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Development of an efficient transgenic method bu using GFP-reporter gene and nuclear transfer

使用 GFP 报告基因和核移植开发有效的转基因方法

基本信息

批准号：
10556065
负责人：
FUNAHASHI Hiroaki
金额：
$ 7.87万
依托单位：
OKAYAMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10556065/
关键词：
cattle transgenic GFP nuclear transfer early development in vitro fertilization swine polyspermy

项目摘要

The effect of timing of microinjection of DNA constructs on the efficiency of transgenic embryo production and improved efficiency and quality through combining EGFP as a reporter gene with nuclear transfer techniques were examined. From 12-24 h after insemination, constructs of pCXNeo-EGFP were microinjected into a pronucleus of bovine IVM-IVF zygotes. Due to difficulty in visualizing the pronucleus, the incidence of successful injection of linear DNA into pronucleus was higher when injected from 20 to 24 h, as compared with an early period from 12 to 16 h after insemination. However, developmental competence of DNA-injected zygotes and the EGFP-expression rate were not different among the variously-timed microinjections. A majority of the embryos expressing EGFP signal were mosaic. Following nuclear transfer of blastomeres expressing EGFP, 4.5 % of morulae that developed from the NT-eggs had a strong EGFP signal in all live blastomeres. In other embryos, EGFP signal had been lost. When cells derived from the EGFP-positive NT morulae were subcultured, all the cells expressed strong EGFP signal at the second passage and demonstrated neomycin resistance. These results show that transient expression of non-integrated EGFP appears frequently in EGFP-positive bovine embryos and that additional selection of EGFP-positive morulae after nuclear transfer of EGFP-positive blastomeres would facilitate selection of transgenic embryos.Efficient methods to produce porcine zygotes in vitro for the microinjection of DNA constructs were also developed. Replacement of caffeine with adenosine or fertilization promoting peptide, which induced capacitation but prevented acrosome reaction of boar spermatozoa, reduced the incidence of polyspermic penetration of porcine oocytes without any reduction in penetration.

研究了显微注射 DNA 构建体的时间对转基因胚胎生产效率的影响，以及通过将 EGFP 作为报告基因与核移植技术相结合来提高效率和质量。授精后 12-24 小时，将 pCXNeo-EGFP 构建体显微注射到牛 IVM-IVF 受精卵的原核中。由于难以观察原核，与授精后 12 至 16 小时的早期相比，在 20 至 24 小时内注射线性 DNA 成功注射到原核的发生率较高。然而，注射 DNA 的受精卵的发育能力和 EGFP 表达率在不同时间的显微注射之间没有差异。大多数表达 EGFP 信号的胚胎是嵌合体。对表达 EGFP 的卵裂球进行核移植后，从 NT 卵发育而来的桑葚胚中有 4.5% 在所有活卵裂球中都具有强 EGFP 信号。在其他胚胎中，EGFP 信号已丢失。当对源自EGFP阳性NT桑葚的细胞进行传代培养时，所有细胞在第二代时均表达强EGFP信号并表现出新霉素抗性。这些结果表明，非整合EGFP的瞬时表达频繁出现在EGFP阳性牛胚胎中，并且在EGFP阳性卵裂球核移植后额外选择EGFP阳性桑葚将有利于转基因胚胎的选择。还开发了在体外产生用于显微注射DNA构建体的猪受精卵的有效方法。用腺苷或促受精肽替代咖啡因，可诱导获能但阻止公猪精子顶体反应，降低猪卵母细胞多精穿透的发生率，但穿透力没有任何降低。