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1.
为了构建四环素调控的人A30P突变α-synuclein转基因小鼠模型,将外源基因pTRE2-syn和pBC-rtTA同时显微注射到FVB小鼠(Mus muscculus)受精卵的雄原核中,将注射后存活的受精卵移植到同期发情的假孕受体鼠输卵管中,出生个体经PCR检测,获得rtTA和A30P突变α-synuclein双阳性转基因雌鼠1只,A30P单基因阳性雄鼠13只并传代.强力霉索诱导后双阳性后代脑区各部分A30P突变α-synucleinmRNA均有表达,而在诱导满4周后,脑干α-synuclein蛋白表达明显增加,8周后增加更明显.结果表明,通过强力霉素诱导后,可在小鼠小脑、脑干、海马、皮层检测到A30P mRNA表达,脑干α-synuclein表达量显著增加. 相似文献
2.
目的建立Dicer1转基因小鼠模型。方法构建pcDNA3.1-Dicer1转基因构件,经酶切、纯化后通过显微注射方法导入BDF1小鼠受精卵原核并移植到同期受孕的ICR受体母鼠输卵管内。出生后仔鼠用PCR和Southern方法检测鼠尾DNA鉴定基因型,通过免疫组化检测Dicer1基因表达。结果显微注射172枚卵,移植119枚卵于3只受体输卵管中,2只怀孕,共产仔15只,经PCR检测获得6只阳性鼠,Southern检测6只均为阳性。对Southern检测阳性转基因小鼠子代进行RT-PCR检测和免疫组化分析证明Dicer1基因在肝脏、肾脏、肺内均有表达。对腹腔肿胀的转基因阳性1号鼠解剖发现肝脏、脾脏明显增大,胚胎发育异常。结论成功建立Dicer1基因表达的转基因小鼠模型,该模型为进一步研究DICER1基因功能及miRNA的表达及功能等奠定基础。 相似文献
3.
人促红细胞生成素在转基因小鼠乳汁中表达 总被引:10,自引:0,他引:10
将山羊的β乳球蛋白启动子连接人促红细胞生成素基因,构建转基因表达载体。通过显微注射的方法转基因小鼠。经PCR斑点杂交和Southern杂交检测验证,获得4只转基因阳性小鼠,其中3只母鼠哺乳期收集乳汁,经EpoELISA检测为阳性。对4组转基因阳性小鼠的部分子代母鼠,经PCR和Southern杂交分析,又鉴定出6只获得遗传的阳性G1代母鼠以Dot-ELISA的方法检测,其中两只G1代母鼠乳汁中的Epo含量为0.5μg/mL。实验证实,867bp的β乳球蛋白启动子可以指导人促红细胞生成素在小鼠乳汁中表达。 相似文献
4.
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。 相似文献
5.
人乙肝病毒增强子ⅡB1结合因子(hB1F)系Ftz—F1(NR5A)亚家族的新成员。经基因重组法将人hb1 fcDNA置于小鼠白蛋白增慢子/启动子序列下游构建成肝特异重组载体,通过原核显微注射将该载体导入小鼠受精卵原核,经注射且状态良好的卯回输至假孕母鼠输卯管。产下仔鼠经PCR和Southern blotting鉴定,同时RT—PCR和Western blotting分析转基因的表达。阳性Founder鼠与正常C57鼠交配以建立转基因纯系小鼠,F1代以PCR法鉴定。结果共获得4只PCR鉴定转基因阳性Founder鼠,其中一只同时经Southern blotting鉴定为阳性。RT—PCR和Western blotting结果显示,外源基因在转基因小鼠的肝组织成功表达。遗传学分析表明,转基因已整合入小鼠基因组并可稳定溃传。 相似文献
6.
目的建立TNNT3(R69H)突变转基因小鼠模型。方法构建pEGFP-TNNT3(R69H)转基因构件,通过原核显微注射方法将线性化、纯化后的外源质粒pEGFP-TNNT3(R69H)注射入BDF1小鼠受精卵中,胚胎移植至同期发情的假孕受体母鼠输卵管内,获得子代小鼠。用PCR和Southern blot方法检测子代鼠尾基因组DNA,通过RT-PCR及Western blot的方法检测TNNT3基因表达。结果 8只假孕小鼠共移植注射后的胚胎82枚,出生40只子代鼠,经PCR和Southern方法检测得到5只转基因阳性小鼠。对其子代小鼠进行RT-PCR、Western blot检测结果显示,TNNT3在转基因小鼠心脏和骨骼肌中表达量明显增多。结论通过显微注射法使外源基因pEGFP-TNNT3(R69H)在小鼠基因组中整合,成功建立了TNNT3(R69H)突变转基因小鼠模型。 相似文献
7.
制备hβ2m HLA B2 70 4双转基因小鼠 ,研究hβ2m对HLA B2 70 4表达的影响 .通过hβ2m转基因小鼠和HLA B2 70 4转基因小鼠交配 ,出生动物及后代经PCR初步筛选 ,采用Southern杂交对经PCR初步筛选的hβ2m HLA B2 70 4双转基因阳性小鼠基因组DNA标本作进一步鉴定 .阳性者进行RT PCR和流式细胞光度术检测其在mRNA和蛋白水平的表达 .hβ2m阳性小鼠和HLA B2 70 4阳性小鼠交配 ,生仔鼠 6 7只 ,其中 2 8只仔鼠同时整合hβ2m和HLA B2 70 4基因 .Southern杂交证实 ,阳性鼠同时都含有hβ2m和HLA B2 70 4基因 .阳性小鼠的皮肤、结肠、睾丸和脾脏组织中均有HLA B2 70 4和hβ2m基因的mRNA表达 ,其外周血淋巴细胞膜上HLA B2 70 4基因的蛋白表达为 35 87% ,在HLA B2 70 4单转基因小鼠外周血淋巴细胞膜上HLA B2 70 4基因的蛋白表达为 7 87% (Histogramsta tistics) .成功地制备了hβ2m HLA B2 70 4双转基因小鼠 .在hβ2m HLA B2 70 4双转基因小鼠的外周血淋巴细胞膜上 ,HLA B2 70 4基因在蛋白水平表达较高 ,而在HLA B2 70 4单转基因小鼠中则表达不明显 .hβ2m可与HLA B2 70 4重链分子结合 ,稳定和增高其在淋巴细胞膜上的表达 相似文献
8.
目的建立TNNI2突变转基因小鼠模型。方法构建pEGFP-tnni2转基因构件,TNNI2基因的第175个氨基酸缺失,通过原核显微注射法。把线性化、纯化后的外源基因pEGFP-tnni2注射入BDF1小鼠受精卵中。胚胎移植给同期发情的假孕受体母鼠,获得子代小鼠。用PCR和Southern方法检测子代鼠尾DNA鉴定基因型。通过RT-PCR方法检测tnni2基因表达。结果移植注射胚胎115枚给4只假孕小鼠共出生了23只后代鼠。经PCR和Southern方法检测得到4只阳性小鼠。对其子代进行RT-PCR检测,tnni2基因在肌肉、心脏内表达。结论通过显微注射法使外源基因pEGFP-tnni2(TNNI2基因的第175个氨基酸缺失)在小鼠基因组中得到整合,建立了转pEGFP-tnni2的转基因小鼠模型。 相似文献
9.
目的建立5-脂氧化酶(5-LO)转基因小鼠进行动脉粥样硬化的发病分子机制的研究。方法通过显微注射的方法,将5-脂氧化酶基因片段(6.8 kb)导入BDF1受精卵雄原核并移植到同期受孕的假孕母鼠输卵管中,对产出仔鼠的鼠尾组织DNA进行PCR、Southern blot检测,对9、20、24号转基因小鼠分别提取腹腔细胞、骨髓细胞及脾、肾组织总RNA和蛋白,并采用RT-PCR、Western blot方法进行转录水平检测和蛋白表达检测。结果共产生25只子代小鼠,经PCR和Southern检测获得7只阳性小鼠,经RT-PCR和Western blot检测结果表明,9、20、24号转基因小鼠腹腔细胞、骨髓细胞、脾、肾5-LO和5-脂氧化酶激活蛋白(FLAP)在RNA和蛋白水平表达均高于正常BDF1对照小鼠,且统计学分析腹腔细胞、骨髓细胞表达均具有显著差异(P0.05)。结论成功建立5-LO转基因小鼠模型。 相似文献
10.
体内精原干细胞转染法建立转基因小鼠 总被引:1,自引:0,他引:1
将人Bcl-2 cDNA与小鼠乳清酸蛋白(WAP)5’上游调控序列融合后,与脂质体按一定比例混合,再加入适量的台盼蓝制成转染液,注入到小鼠睾丸中的曲细精管中,转染精原干细胞以探讨建立转基因小鼠的可行性。共注射了3只公鼠,4天后将公鼠与发情母鼠合笼交配,共生仔鼠20只。检测结果表明,有3只呈PCR阳性,Southern blot检测,阳性鼠2只,1只公鼠,1只母鼠,其中,公鼠意外死亡;Western blot证实,1只母鼠的乳腺组织表达了Bcl-2蛋白,其F1代的16只小鼠中,有7只呈PCR阳性。证实了体内精原干细胞转染建立转基因动物的可行性。 相似文献
11.
两种含SV40T不同区段的基因构建及其转基因小鼠的建立 总被引:1,自引:1,他引:0
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。 相似文献
12.
Lack of an immune response against the tetracycline-dependent transactivator correlates with long-term doxycycline-regulated transgene expression in nonhuman primates after intramuscular injection of recombinant adeno-associated virus
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Favre D Blouin V Provost N Spisek R Porrot F Bohl D Marmé F Chérel Y Salvetti A Hurtrel B Heard JM Rivière Y Moullier P 《Journal of virology》2002,76(22):11605-11611
We previously documented persistent regulation of erythropoietin (Epo) secretion in mice after a single intramuscular (i.m.) injection of a recombinant adeno-associated virus (rAAV) vector harboring both the tetracycline-dependent transactivator (rtTA) and the Epo cDNA (D. Bohl, A. Salvetti, P. Moullier, and J. M. Heard, Blood 92:1512-1517, 1998). Using the same vector harboring the cynomolgus macaque Epo cDNA instead, the present study evaluated the ability of the tetracycline-regulatable (tetR) system to establish long-term transgene regulation in nonhuman primates. The vector was administered i.m., after which 5-day induction pulses were performed monthly for up to 13 months by using doxycycline (DOX), a tetracycline analog. We show that initial inductions were successful in all individuals and that there was a tight regulation and a rapid deinduction pattern upon DOX withdrawal. For one macaque, regulation of Epo secretion was maintained during the entire experimental period; for the five remaining macaques, secreted Epo became indistinguishable from endogenous Epo upon repeated DOX inductions. We investigated the mechanism involved and showed that, except in the animal in which secretion persisted, delayed humoral and cellular immune responses were directed against the rtTA transactivator protein associated with the reduction of vector DNA in transduced muscles. This study provides some evidence that, when the immune system is not mobilized against the rtTA transactivator, the tetR-regulatable system is able to support long-term transgene regulation in the context of an rAAV in nonhuman primates. In addition, our results suggest potential improvements for vector design. 相似文献
13.
Bendiksen S Van Ghelue M Winkler T Moens U Rekvig OP 《Journal of immunology (Baltimore, Md. : 1950)》2004,173(12):7630-7640
The mechanism(s) responsible for autoimmunity to DNA and nucleosomes in SLE is largely unknown. We have demonstrated that nucleosome-polyomavirus T-Ag complexes, formed in context of productive polyomavirus infection, activate dsDNA-specific B cells and nucleosome-specific CD4(+) T cells. To investigate whether de novo expressed T-Ag is able to terminate nucleosome-specific T cell tolerance and to maintain anti-dsDNA Ab production in nonautoimmune mice, we developed two binary transgenic mouse variants in which expression of SV40 large T-Ag is controlled by tetracycline, MUP tTA/T-Ag (tet-off), and CMV rtTA/T-Ag (tet-on) mice. Data demonstrate that MUP tTA/T-Ag mice, but not CMV rtTA/T-Ag mice, are tightly controlling T-Ag expression. In MUP tTA/T-Ag transgenic mice, postnatal T-Ag expression activated CD8(+) T cells but not DNA-specific B cells, while immunization with T-Ag and nucleosome-T-Ag-complexes before T-Ag expression resulted in elevated and remarkably stable titers of anti-T-Ag and anti-dsDNA Abs and activation of T-Ag-specific CD4(+) T cells. Immunization of nonexpressing MUP tTA/T-Ag mice resulted in transient anti-T-Ag and anti-dsDNA Abs. This system reveals that a de novo expressed DNA-binding quasi-autoantigen maintain anti-dsDNA Abs and CD4(+) T cell activation once initiated by immunization, demonstrating direct impact of a single in vivo expressed molecule on sustained autoimmunity to DNA and nucleosomes. 相似文献
14.
Zheng X Gao JX Zhang H Geiger TL Liu Y Zheng P 《Journal of immunology (Baltimore, Md. : 1950)》2002,169(9):4761-4769
In addition to their overexpression in cancer cells, most of the tumor-associated Ags are expressed at low but detectable levels in normal tissues. It is not clear whether the repertoire of T cells specific for unmutated tumor Ags is shaped by negative selection during T cell development. The transgenic adenocarcinoma of mouse prostate (TRAMP) model is transgenic for the SV40 large T Ag (Tag) under the control of the rat probasin regulatory elements. Although it has been established that T lymphocytes from TRAMP mice are tolerant to SV40 Tag, the mechanism of the tolerance is largely unknown. To examine whether the T cell clonal deletion is responsible for the tolerance, we crossed the TRAMP mice with mice transgenic for a rearranged TCR specific for SV40 Tag presented by the H-2K(k). Double transgenic TRAMP/TCR mice showed profound thymic deletion of SV40 Tag-reactive T cells, including a 6- to 10-fold reduction in the total thymocyte numbers and a >50-fold reduction in phenotypically mature T cells. Consistent with this finding, we observed that the SV40 Tag and endogenous mouse probasin genes are expressed at low levels in the thymus. These results demonstrate that clonal deletion is a major mechanism for tolerance to Ags previously regarded as prostate-specific, and provide direct evidence that the T cell repertoire specific for an unmutated tumor Ag can be shaped by clonal deletion in the thymus. 相似文献
15.
Cytotoxic T-Lymphocyte Epitope Immunodominance in the Control of Choroid Plexus Tumors in Simian Virus 40 Large T Antigen Transgenic Mice
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Todd D. Schell Lawrence M. Mylin Ingo Georgoff Angelica K. Teresky Arnold J. Levine Satvir S. Tevethia 《Journal of virology》1999,73(7):5981-5993
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. 相似文献
16.
Beermann F Hunziker A Foletti A 《Pigment cell research / sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society》1999,12(2):71-80
Cutaneous and ocular melanomas are due to malignant transformation of neural crest-derived melanocytes. The rising incidence of this tumor in humans has stimulated experiments to devise suitable mouse models. In the past years, transgenic mouse lines have been generated using different oncogenes - Ha-ras, SV40 T antigen (Tag), ret - which develop benign lesions of melanocytes, melanoma, and/or eye tumors. Pigment cell tumors in humans, although rather rare, can also develop from the retinal pigment epithelium (RPE), a cell layer of neuroectodermal origin. We, therefore, established transgenic models for this ocular tumor. Regulated by the promoter of tyrosinase-related protein-1 (TRP-1), two oncogenes, ret and SV40 Tag, were targeted to the developing RPE in transgenic mice. The TRP-1/ret transgenic mice displayed microphthalmia and benign tumors of the RPE. Expression of SV40 T antigen (TRP-1/Tag) led to malignant tumors, which were invasive and metastasized to inguinal lymph node and spleen. 相似文献
17.
Conditional gene expression in the respiratory epithelium of the mouse 总被引:12,自引:0,他引:12
Transgenic mouse models mediating conditional temporal and spatial regulation of gene expression to the respiratory epithelium were developed utilizing the reverse tetracycline transactivator (rtTA) expressed under the control of SP-C and CCSP promoters. Luciferase activity was detected in the lungs of fetal and adult double transgenic mice but was not detected in other tissues or in single transgenic mice. In adult mice, maximal luciferase activity was detected 16h after the administration of doxycycline in the drinking water, or 2h after the injection of doxycycline. Activation of the transgene was observed after the administration of doxycycline in food pellets. After prolonged exposure to doxycycline, luciferase activity decreased slowly following removal of doxycycline, suggesting the importance of tissue pools which maintained expression of the transgene. In SP-C-rtTA mice, exposure of the pregnant dam to doxycycline induced luciferase activity in fetal lung tissue as early as E10.5. Luciferase activity was maintained in the lung tissue of pups during the period of lactation when the mother received doxycycline in the drinking water. In the CCSP-rtTA mice, luciferase was not detected in the absence of doxycycline. In the SP-C-rtTA mice, luciferase activity was detected in the absence of doxycycline but was enhanced approximately 10-fold by administration of drugs. The SP-C-rtTA and CCSP-rtTA activator mice control the expression of transgenes in the developing and mature respiratory epithelium, and will be useful for the study of gene function in the lung. 相似文献
18.
Traykova-Brauch M Schönig K Greiner O Miloud T Jauch A Bode M Felsher DW Glick AB Kwiatkowski DJ Bujard H Horst J von Knebel Doeberitz M Niggli FK Kriz W Gröne HJ Koesters R 《Nature medicine》2008,14(9):979-984
We describe a transgenic mouse line, Pax8-rtTA, which, under control of the mouse Pax8 promoter, directs high levels of expression of the reverse tetracycline-dependent transactivator (rtTA) to all proximal and distal tubules and the entire collecting duct system of both embryonic and adult kidneys. Using crosses of Pax8-rtTA mice with tetracycline-responsive c-MYC mice, we established a new, inducible model of polycystic kidney disease that can mimic adult onset and that shows progression to renal malignant disease. When targeting the expression of transforming growth factor beta-1 to the kidney, we avoided early lethality by discontinuous treatment and successfully established an inducible model of renal fibrosis. Finally, a conditional knockout of the gene encoding tuberous sclerosis complex-1 was achieved, which resulted in the early outgrowth of giant polycystic kidneys reminiscent of autosomal recessive polycystic kidney disease. These experiments establish Pax8-rtTA mice as a powerful tool for modeling renal diseases in transgenic mice. 相似文献