共查询到18条相似文献，搜索用时 171 毫秒
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鼻咽癌来源潜伏膜蛋白1的表达诱发转基因小鼠鼻咽不典型增生 总被引：2，自引：0，他引：2
为建立鼻咽癌来源潜伏膜蛋白1（N－LMP1）转基因小鼠模型，从整体的角度研究N－LMP1的功能，进一步阐明EB病毒在鼻咽癌变过程中的作用，应用DNA重组技术将角质细胞特异性启动子EDL2与N－LMP1连接，构建转基因EDL2－N－LMP1；并用显微注射技术，将转基因EDL2－N－LMP1注射入小鼠受精卵前核，再将注射后的受精卵植入假孕母鼠，获得转基因鼠，然后观察目的的基因在鼻咽部的表达和病理变化。本实验共获得53只转基因鼠，PCR法证明其中6只小鼠有N－LMP1基因扩增带，Suothern杂交显示PCR阳性小鼠有特异的杂交信号，整合率为11．3％，1只4月龄的转基因鼠出现了鼻咽部不典型增生，说明N－LMP1单独就能诱发小鼠鼻咽部发生癌前病变，为EB病毒可能是鼻咽癌的主要病因提供了有力的证据。 相似文献
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跨膜蛋白66(TMEM66)是与细胞凋亡以及癌症发生密切相关的重要功能基因,为了在个体水平研究其生物学功能,我们进行了TMEM66基因突变体(TMEM66V)转基因小鼠的构建。本研究通过原核注射法进行转基因操作,将获得的312个受精卵移植到13只代孕母鼠中,运用PCR、Southern blotting对出生的小鼠进行转基因鉴定,对于转基因阳性小鼠通过传代试验研究外源基因是否稳定整合,并通过反向PCR方法研究外源基因的整合方式。结果显示在出生的55只小鼠中有6只为转基因阳性,其中3只转基因小鼠可以稳定传代,表明这些小鼠中外源基因发生了稳定整合。反向PCR检测结果发现外源片段是以串联重复的方式整合到转基因小鼠的基因组中。本研究成功构建了TMEM66基因突变体转基因小鼠,为进一步研究TMEM66基因的功能奠定了基础。 相似文献
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人白细胞抗原 -B2 7( HLA-B2 7)与强直性脊柱炎 ( AS)有强相关性 ,为构建 AS动物模型以用于其发病机理和治疗的研究 ,将 HLA-B2 7基因与 β2 -微球蛋白基因等量混合后 ,利用显微注射技术注入昆明小鼠的1 3 70枚受精卵中 ,再将注射后存活的 1 0 50枚卵移植到 58只假孕母鼠体内 ,产仔 97只。PCR检测上述 97只转基因仔鼠 ,结果 2 7只为阳性。Southern blotting检测上述 2 7份 PCR阳性小鼠的基因组 ,结果 8只为阳性 ,表明已成功地建立了 HLA-B2 7转基因小鼠 相似文献
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《畜牧兽医学报》2015,(10)
旨在研究和分析牛FABP3基因在小鼠体内遗传和表达特性,对培育优质高产转基因肉牛新品种具有重要意义。本研究利用前期获得的4只FABP3转基因小鼠,通过制定的4组交配方案获得子代小鼠,并利用PCR、实时荧光定量PCR和组织原位表达技术对转基因牛FABP3基因小鼠的子代遗传特征,以及FABP3基因在G0代和G1代小鼠心、肝、肾、骨骼肌组织的表达情况进行了检测。RT-PCR检测结果表明,在G0代转基因小鼠中,牛FABP3基因能够表达;牛FABP3基因在转基因小鼠中可以遗传,G1代转基因小鼠平均阳性率达68.4%;相对定量PCR结果表明,转基因小鼠总FABP3基因在G1代小鼠心肌和骨骼肌中表达量相对于阴性个体有不同程度的增加;组织原位检测表明,在G0代个体中表达量较高,G1代个体中虽能看到绿色荧光,但略有降低。本研究表明,牛FABP3基因存在于小鼠基因组中,并能够被表达,且可以遗传给后代,这为后期进一步研究牛FABP3基因对小鼠骨骼肌脂肪含量的影响奠定坚实基础。 相似文献
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美国科学家已成功得到第只转基因灵长类动物罗猴 《中国兽医学报》2001,21(3):262
美国科学家已成功得到第 1只转基因灵长类动物罗猴 ( rhesus)。这只猴被命名为 ANDi(即 inserted DNA的倒拼 ) ,是用反转录病毒载体介导的转基因方法产生的 ,所转的外源基因是作为标记基因的绿色荧光蛋白基因。在过去 ,转基因小鼠为人类的许多疾病提供了研究模型 ,但由于小鼠与人的亲缘关系较远 ,因此转基因猴的建立可望弥补转基因小鼠动物模型的一些不足 ,加快某些人类顽疾的研究进程第1只转基因灵长类动物产生@郭志儒 相似文献
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本研究旨在构建Wip1过表达转基因小鼠,并对Wip1过表达转基因小鼠进行RNA水平上的筛选,为研究动脉粥样硬化的发生机制提供一种动物模型。通过逆转录聚合酶链式反应（RT-PCR）获得小鼠组织的cDNA,并以其为模板进行PCR扩增,对扩增片段进行胶回收,然后进行扩增片段和空载体的双酶切,将酶切后胶回收得到的Wip1基因全长与载体pcDNA3.1（+）进行连接,获得Wip1-pcDNA3.1（+）过表达载体,最终得到Wip1过表达转基因原代小鼠,并利用Southern blotting方法和实时荧光定量PCR方法对小鼠进行鉴定筛选。试验获得Wip1过表达转基因原代小鼠30只,利用Southern blotting方法鉴定出阳性小鼠11只,阳性率为36.67%,实时荧光定量PCR筛选Wip1过表达转基因小鼠阳性率为32.84%。以上结果表明,试验成功构建了Wip1-pcDNA3.1（+）过表达载体,并且经过Wip1基因在小鼠组织DNA和RNA水平上的鉴定筛选,成功获得了Wip1过表达转基因小鼠。 相似文献
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选择ICR小鼠为受试动物,应用显微注射仪,将线状人β干扰素基因,按2940～10000拷贝,1～3pL的剂量注射到供体受精卵的雄前核,然后移植到假孕受体的输卵管。实验先后注射了301个受精卵,移植到20只假孕受体,其中5只妊娠,产仔25只。从所获得的25只亲代小鼠的尾组织提取DNA,用内切酶BamHI消化,以[α-~(32)P]-dCTP标记的上述外源基因为特异性核酸探针,应用DNA印迹法检测这些小鼠DNA内外源基因的整合状况.结果表明,亲代小鼠中有人β干扰素基因重组子的转基因小鼠,杂交带在6.8kb位置。应用上述同样方法对亲代转基因小鼠的子1代组织DNA检测发现,绝大多数小鼠的标本除见到1条1.8kb人β干扰素基因的较弱杂交带外,还有数条5.0kb以上强弱各异的杂交带,表明这种外源基因不仅整合到了亲代小鼠的遗传物质内,而且可经垂直方式传给子代。人β干扰素基因的鼠体水平表达检测发现,1只子1代转基因小鼠的肿瘤组织浸出液中含有人β干扰素,滴度达160U/mL,表明整合在小鼠DNA内的人β干扰素外源基因不经诱导而有一定表达。 相似文献
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将缺失糖基磷脂酰肌醇（glycosyl phosphatidyl inositol,GPI）的Doppel的质粒（pDoppel 1-155）,用Not Ⅰ酶切,回收并纯化目的基因片段,通过显微注射,导入BALB/c小鼠受精卵的雄原核内,结果32只仔鼠出生,其中有6只PCR检测阳性。将其中1只F0代小鼠与正常小鼠交配,共获得21只F1代小鼠,经PCR检测,有10只仔鼠为阳性。对2只F1代PCR阳性小鼠脑组织进行RT-PCR检测,其中1只为阳性。采集3只F1代PCR阳性小鼠脑组织进行Western blotting检测,在小鼠脑组织能够表达17 ku的Doppel 1-155蛋白。本研究为进一步深入研究Doppel蛋白在体内的生物学特性提供了转基因动物模型。 相似文献
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为探究FoxO1对瘦素信号传导的调节机制，本研究构建了重组质粒pEF1α-myc-FoxO1ΔDBD，并将其转染于293Rb细胞，应用Western blotting检测突变体FoxO1ΔDBD的表达；然后采用DNA原核显微注射的方法制备含有突变体FoxO1ΔDBD的转基因小鼠模型，以鼠尾基因组DNA为模板，PCR扩增鉴定基因型；采用实时荧光定量PCR和免疫共沉淀方法检测FoxO1ΔDBD在转录水平和翻译水平的表达，并对FoxO1ΔDBD转基因鼠进行表型分析。结果显示，试验成功构建了重组质粒pEF1α-myc-FoxO1ΔDBD，并在293Rb细胞中检测到了突变体FoxO1ΔDBD的表达，继而获得了10只首建鼠，建立了10个繁育系。试验建立了高G-C含量PCR扩增产物的基因型鉴定方法，并成功地应用于转基因小鼠中。实时荧光定量PCR和免疫共沉淀结果显示，在转录水平和翻译水平上成功地检测出FoxO1ΔDBD的表达，表型分析结果显示，转基因小鼠体重显著低于野生型小鼠（P < 0.05），提示瘦素的作用增强。综上所述，本试验成功构建了FoxO1ΔDBD转基因小鼠，为探究FoxO1对瘦素信号传导的调节机制提供研究模型。 相似文献
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Manabu OZAWA Yukiko YAMASAKI Miho HIRABAYASHI Yukio KANAI 《Animal Science Journal》2003,74(3):181-185
Mammalian preimplantation embryos are susceptible to heat stress. This present study examined how maternal heat stress affects the development of mouse zygotes in vivo and in vitro. In Experiment 1, zygotes collected from female mice that were heat‐stressed for 12 h on day 1 of pregnancy were cultured in vitro. Maternally heat‐stressed zygotes developed normally to the two‐cell stage, but the majority of embryos failed to develop into morulae or blastocysts. In Experiment 2, pregnant mice were heat‐stressed on day 1 or from day 1 to day 3 of pregnancy. The number of living fetuses on day 14 of pregnancy was lower in heat‐stressed mice than in non‐stressed mice, but the difference was significant only in successively heat‐stressed mice. These results demonstrate that maternally heat‐stressed zygotes have reduced in vitro viability, but this phenomenon does not necessarily lead to embryo loss in the maternal environment. 相似文献
12.
由于传统显微注射法具有操作繁琐、技术要求高和外源基因导入率低等局限性,本试验对常规显微注射法进行改进,旨在建立一套比较完善的水平显微注射方法。采用水平微注射系统,将外源DNA片段导入胚胎的雄原核中,获得子代鼠,分娩后3周提取鼠尾基因组DNA,PCR法筛选转基因阳性鼠。本试验采用Puc/BLG IN S和Chym os in基因,共使用710枚鼠原核胚进行研究,胚胎经注射后移植了25只受体,产下仔鼠77只;妊娠率分别为33.3%(5/15)和50%(5/10),胚胎成活率分别为9.47%(41/433)和l3.00%(36/277),阳性率分别为4.88%(2/41)和8.33%(3/36)。将阳性雌鼠配种妊娠后,对外源基因整合情况进行检测,结果表明初步获得了转基因阳性小鼠。 相似文献
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Hirabayashi M Hirao M Takahashi R Kimura K Hirasawa K Ueda M Hochi S 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2000,62(10):1047-1052
Superovulation of female rabbits was induced by subcutaneous injection(s) of porcine FSH. Zygotes were recovered 17 to 19 hr after hCG injection and were classified into two categories under a microscope equipped with Nomarski interference-contrast optics at x 200 magnification: (A) zygotes with clearly visible pronuclei, or (B) zygotes with visualized pronuclei after 10 min centrifugation at 12,000 x g. No significant difference between strains was found in the proportion of category-A zygotes (JW 72.6% vs NZW 79.3%). Pronuclei of category-A zygotes were located in the center of the cytoplasm, and the pronuclei of category-B zygotes were slightly moved by centrifugation toward the mass of cytoplasmic lipid droplets. Exogenous DNA solution (5 microg/ml of fusion gene composed of bovine alphaS1-casein promoter and human growth hormone structural gene) was microinjected into the pronucleus of the JW zygotes. The pronucleus of category-A zygotes with a mean volume of 7.4 pl swelled up to 16.6 pl (132% increase), while that of category-B zygotes with a mean volume of 6.1 pl swelled up to 15.9 pl (148% increase). Nevertheless, similar proportions of category-A and category-B zygotes developed into offspring after transfer to recipient females (11.1 and 11.2%, respectively). The efficiency to produce hGH-carrying transgenic rabbits was 0.9% (2/235) from category-A zygotes and 0.5% (1/215) from category-B zygotes (P>0.05). To date, transgenic rabbits have been produced without centrifugation of pronuclear zygotes. However approximately 25% of fertilized rabbit zygotes can be used for DNA microinjection after they have been centrifuged to visualize their pronuclei. 相似文献
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Developmental competence and glutathione content of maternally heat-stressed mouse oocytes and zygotes 总被引：4，自引：0，他引：4
Takaya MATSUZUKA Manabu OZAWA Miho HIRABAYASHI Atsuko USHITANI Yukio KANAI 《Animal Science Journal》2004,75(2):117-124
The loss of developmental competence and the glutathione (GSH) content of maternally heat‐stressed mouse oocytes and zygotes were determined. In experiment 1, zygotes were collected from female mice that were heat‐stressed at 35°C for 10 h after hCG injection (oocyte maturation stage), or for 12 h on Day 1 of pregnancy (zygote stage), followed by in vitro culture. To minimize the effects of heat stress on the fertilization process, heat‐stressed oocytes that were fertilized in vitro were also included in this experiment. In experiment 2, heat‐stressed oocytes and zygotes were assayed for GSH content. The application of heat stress to the oocytes resulted in a significant decrease in the percentage of zygotes that developed to morulae or blastocysts, both for naturally fertilized oocytes (56.9% for heat‐stressed vs 85.4% for control) or in vitro‐fertilized oocytes (54.5%vs 73.6%). In the heat‐stressed zygotes, the disruption of embryonic development was more drastic (24.3%vs 90.3%), with the majority of zygotes being arrested at the two‐cell stage. In contrast, the GSH content decreased significantly in heat‐stressed zygotes, but not in heat‐stressed oocytes. These results demonstrate that the loss of developmental competence of early embryos is associated with a decrease in the GSH content of maternally heat‐stressed zygotes, but not of maternally heat‐stressed oocytes. 相似文献
16.
Kato M Ishikawa A Hochi S Hirabayashi M 《The Journal of reproduction and development》2004,50(2):191-195
The present study was conducted to examine the developmental potential to offspring of rat embryos cultured from 1-cell to morula/blastocyst stage. Pronuclear zygotes from Wistar x Wistar or (SD x DA) x Wistar strains were cultured in modified rat 1-cell embryo culture medium (mR1ECM) for 96 h in 5% CO(2) in air at 37 C. The proportion of the 3-way cross hybrid zygotes developing into morula/blastocyst stage (74%) was higher than that of the Wistar zygotes (66%). Day-5 morulae/blastocysts developed in vitro were transferred into Day-3 or -4 pseudopregnant recipients of Wistar or SD x DA strain. The transfer of cultured embryos resulted in the birth of offspring at 13-59%, while that of non-cultured control blastocysts showed birth rates of 35-65%. The best offspring rate of cultured embryos (59%) was obtained when the hybrid 1-cell zygotes were cultured in mR1ECM medium and transferred into the 2-days earlier uteri of SD x DA recipients. These results suggest that genetic background of recipients as well as donors is a possible factor affecting full-term development of rat morulae/blastocysts derived from 1-cell stage zygotes cultured in vitro. 相似文献
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作者综述了制备富含ω-3多不饱和脂肪酸克隆猪的研究意义和国内外的研究现状，并介绍了自己的研究进展。用化学合成的方法获得了C briggsae的ω-3脂肪酸去饱和酶基因sFat-1，并构建了哺乳动物细胞表达载体。通过显微注射的方法制备了转基因小鼠，sFat-1基因能够在转基因小鼠中产生更多更长链的而且更有价值的DHA以及DPA。在确认sFat-1基因功能的基础上，将载体转染到猪胎儿成纤维细胞，利用阳性的细胞克隆通过核移植方式制备富含ω-3多不饱和脂肪酸克隆猪。 相似文献
18.
Effects of voltage strength during electroporation on the development and quality of in vitro‐produced porcine embryos 
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下载免费PDF全文 K Nishio F Tanihara T‐V Nguyen T Kunihara M Nii M Hirata T Takemoto T Otoi 《Reproduction in domestic animals》2018,53(2):313-318
This study was conducted to determine suitable conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro‐produced porcine zygotes by electroporation. In the first experiment, when putative zygotes derived from in vitro fertilization (IVF) were electroporated by either unipolar or bipolar pulses, keeping the voltage, pulse duration and pulse number fixed at 30 V/mm, 1 msec and five repeats, respectively, the rate of blastocyst formation from zygotes electroporated by bipolar pulses decreased compared to zygotes electroporated by unipolar pulses. In the second experiment, the putative zygotes were electroporated by electroporation voltages ranging from 20 V/mm–40 V/mm with five 1‐msec unipolar pulses. The rate of cleavage and blastocyst formation of zygotes electroporated at 40 V/mm was significantly lower (p < .05) than that of zygotes electroporated at less than 30 V/mm. Moreover, the apoptotic nuclei indices of blastocysts derived from zygotes electroporated by voltages greater than 30 V/mm significantly increased compared with those from zygotes electroporated by voltages less than 25 V/mm (p < .05). When zygotes were electroporated with Cas9 mRNA and single‐guide RNA (sgRNA) targeting site in the FGF10 exon 3, the proportions of blastocysts with targeted genomic sequences were 7.7% (2/26) and 3.6% (1/28) in the embryos derived from zygotes electroporated at 25 V/mm and 30 V/mm, respectively. Our results indicate that electroporation at 25 V/mm may be an acceptable condition for introducing Cas9 mRNA and sgRNA into pig IVF zygotes under which the viability of the embryos is not significantly affected. 相似文献