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卵母细胞胞浆内单精子注射(ICSI)技术作为辅助受精的一种手段,自从出现以来,就显示出广泛的应用前景.ICSI将体外受精和胚胎的显微操作技术相结合,大大降低对精子质量的要求,在畜牧业生产实践和哺乳动物生殖生理的基础研究中都有着十分重要的意义.然而,ICSI过程中的每一个环节都可能影响到其效果,最主要和最根本的影响因素是精子和卵子本身的状态.论文就精子有关方面的因素,简要综述了精子顶体与核周鞘、细胞骨架与中心粒、DTT处理和卵母细胞激活因子(SOAF)等对ICSI的影响. 相似文献
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单精子细胞质内注射(ICSI),是显微受精的一种,以其高效性已广泛应用于生产、科研和医学的诸多领域。本文从关于第1极体、精子制动、精子顶体、与IVF的关系、安全性等5方面,探讨了ICSI技术的研究进展,并简要地展望了其发展前景。 相似文献
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试验旨在探讨单精子胞浆内注射（intracytoplasmic sperm injection,ICSI）法生产猪体外受精卵和应用猪ICSI受精卵进行胞质注射生产转基因胚胎的可行性。首先对比了猪体外受精（in vitro fertilization,IVF）受精卵与ICSI受精卵的胚胎发育效率;然后观察了猪ICSI受精卵的双原核形成时间及效率,对精子注射到胞质后6~18 h分6个时间段进行地衣红染色,对比精子进入卵胞质后的状态及原核形成;最后对猪IVF受精卵受精后8~10 h及ICSI受精卵受精后12~14 h进行EGFP-N1质粒（20 ng/μL）胞质注射,观察胚胎发育效率及转基因效率。结果表明,ICSI受精卵的胚胎发育率（卵裂率89.4%和67.9%、囊胚率36.5%和16.1%）显著优于IVF组（P<0.05）,适合用于猪的体外受精卵试验;猪ICSI受精卵双原核在精子注射到卵胞质后12~14 h形成,双原核形成率为54.90%,显著高于其余5个试验组（P<0.05）;ICSI受精卵胞质注射组胚胎卵裂率（86.2%和66.3%）、囊胚率（30.0%和13.6%）及转基因效率（18.5%和0）均显著高于IVF受精卵胞质注射试验组（P<0.05）。本试验结果为采用ICSI受精卵进行胞质注射生产转基因猪的研究奠定了基础。 相似文献
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人Bcl-2乳腺定位表达转基因小鼠的建立 总被引：3，自引：1，他引：2
以 p CAT、p WB和 p WC为原始质粒 ,构建 WAP启动子调控的 Bcl- 2乳腺组织特异性表达载体 p WBS。载体用Bgl +Sal +Pvu 酶切 ,回收 3.0 kb的基因片段 WAP- Bcl- 2 - SV40 Poly A,通过显微注射方法导入 C5 7BL / 6 J×DBA/ 2 JF1代小鼠受精卵的雄原核内。共注射 10 0 0枚卵 ,将存活的约 6 0 0枚卵分别移植至 2 9只假孕母鼠输卵管内 ,获得仔鼠 5 5只。PCR检测 ,阳性鼠 12只。Southern blot检测 ,阳性鼠 4只 ,其中公、母鼠各 2只 ,在饲养过程中 ,2只公鼠均意外死亡。Western blot证实 ,1只母鼠 Bcl- 2表达阳性 ,其 F1代的 40只小鼠中 ,有 18只呈 PCR阳性 ,其中母鼠8只 ,在 5个月的反复怀孕过程中 ,未观察到乳房有任何肿瘤或肿块的出现 ,证实 Bcl- 2单独在转基因鼠乳腺中表达不会引起转基因鼠乳腺癌的发生。 相似文献
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以含有 1.6 kb的小鼠乳清酸蛋白 (WAP)上游调序列的 p CAT- WAP和含有人 c- myc c DNA的 p WM为原始质粒 ,构建了 WAP启动子调控下的 c- myc乳腺定位表达载体 p WCS。载体用 Bgl Bam H 酶切 ,回收 3.5 kb的基因片段 WAP- c- myc- SV40 Poly A,通过显微注射方法导入 C5 7BL/ 6 J× DBA/ 2 JF1 代小鼠受精卵的雄原核内。共注射10 0 0枚卵 ,将存活的约 6 0 0枚卵分别移植至 2 9只假孕母鼠输卵管内 ,获得仔鼠 45只。PCR检测 ,阳性鼠 9只 ,South-ern blot检测 ,阳性鼠 3只 ,其中 1只母鼠 ,2只公鼠 ,在饲养过程中 ,1只母鼠意外死亡。对 2只转基因公鼠的 F1代PCR检测表明 ,仅 1只公鼠具有遗传性 ,在所生的 2 7只 F1代小鼠中有 13只为 PCR阳性。 相似文献
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从Heta细胞RNA中通过RT-PCR扩增出c-myc基因的cDNA,将c-myc cDNA克隆入Tet-on系统的反应元件pTRE2载体,与含有Tet-on系统的调控元件pBC-rtTA基因混合显微注射到FVB小鼠受精卵的雄原核中,共注射603枚卵,将注射后存活的263枚卵移植到21只假孕母鼠输卵管内,有17只怀孕,共产仔59只.PCR和Southern blotting检测有2只双基因阳性公鼠和1只c-myc单基因阳性公鼠.3只公鼠的F1代PCR检测表明,其携带的c-myc外源基因均具有遗传性. 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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tPA乳腺组织特异性表达载体构建及转基因小鼠建立 总被引：3，自引：1，他引：2
以pKB、pcDNA3和pCPA为原始质粒，构建BLG启动子调控的组织型纤溶酶原激活剂（tPA）乳腺组织特异性表达载体pBTA。以SalⅠ酶切质粒pBTA，回收5.45kb基因片段BLG－tPA-bGHpolyA，通过显微注射，导入昆明小鼠受精卵的雄原核内。共注1365枚，将存活的1053枚移植的50只同期发情假孕母鼠的输卵管内，怀孕24只，共产仔79只，上述仔鼠通过PCR检测，结果19只阳性。Southern blotting检测上述19份PCR阳性小鼠基因组，其中4只为整合阳性，说明已获得了乳球蛋白启动子调控下的tPA转基因小鼠，为tPA在小鼠体内表达研究提供了必要条件。 相似文献
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Production of a transgenic pig expressing human albumin and enhanced green fluorescent protein 总被引：2，自引：0，他引：2
Naruse K Ishikawa H Kawano HO Ueda H Kurome M Miyazaki K Endo M Sawasaki T Nagashima H Makuuchi M 《The Journal of reproduction and development》2005,51(4):539-546
We introduced a fusion gene of human albumin and enhanced green fluorescent protein (EGFP) into porcine oocytes using the sperm vector method, and produced a piglet that showed clear expression of GFP in the hooves and skin. PCR and Southern blotting analysis of genomic DNA extracted from the piglet's tissues, including the liver, showed that the tissues carried the transgene. RT-PCR analysis demonstrated that both the human albumin and EGFP genes were expressed in the tissues. The fact that human albumin gene was integrated and expressed in the liver of the transgenic pig opened a way for us to achieve our goal, which was the use of transgenic pigs for the bioartificial liver support system. 相似文献
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This review describes the study of freeze-dried mouse sperm for practical application in preserving and transporting genetic resources. Freeze-dried sperm can be used to preserve and transport genetic resources; however, there still remain many areas which need to be studied. In particular, it is essential to assure long-term preservation over several decades or centuries. Recently, the theory of accelerated degradation kinetics to freeze-dried mouse sperm has been applied, and found that long-term preservation by conventional methods requires temperatures lower than -80 C. When the relationship between the pressure at primary drying and the preservation potential of freeze-dried mouse sperm was examined, a pressure of 0.37 mbar at primary drying significantly improved the developmental rate to the blastocyst stage. In addition, it has been shown that freeze-dried sperm stored at -80 C with and without transportation can retain their ability to generate viable offspring after storage for up to 2 years. Sperm chromatin structure assay (SCSA) was applied to mouse sperm freeze-dried under several conditions and compared the results with the embryonic developmental rates of freeze-dried sperm after intracytoplasmic sperm injection (ICSI) and with comet assay results. Furthermore, SCSA might be useful for estimation of developmental potential of fertilized eggs derived from ICSI using freeze-dried sperm in mice. 相似文献
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Anzai M Nishiwaki M Yanagi M Nakashima T Kaneko T Taguchi Y Tokoro M Shin SW Mitani T Kato H Matsumoto K Nakagata N Iritani A 《The Journal of reproduction and development》2006,52(5):601-606
Development of assisted reproductive technologies is necessary to obtain fertilized oocytes in a subfertile transgenic mouse strain. Here, we showed the application of laser-assisted drilling of the zona pellucida to in vitro fertilization of cryopreserved mouse oocytes with sperm from subfertile transgenic mice (C57BL/6N-Tg(UCP/FAD2)U8 strain). After cryopreservation by vitrification, the recovery and survival rates of the zona-drilled mouse oocytes were 97% (97/100) and 94% (91/97), respectively. In vitro fertilization of the cryopreserved zona-drilled mouse oocytes with sperm from the subfertile transgenic mice was greatly facilitated (60%, 55/91) compared to that of the cryopreserved zona-intact mouse oocytes (11%, 81/768). In vitro fertilized embryos that developed to the 2-cell stage were again cryopreserved by vitrification, and after warming they were transferred into recipient females. Subsequently, six viable offspring were delivered, and all were confirmed to be transgenic mice. These results indicate that laser-assisted zona drilling of oocytes combined with cryopreservation by vitrification may be a useful approach for large-scale production of in vitro fertilized embryos for managing transgenic mouse strains with reproductive disabilities such as subfertile sperm. 相似文献
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Pedro MOREIRA Serafín PéREZ-CEREZALES Ricardo LAGUNA Raúl FERNáNDEZ-GONZALEZ Belén Pintado SANJUANBENITO Alfonso GUTIéRREZ-ADáN 《The Journal of reproduction and development》2016,62(1):37-42
The production of transgenic animals is an important tool for experimental and applied biology. Over the
years, many approaches for the production of transgenic animals have been tried, including pronuclear
microinjection, sperm-mediated gene transfer, transfection of male germ cells, somatic cell nuclear transfer
and the use of lentiviral vectors. In the present study, we developed a new transgene delivery approach, and
we report for the first time the production of transgenic animals by co-injection of DNA and round spermatid
nuclei into non-fertilized mouse oocytes (ROSI). The transgene used was a construct containing the human CMV
immediate early promoter and the enhanced GFP gene. With this procedure, 12% of the live offspring we obtained
carried the transgene. This efficiency of transgenic production by ROSI was similar to the efficiency by
pronuclear injection or intracytoplasmic injection of male gamete nuclei (ICSI). However, ICSI required fewer
embryos to produce the same number of transgenic animals. The expression of Egfp mRNA and
fluorescence of EGFP were found in the majority of the organs examined in 4 transgenic lines generated by
ROSI. Tissue morphology and transgene expression were not distinguishable between transgenic animals produced
by ROSI or pronuclear injection. Furthermore, our results are of particular interest because they indicate
that the transgene incorporation mediated by intracytoplasmic injection of male gamete nuclei is not an
exclusive property of mature sperm cell nuclei with compact chromatin but it can be accomplished with immature
sperm cell nuclei with decondensed chromatin as well. The present study also provides alternative procedures
for transgene delivery into embryos or reconstituted oocytes. 相似文献
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Moisyadi S Kaminski JM Yanagimachi R 《Comparative immunology, microbiology and infectious diseases》2009,32(2):47-60
Even though intracytoplasmic sperm injection (ICSI) has been widely used for the production of offspring in human infertility clinics and in reproductive research laboratories using mice, many researchers engaged in animal transgenesis still consider it somewhat cumbersome. The greatest advantage of ICSI-mediated transgenesis is that it allows introduction of very large DNA transgenes (e.g., yeast artificial chromosomes), with relatively high efficiency into the genomes of hosts, as compared to pronuclear injection. Recently, we have developed an active form of intracytoplasmic sperm injection-mediated transgenesis (ICSI-Tr) with fresh sperm utilizing transposons. The transgenic efficiencies rival all transgenic techniques except that of lentiviral methods. 相似文献