...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>International Journal of Molecular Sciences >Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations
【24h】

Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations

机译：将CRISPR / Cas9相关的mRNA直接注射到孤雌激活猪卵母细胞的细胞质中导致Indel突变的频繁镶嵌

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B4 (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types.
机译：一些报道表明，通过一步合子显微注射CRISPR / Cas9相关成分的mRNA在猪中成功进行了基因组编辑。考虑到相对较长的妊娠期和高昂的住房成本，需要建立一个基于胚泡的检测方法来快速优化上述系统。作为概念验证，我们尝试使用孤雌激素激活的猪卵母细胞破坏编码α-1,3-半乳糖基转移酶的基因（GGTA1），该基因合成α-Gal表位。可以通过用荧光标记的异凝集素BS-I-B 4 （IB4）染色来监测α-Gal表位表达的缺乏，该标记特异性结合α-Gal表位。当向卵母细胞注射特异于GGTA1的引导RNA以及增强的绿色荧光蛋白（EGFP）和人Cas9 mRNA时，尽管几乎所有（96％，23/24），发育中的胚泡中有65％（24/37）表现出绿色荧光。显示出马赛克荧光图案。用IB4染色表明绿色荧光区域通常具有降低的与IB4的结合活性。在测试的16个样品中，六个（五个荧光和一个非荧光胚泡）具有indel突变，表明EGFP表达与突变诱导之间存在相关性。此外，提示合子显微注射mRNAs可能导致仔猪产生带有各种突变类型的细胞。

著录项

相似文献

  • 外文文献
  • 中文文献
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号