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。     

Tet-on诱导表达c-myc和SV40Tag的转基因小鼠肿瘤模型

目的研究Tet-on诱导表达c-myc和SV40Tag小鼠肿瘤模型的肿瘤发生和基因表达情况,探讨c-myc基因的作用。方法用pTRE2-c-myc单阳性转基因小鼠和Tet-on、pTRE2-SV40Tag双阳性转基因小鼠交配,后代检测得到Tet-onp、TRE2-SV40Tag、pTRE2-c-myc三阳性转基因小鼠,经强力霉素诱导一段时间以后,观察肿瘤的发生;通过RT-PCR、病理组织切片和磁共振等方法对肿瘤的发生部位和时相进行研究。结果Tet-on、pTRE2-SV40Tag、pTRE2-c-myc三阳性转基因小鼠①经诱导后发生肿瘤,且发瘤率和发瘤时间高于和短于Tet-on、pTRE2-SV40Tag双阳性转基因小鼠;②c-myc和SV40Tag基因在表达部位上有所不同。结论c-myc和SV40Tag基因同时表达与SV40Tag基因单独表达时相比,肿瘤发生明显增强,提示c-myc基因与肿瘤的发生有着密切关系。     

广泛表达人载脂蛋白E3转基因小鼠的建立

目的建立人载脂蛋白E3（apolipoprotein E3,ApoE3）转基因小鼠,研究该基因在多种组织中表达水平的变化对动物的影响,探索该基因的功能。方法RT-PCR法克隆人ApoE3基因,把该基因插入CMV启动子下游,构建转基因表达载体,通过显微注射法建立转ApoE3基因C57BL／6J小鼠。并利用特异引物PCR法鉴定转基因小鼠的基因型,Western blot检测基因表达水平。通过生化指标分析初步鉴定ApoE3基因的功能。结果建立了2个系的高表达人ApoE3转基因小鼠。结论成功建立了CMV启动子启动的高表达人ApoE3基因转基因小鼠,为进一步探索该基因的功能奠定了基础。     

恒河猴外周血及肾组织SV40病毒基因分析

SV40即猿猴病毒40(simian virus 40),是DNA肿瘤病毒的原型代表,其基因结构为共价闭合环状双股DNA分子,标准参考株SV40-776含5243个核甘酸,不同分离株bp数略有差异.     

小鼠细小病毒非结构基因转基因小鼠的建立

为探讨小鼠细小病毒（ＭＶＭ）非结构蛋白在ＭＶＭ感染中的抗肿瘤作用,酶切表达质粒ｐＵＬＢ３２３８获取该非结构基因,通过显微注射法接种入小鼠受精卵内制备转基因鼠。共注射受精卵７２０枚,选取存活受精卵２２５枚植入假孕小鼠输卵管,产仔１４只。转基因小鼠尾部组织ＰＣＲ法ＤＮＡ检测证明,其中４只整合靶基因。整合转基因的４只Ｇ０代小鼠与正常Ｃ５７ＢＬ／５Ｊ小鼠配种均可生产整合靶基因的小鼠。ＲＴ－ＰＣＲ法ｍＲＮＡ     

肥大软骨细胞特异性表达Cre重组酶转基因小鼠的建立

肥大软骨细胞是软骨细胞的终末分化形式,在软骨内成骨过程中发挥十分关键的作用。为了研究肥大软骨细胞在骨骼发育过程中的功能,我们构建了在8.2 kb小鼠X型胶原基因（Col10a1）启动子控制下表达Cre重组酶的转基因小鼠品系（Col10a1-8.2-Cre）。采用显微注射法将11.5 kb的转基因片段引入小鼠基因组,共注射受精卵328枚,获得子代鼠51只,经PCR基因型鉴定有3只在基因组上整合有Cre重组酶基因。PCR检测发现Col10a1-8.2-Cre转基因在含有肥大软骨细胞的组织中表达。为了检测Cre重组酶表达的强度和组织特异性,转基因小鼠与ROSA26报告小鼠交配。子代ROSA26;Col10a1-8.2-Cre双转基因小鼠LacZ染色检测的结果显示,Cre重组酶在所有的肥大软骨细胞中表达。原位杂交的结果验证Col10a1-8.2-Cre转基因表达在肥大区的上端。以上结果表明,我们建立的肥大软骨细胞特异性表达Cre重组酶的转基因小鼠品系可以作为一种遗传学工具,介导目的基因在肥大软骨细胞中的敲除。     

角质细胞特异性表达Cre重组酶转基因小鼠的建立

构建了含有角质细胞特异性角质素5启动子、Cre重组酶基因和人生长激素基因plyA的转基因载体pK5-Cre-hGH。以显微注射的方法将4．2kb的转基因片段K5-Cre-hGH引入小鼠基因组,共注射720枚受精卵,其中龄5枚移植至29只假孕母鼠的输卵管中发育,获得子代小鼠48只,经基因型鉴定有12只小鼠在其基因组上整合有Cre基因,整合率为25％。将带有cre重组酶基因的小鼠与基因组上携带loxP位点的smad4条件基因打靶小鼠杂交以检测Cre重组酶组织特异性表达情况以及介导重组的功能。结果表明,K5-Cre转基因小鼠只在皮肤组织中表达Cre重组酶并能在体内成功地介导loxP位点的重组。     

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。     

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。     

Development of Oral Epithelial Cell Line ROE2 with Differentiation Potential from Transgenic Rats Harboring Temperature-Sensitive Simian Virus40 Large T-Antigen Gene

We have developed an immortalized oral epithelial cell line, ROE2, from fetal transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene. The cells grew continuously at either a permissive temperature of 33°C or an intermediate temperature of 37°C. At the nonpermissive temperature of 39°C, on the other hand, growth decreased significantly, and the Sub-G1 phase of the cell cycle increased, indicating that the cells undergo apoptosis at a nonpermissive temperature. Histological and immunocytochemical analyses revealed that ROE2 cells at 37°C had a stratified epithelial-like morphology and expressed cytokeratins Krt4 and Krt13, marker proteins for oral nonkeratinized epithelial cells. Global-scale comprehensive microarray analysis, coupled with bioinformatics tools, demonstrated a significant gene network that was obtained from the upregulated genes. The gene network contained 16 genes, including Cdkn1a, Fos, Krt13, and Prdm1, and was associated mainly with the biological process of skin development in the category of biological functions, organ development. These four genes were validated by quantitative real-time polymerase chain reaction, and the results were nearly consistent with the microarray data. It is therefore anticipated that this cell line will be useful as an in vitro model for studies such as physiological functions, as well as for gene expression in oral epithelial cells.     

Efficient Establishment of Pig Embryonic Fibroblast Cell Lines with Conditional Expression of the Simian Vacuolating Virus 40 Large T Fragment

The pig is an important animal for both agricultural and medical purposes. However, the number of pig-derived cell lines is relatively limited when compared with mouse- and human-derived lines. We established in this study a retroviral conditional expression system for the Simian vacuolating virus 40 large T fragment (SV40T) which allowed us to efficiently establish pig embryonic fibroblast cell lines. The established cell lines showed high levels of cell proliferation and resistance to cellular senescence. A chromosome analysis showed that 84% of the cells had the normal karyotype. Transient expression of the Cre recombinase allowed us to excise the SV40T fragment from the genome. The development of this research tool will enable us to quickly establish new cell lines derived from various animals.     

Relationship between expression of epidermal growth factor and simian virus 40 T antigen in a line of transgenic mice

The pattern of expression of the simian virus 40 (SV40) T antigen gene and resultant dysplasia were re-examined in a line of transgenic mice in which the T antigen gene was under the control of the SV40 early promoter. We found that T antigen expression in the kidney, and resulting dysplastic lesions, occurred exclusively in the distal convoluted tubules and the ascending limbs of Henle. Epidermal growth factor (EGF) expression in the kidney of normal mice was similarly immunolocalized. The correlation between high EGF immunoreactivity in normal mouse tissues and T antigen expression in the transgenic counterpart was also seen in the choroid plexus epithelium and in the submandibular glands of male mice. T antigen was not found in the submandibular gland of transgenic females. Similarly, EGF was only rarely detected in the normal female submandibular gland. In contrast to the correlation between T antigen expression in the transgenic mice and EGF expression in the corresponding tissues of the normal mice, within the dysplastic lesions of the transgenic mice EGF expression was severely diminished. Adenocarcinomas of the male submandibular gland from another line of transgenic mice that expresses theInt-1 transgene, showed similarly reduced levels of immunostaining for EGF. Thus, reduced expression of EGF might be a general feature of dysplasia and tumorigenesis in those tissues that normally express EGF.     

SV40 T antigen alone drives karyotype instability that precedes neoplastic transformation of human diploid fibroblasts

To define the role of SV40 large T antigen in the transformation and immortalization of human cells, we have constructed a plasmid lacking most of the unique coding sequences of small t antigen as well as the SV40 origin of replication. The promoter for T antigen, which lies within the origin of replication, was deleted and replaced by the Rous sarcoma virus promoter. This minimal construct was co-electroporated into normal human fibroblasts of neonatal origin along with a plasmid containing the neomycin resistance gene (neo). Three G418-resistant, T antigen-positive clones were expanded and compared to three T antigen-positive clones that received the pSV3neo plasmid (capable of expressing large and small T proteins and having two origins of replication). Autonomous replication of plasmid DNA was observed in all three clones that received pSV3neo but not in any of the three origin minus clones. Immediately after clonal expansion, several parameters of neoplastic transformation were assayed. Low percentages of cells in T antigen-positive populations were anchorage independent or capable of forming colonies in 1% fetal bovine serum. The T antigen-positive clones generally exhibited an extended lifespan in culture but rarely became immortalized. Large numbers of dead cells were continually generated in all T antigen-positive, pre-crisis populations. Ninety-nine percent of all T antigen-positive cells had numerical or structural chromosome aberrations. Control cells that received the neo gene did not have an extended life span, did not have noticeable numbers of dead cells, and did not exhibit karyotype instability. We suggest that the role of T antigen protein in the transformation process is to generate genetic hypervariability, leading to various consequences including neoplastic transformation and cell death.     

软骨组织特异性表达Cre重组酶转基因小鼠的研制和鉴定

构建了含有软骨组织特异性Ⅱ型胶原A1启动子和Cre重组酶基因的转基因载体pcol2Al-Cre。323枚小鼠受精卵经显微注射引入转基因片段后,分别移植至14只假孕母鼠的输卵管使其发育。共得到仔鼠52只,PCR结果显示其中10只小鼠基因组上有Cre基因的整合,整合率为19．2％。用整合有Cre基因的转基因小鼠与基因组上携带LoxP位点的条件基因打靶小鼠交配,以检测Cre酶介导的重组及其组织特异性。PCR结果表明：col2Al-Cre转基因小鼠软骨组织中表达的Cre重组酶成功地介导了LoxP之间的重组。此结果通过Southern杂交得到了进一步的证实。     

Cytotoxic T-Lymphocyte Epitope Immunodominance in the Control of Choroid Plexus Tumors in Simian Virus 40 Large T Antigen Transgenic Mice

The simian virus 40 (SV40) large tumor antigen (Tag) is a virus-encoded oncoprotein which is the target of a strong cytotoxic T-lymphocyte (CTL) response. Three immunodominant H-2(b)-restricted epitopes, designated epitopes I, II/III, and IV, have been defined. We investigated whether induction of CTLs directed against these Tag epitopes might control Tag-induced tumors in SV11(+) (H-2(b)) mice. SV11(+) mice develop spontaneous tumors of the choroid plexus due to expression of SV40 Tag as a transgene. We demonstrate that SV11(+) mice are functionally tolerant to the immunodominant Tag CTL epitopes. CTLs specific for the H-2Kb-restricted Tag epitope IV were induced in SV11(+) mice following adoptive transfer with unprimed C57BL/6 spleen cells and immunization with recombinant vaccinia viruses expressing either full-length Tag or the H-2Kb-restricted epitope IV as a minigene. In addition, irradiation of SV11(+) mice prior to adoptive transfer with unprimed C57BL/6 spleen cells led to the priming of epitope IV-specific CTLs by the endogenous Tag. Induction of epitope IV-specific CTLs in SV11(+) mice by either approach correlated with increased life span and control of the choroid plexus tumor progression, indicating that CTLs specific for the immunodominant Tag epitope IV control the progressive growth of spontaneous tumors induced by this DNA virus oncogene in transgenic mice.     

野生及笼养猕猴SV40T抗原基因检测方法的建立

目的建立猴外周血单核细胞SV40DNA的PCR检测方法,对猕猴SV40T抗原基因进行检测。方法使用PCR方法对分别来自野外（云南宁蒗71只、景东60只）及笼养猕猴（64只）的SV40大T抗原基因进行检测,同时对扩增出的阳性结果进行测序。结果在195份样本中,有4只来自野生猴样本扩增出SV40大T抗原基因（3．1％,4／131）,1只来自笼养猴样本扩增出SV40大T抗原基因（1．6％,1／64）。测序结果显示：猴血样本扩增片段序列与GenBank中的SV40大T抗原C-末端的基因序列片段有15个核苷酸不同（3．3％差异）,与SV40．776标准株的序列基本一致,但SV40—776在nt3020处有一缺口。结论云南野生及笼养猕猴猴群均能检出SV40T抗原基因。因此建立SV40病毒的DNA检测技术,对用于科研及疫苗生产的实验猕猴的病毒学质量控制具有重要意义。     

Lactation-induced WAP-SV40 Tag Transgene Expression in C57BL/6J Mice Leads to Mammary Carcinoma

Two transgenic lineages were generated by directing the expression of SV40 T antigen to the mammary gland of inbred C57BL/6J mice using the whey acidic protein (WAP) promoter. In one lineage, WAPTag 1, multiparous female mice developed mammary adenocarcinoma with an average latency period of 13 months. The histopathological phenotype was heterogeneous, tumours occurred in a stochastic fashion, normal tissue was located next to neoplastic tissue, the mammary tumours usually developed and were remarkably similar to that observed in human cases. In addition, male and virgin females developed a poorly differentiated SV40 T antigen-positive soft tissue sarcoma, also at 13 months of age. In the other lineage, WAPTag 3, some parous females developed mammary tumours, but most mice succumbed to osteosarcomas arising from the os petrosum at 5.5 to 6 months of age and on necropsy, renal adenocarcinomas were also found. Appearance of these unexpected tumour types demonstrates the non-specific e...     

Infection of CV1 cells expressing the polyoma virus middle T antigen or the SV40 agnogene product with simian virus 40 host-range mutants

Summary SV40 viruses bearing mutations at the carboxy-terminus of large T antigen exhibit a host-range phenotype: such viruses are able to grow in BSC monkey kidney cells at 37° C, but give at least 10 000-fold lower yields than wild type virus in BSC cells at 32° C or in CV1 monkey kidney cells at either temperature. The block to infection in the nonpermissive cell type occurs after the onset of viral DNA replication. Infectious progeny virions are produced at very low efficiency. Although capsid proteins are synthesized at decreased levels, this does not account for the magnitude of the defect. Presumably some step of virion assembly or maturation is affected in these mutants. We have previously reported that the viral agnogene product, a protein throught to be involved in viral assembly or release, fails to accumulate in CV1 cells infected with host-range mutants. In polyoma virus the middle T antigen plays a role in virion maturation by influencing the phosphorylation of capsid proteins. In this communication we show that host-range mutants fail to undergo productive infection of CV1 cells expressing middle T antigen. These mutants do form plaques on an agnoprotein-expressing cell line. However, the agnoprotein does not seem to act by correcting the mutational block but rather increases the efficiency of plaque formation. This work was supported by grants CA40586 and BRSG 2S07RR07084-23 to J. M. P. and grant CA33079 to L. T., from the National Institutes of Health, Bethesda, MD.