SV40 TAg转基因小鼠模型的建立及其表达

为了建立中枢神经系统肿瘤小鼠模型,构建了大鼠神经元特异性烯醇化酶(ratneu-ron-specificenolase,NSE)基因启动子调控下的猿猴病毒40大T抗原基因(simianvirus40largeTantigengene,SV40TAg)转基因载体,通过受精卵雄原核显微注射的方法制备转基因小鼠。PCR鉴定转基因小鼠的基因型;RT-PCR和Northern印迹检测转基因阳性鼠中SV40TAgRNA水平的表达及其组织特异性;免疫组化检测其蛋白质水平的表达。经显微注射共获得9只首代转基因阳性鼠(首建者,Founder小鼠),其中2例出生时即发生神经干细胞来源的肿瘤,其他Founder小鼠经繁育后共建立了5个SV40TAg转基因小鼠系,其中有4个系检测到SV40TAgRNA水平的表达且特异性地表达于脑组织,但未检测到蛋白质水平的表达。研究表明NSE启动子活性具有较强的组织特异性,并起始于小鼠胚胎发育期;SV40TAg具有明显的致癌作用,且SV40TAg诱发的神经系统肿瘤易造成转基因小鼠早期死亡。     

四环素调控SV40Tag转基因小鼠模型的建立

目的构建四环素调控的SV40T转基因小鼠模型。方法同时显微注射外源基因p205-rtTA-C3和pTRE-Tag至FVB小鼠原核,注射受精卵移植到同期发情的假孕受体出生个体,经PCR和Southern检测获得阳性转基因小鼠。结果经PCR结合Southern检测得到rtTA和Tag双阳性转基因小鼠一只,rtTA单阳性两只和Tag单阳性一只。结论通过饮水给与四环素的双阳性小鼠可在卵巢中检测到Tag mRNA的表达。     

Pin1在皮肤组织中的表达以及可诱导转基因小鼠模型的建立

目的观察Pin1在皮肤中的表达情况,构建Pin1在皮肤中可诱导表达的转基因小鼠模型。方法将小鼠Pin1基因克隆到改造过的可与Myc标签蛋白融合的p TRE2载体中,并将线性化的DNA通过显微注射的方式构建TRE-Pin1小鼠。结果成功获得TRE-Pin1转基因首建鼠,该小鼠与上皮特异的K14-rt TA转基因小鼠配繁,获得Pin1在皮肤上皮特异性可诱导表达的双转基因鼠;通过将多西霉素(又名强力霉素,Doxycycline)加入饮水的方式诱导Pin1基因的表达,并通过Western blot,免疫组织化学等方式证明了Pin1蛋白在皮肤上皮中能特异性地过表达。我们还发现内源的Pin1在皮肤中主要表达于上皮细胞。结论成功构建了Pin1在皮肤中可诱导表达的转基因小鼠模型,为后续研究Pin1在皮肤中的功能奠定基础。     

人sCD40L转基因小鼠模型的构建

目的研究阻断CD40-CD40L共刺激信号通路对移植皮肤免疫排斥反应的影响。方法通过RT-PCR技术克隆了呈可溶性表达的CD40L分子胞外区(sCD40L),利用K14启动子构建了sCD40L皮肤特异性表达载体,并利用该载体制备了转基因小鼠。结果所克隆的CD40L胞外区片段其大小及序列符合预期;以哺乳动物表达载体PCI为骨架,通过DNA重组,获得了含K14启动子和sCD40L编码区的皮肤特异性表达载体K14-sCD40L;通过显微注射和胚胎移植,PCR筛选检测:49只G0代小鼠有1只小鼠扩增出特异性条带,阴性对照无条带。结论成功建立sCD40L转基因阳性小鼠。     

小鼠转基因的可诱导表达

转基因小鼠是研究基因表达调控及表达产物生物学效应的最佳体系之一。小鼠转基因可诱导表达系统的研究和应用为实现对转基因表达在时间和空间上进行严格调控。深入研究单一基因在生物体不同发育阶段。不同生理条件和不同病理状态下不同组织中的功能作用提供了有力的手段。并为进一步阐明人类生理机能和疾病的分子机制及基因治疗的研究开辟了新的前景。     

人c-myc转基因细胞的建立

目的:建立人c-myc转基因细胞。方法:通过成功构建c-myc逆转录病毒表达载体,并经脂质体介导转染包装细胞293T,收集产重组病毒的293T培养上清,运用NIH3T3细胞测定了病毒滴度,用适当浓度的病毒感染L929细胞,经用Zeocin选择性培养基筛选细胞。结果:得到稳定高表达c-myc基因的L929转基因细胞。结论:运用逆转录病毒转染法可得到高表达的转基因细胞。     

Tet system的调控原理及其在转基因小鼠模型上的应用

基因表达的调控是分子生物学研究的一个重要问题,也是基因治疗和基因功能研究的重要手段。诱导性基因表达系统可以从时间上调控基因的表达,是基因治疗和基因功能研究的重要工具之一。其中,四环素诱导基因表达系统(tetracycline inducible expression system,Tet system)是应用最广泛的一种,它可以在时间和空间上对基因进行严谨和高效地诱导表达。基于该系统获得了不同用途的转基因动物,这些模型动物的建立为研究特定基因的功能及其在疾病发生中的作用打下了实验基础。现就四环素诱导表达系统的原理和在小鼠模型上的研究应用做一综述。     

DNA肿瘤病毒SV40的细胞转化特性

SV40是被广泛应用于肿瘤研究的DNA肿瘤病毒。本文简述了它的一般生物学特性,根据大量研究报道着重归纳了SV40在体外转化细胞的特性及其转化机制,并展望了SV40转化蛋白在癌变机理、细胞分化等研究中的进一步应用。     

Tet-on和Cre/loxP系统双重调控HCV NS5B真核表达载体的设计与构建

目的:构建可受Tet-on和Cre/loxP系统双调控的HCVNS5B真核表达载体,为建立可严格调控HCV NS5B蛋白表达的转基因小鼠奠定基础.方法:以真核表达载体pBI-3为载体构建骨架,在其启动子下游依次插入luc报告基因、BGH pA和NS5B基因片段,并分别在luc报告基因上游和BGH pA尾下游引入一个loxP位点.结果:成功构建了可受Tet-on和Cre/loxP系统双调控的HCV NS5B真核表达载体pBI-3/luc-BGH pA-NS5B.结论:pBI-3/luc-BGH pA-NS5B真核表达载体的成功构建为可严格调控HCV NS5B蛋白表达转基因小鼠的建立打下了良好的基础.     

两种含SV40T不同区段的基因构建及其转基因小鼠的建立

目的　为解决SV4 0T转基因小鼠高发瘤难保种的问题 ,构建了两种含有SV4 0T不同区段的外源基因 :Rb结合域点突变的SV4 0T基因 (SV4 0T DRb)和无p5 3结合域的SV4 0T基因 (SV4 0T Dp5 3)。方法　利用分子生物学的基因克隆手段 ,将改造的SV4 0T基因片段克隆测序 ,最终将这两个改造后的基因克隆进乳腺特异性的真核表达载体p2 0 5C3中 ,运用雄原核显微注射法制备转基因小鼠。结果　利用PCR方法检测出 6只双阳性转基因 (同时检测到SV4 0T DRb和SV4 0T Dp5 3两种基因 )小鼠 ,为避免检测结果中假阳性的发生 ,应用Southern blot方法检测出 1只双阳性转基因小鼠。结论　本试验的结果证明 ,构建的两种含SV4 0T不同区段的策略是成功的 ,其建立的阳性转基因小鼠确实是弱化了SV4 0T转基因小鼠高发瘤的特性。     

SV40灭活疫苗的制备及其对小鼠免疫的研究

猿猴空泡病毒40(Simian vacuolating virus 40,SV40) 属于乳多空病毒科,是一种DNA肿瘤病毒。亚洲猿类特别是恒河猴是SV40的天然宿主。感染SV40病毒可导致猴体急性病变或呈长期带毒状态,此外能诱使幼鼠产生肿瘤,并能使多种培养细胞发生转化。本研究初步建立了SV40 病毒在Vero细胞中的增殖培养方法,并且初步建立了β丙内脂灭活病毒的方法和纯化工艺。使用SV40病毒灭活疫苗对Balb/c小鼠进行了免疫,结果表明该疫苗具有较好的免疫原性。随后对SV40 病毒DNA在免疫小鼠的重要脏器中的整合情况进行了调查,结果表明SV40病毒DNA未在小鼠重要脏器中整合。本研究为SV40病毒灭活疫苗的研制和进一步开展猴体抗SV40 感染实验奠定了良好的基础。     

SV40灭活疫苗的制备及其对小鼠免疫的研究

猿猴空泡病毒40(Simian vacuolating virus 40,SV40) 属于乳多空病毒科,是一种DNA肿瘤病毒.亚洲猿类特别是恒河猴是SV40的天然宿主.感染SV40病毒可导致猴体急性病变或呈长期带毒状态,此外能诱使幼鼠产生肿瘤,并能使多种培养细胞发生转化.本研究初步建立了SV40病毒在Vero细胞中的增殖培养方法,并且初步建立了β-丙内脂灭活病毒的方法和纯化工艺.使用SV40病毒灭活疫苗对Balb/c小鼠进行了免疫,结果表明该疫苗具有较好的免疫原性.随后对SV40病毒DNA在免疫小鼠的重要脏器中的整合情况进行了调查,结果表明SV40病毒DNA未在小鼠重要脏器中整合.本研究为SV40病毒灭活疫苗的研制和进一步开展猴体抗SV40感染实验奠定了良好的基础.     

Complete Genomic Sequence Analysis of a New SV40 Isolate

The genome of a new SV40 strain (SV-IMB) isolated from a rhesus monkey was completely sequenced and compared with other isolates. The results showed that the whole genome contains 5246bp, and the average identity of SV-IMB was 98.1% as compared to other SV40 isolates. Its regulatory region is composed of a complete enhancer and a defective enhancer. Amino acid changes occurred to some extent in both the large T antigen (T-Ag) and VP1 region. The findings demonstrate that the SV-IMB is a new SV40 isolate.     

Complete genomic sequence analysis of a new SV40 isolate

The genome of a new SV40 strain(SV-IMB) isolated from a rhesus monkey was completely sequenced and compared with other isolates. The results showed that the whole genome contains 5246bp, and the aver age identity of SV-IMB was 98.1% as compared to other SV40 isolates. Its regulatory region is composed of a complete enhancer and a defective e enhancer. Amino acid changes occurred to some extent in both the large T antigen (T-Ag) and VP1 region. The findings demonstrate that the SV-IMB is a new SV40 isolate.     

Transcriptionally regulated immortalization overcomes side effects of temperature-sensitive SV40 large T antigen

The temperature-sensitive mutant of the SV40 virus large T antigen (TAg) tsA58 is frequently employed for the conditional immortalization of primary cells. By increasing the temperature to 39 degrees C, the activity of the mutant TAg is reduced and the status of such cells may then resemble more closely that of primary cells. As an alternative, we used a novel immortalization vector with a tetracycline-regulated expression of the wild-type TAg. This enabled us to investigate the effects of the immortalizing gene expression and of temperature shifts independently of each other. Even for wild-type TAg-derived cell lines the elevated temperatures led to various clone-dependent phenotypes. This suggests that in freshly established cell lines temperature-sensitive growth phenotypes can arise spontaneously and independently of a temperature-sensitive immortalizing gene. Similar effects were observed with spontaneously immortalized cells. On the other hand, not all of the ts-TAg-derived cell lines were proliferation arrested at the non-permissive temperature. Therefore, the assumption that temperature-sensitive growth is solely due to the ts-TAg must be verified for each ts-TAg-derived cell line individually. This complexity could be avoided by using the autoregulatory immortalization vector expressing the wild-type TAg.     

Analysis of stable and unstable viral forms in SV40-infected human keratinocytes

We analyzed the state of the genomic DNA of the papovavirus SV40 in human keratinocytes as viral-infected cells gradually acquired a transformed phenotype over time. Initially, the vast majority of the viral DNA is maintained either in a full-length supercoiled form or as truncated subgenomic fragments with little evidence of integration. However, analyses of clonal populations revealed great heterogeneity and instability of the viral DNA, and we were able to isolate one clonal subpopulation in which integrated forms of the virus appeared to predominate. Similarly, uncloned populations eventually ceased production of the "free" viral DNA after several years in culture and instead came to display tandemly repeated SV40 copies at a single host integration site. Interestingly, Bg1 II digestion of host DNA generated restriction fragments containing the integrated SV40 DNA, which were of differing sizes in cultures at the 144th vs the 163rd serial passage suggesting modification or rearrangement of sequences at or near the integration site. Host sequences flanking the integrated viral DNA at the 163rd serial passage have been isolated on restriction fragments generated by Eco RI, Bam HI, and Hpa II digestion. These analyses suggest that the integrated virus is linearized near the Bg1 I site and contains a large deletion in the SV40 early region at one of the viral-host junctions.     

Comparison of spontaneous and ultraviolet-induced mutagenesis on naked SV40 DNA and SV40 virus

Molecular aspects of mutagenesis in mammalian cells have been essentially analyzed using biological probes such as viruses and shuttle vector. Although the main data concerning the specificity of carcinogen-induced mutations are similar, the observed spontaneous mutation frequencies are significantly different when using one or the other model. This frequency is considerably higher with shuttle vectors than with viruses. We have performed an analysis of mutagenesis in order to determine if the obligatory transfection step associated with shuttle vector technology was responsible for the high mutation frequency found with these molecules. For this purpose simian virus 40 (SV40) genome used as virus or as naked DNA was introduced into permissive cells by viral infection or DNA transfection respectively. Our results show that transfection alone does not induce a higher mutation frequency on SV40 DNA the virus infection. Moreover, we have shown that the ultraviolet-light induced mutation spectrum was similar on the SV40 VP1 gene after viral infection or DNA transfection.     

Nucleotide sequence-directed mapping of the nucleosomes of SV40 chromatin

In our previous work we have shown by comparison of experimental and computational data that the positions of the histone octamers bound to the DNA molecule appear to be completely sequence-dependent. This provides a convenient and quick method for locating the nucleosomes along the DNA molecule, as soon as the nucleotide sequence is known. Using this computational approach, the complete nucleosomal map of the SV40 minichromosome has been constructed. The map consists of 25 nucleosomes, with their coordinates (centers) being specified with high accuracy. The map is found to be in remarkable agreement with available experimental data.     

Differentiation and growth potential of human ovarian surface epithelial cells expressing temperature-sensitive SV40 T antigen

The epithelial ovarian carcinomas arise in the ovarian surface epithelium (OSE) which is the mesothelial covering of the ovary. Studies of human USE have been hampered by the small amounts and limited lifespan of this epithelium in culture. OSE cells expressing SV40 large T antigen (Tag) or the HPV genes E6 and E7 have increased growth potentials but lack some of the normal characteristics of OSE. In this study, we used conditional SV40 Tag expression to produce OSE cells with increased proliferative potentials but relatively normal phenotypes. Primary OSE cultures from three women, one of whom had a BRCA1 mutation, were infected with a temperature-sensitive Tag construct (tsTag), and from these, 28 monoclonal and four polyclonal lines were isolated. The effects of temperature changes were examined in two monoclonal and two polyclonal lines. At the permissive temperature (34 degrees C), these cell lines underwent 52-71 population doublings (PD) compared to 15-20 PD for normal OSE. Nuclear SV40-Tag and p53 expression, demonstrated by immunofluorescence, showed that tsTag was uniformly present and biologically active in all lines. At 34 degrees C, culture morphologies ranged from epithelial to mesenchymal. The mean percentage of cells expressing the epithelial differentiation marker, keratin. varied between lines from 20 to 97%. Collagen type III, a mesenchymal marker expressed by OSE in response to explantation into culture, was present in 24-43% of cells. At 39 degrees C, tsTag was inactivated by 2 d while nuclear p53 staining diminished to control levels over 2 wk. Over 3 d. the cells assumed more epithelial morphologies, keratin expression reached 85-100% in all lines and collagen expression increased significantly in two lines. The cultures with the BRCA1 mutation expressed the most keratin and the least collage n III at both temperatures. As indicated by beta-galactosidase staining at pH 6.0, changes leading to senescence were initiated at 39 degrees C by 6 h and were present in all cells after 24 h. However, the cells underwent 1-3 population doublings over up to 1 wk before growth arrest and widespread cell death, thus providing an experimental system where large numbers of OSE cells with different genetic backgrounds and growth potentials can be studied without the concurrent influence of Tag.     

Studies of simian virus 40 DNA. VII. A cleavage map of the SV40 genome

A physical map of the Simian virus 40 genome has been constructed on the basis of specific cleavage of Simian virus 40 DNA by bacterial restriction endonucleases. The 11 fragments produced by enzyme from Hemophilus influenzae have been ordered by analysis of partial digest products and by analysis of an overlapping set of fragments produced by enzyme from Hemophilus parainfluenzae. In addition, the single site in SV40 DNA cleaved by the Escherichia coli R_I restriction endonuclease has been located. With this site as a reference point, the H. influenzae cleavage sites and the H. parainfluenzae cleavage sites have been localized on the map.