL) responses in vitro and the rejection of CEA-transfected MC-38 murine colon carcinoma cells, C15, in vivo (Saha et al.,Cancer Res 2004; 64: 4995-5003). These CTL mediated major histocompatibility complex (MHC) class I-restricted tumour cell lysis, production of interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), and expression of Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL) in response to C15 cells. CTL used perforin-, FasL-, and TRAIL-mediated death pathways to lyse C15 cells, although perforin-mediated killing was the predominant lytic mechanism in vitro. The cytokines IFN-gamma and TNF-alpha synergistically enhanced surface expression of Fas, TRAIL receptor, MHC class I and class II on C15 cells that increased the sensitivity of tumour cells to CTL lysis. CTL activity generated in 3H1-pulsed DC immunized mice was directed against an epitope defined by the idio-peptide LCD-2, derived from 3H1. In vivo lymphocyte depletion experiments demonstrated that induction of CTL response and antitumour immunity was dependent on both CD4+ and CD8+ T cells. The analysis of splenocytes of immunized mice that had rejected C15 tumour growth revealed up-regulated surface expression of memory phenotype Ly-6C and CD44 on both CD4+ and CD8+ T cells. The adoptive transfer experiments also suggested the role of both CD4+ and CD8+ T cells in this model system. Furthermore, mice that had rejected C15 tumour growth, developed tumour-specific immunological memory.     

Regulation of renin gene expression in kidneys of eNOS- and nNOS-deficient mice

Our study aimed to assess the roles of nitric oxide derived from endothelium NO-synthase (eNOS) and macula densa neuronal NO-synthase (nNOS) in the regulation of renal renin expression. For this purpose renin mRNA levels and renin content were determined in kidneys of wild-type (wt), nNOS-deficient (nNOS-/-), and eNOS-deficient (eNOS-/-) mice, in which the renin system was suppressed by feeding a high-salt diet (NaCl 4%), or was stimulated by feeding a low-salt (NaCl 0.02%) diet together with the converting-enzyme inhibitor ramipril (10 mg kg(-1) day(-1)). In all mouse strains, renin mRNA levels were inversely related to the rate of sodium intake. In eNOS-/- mice renin mRNA levels and renal renin content were 50% lower than in wt mice at each level of salt intake, whilst in nNOS-/- mice renin expression was not different from wt controls. Administration of the general NO-synthase inhibitor nitro-L-arginine methyl ester (L-NAME, 50 mg kg(-1) day(-1)) to mice kept on the low-salt/ramipril regimen caused a decrease of renal renin mRNA levels in wt and nNOS-/- mice, but not in eNOS-/- mice. These observations suggest that neither eNOS nor nNOS is essential for up- or downregulation of renin expression. eNOS-derived NO appears to enhance renin expression, whereas nNOS-derived NO does not.