| Generation of Mutant Pigs by Direct Pronuclear Microinjection of CRISPR/Cas9 Plasmid Vectors
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Author:
Date:
2017-06-05
[Abstract] A set of Cas9 and single guide CRISPR RNA expression vectors was constructed. Only a very simple procedure was needed to prepare specific single-guide RNA expression vectors with high target accuracy. Since the de novo zygotic transcription had been detected in mouse embryo at the 1-cell stage, the plasmid DNA vectors encoding Cas9 and GGTA1 gene specific single-guide RNAs were micro-injected into zygotic pronuclei to confirm such phenomenon in 1-cell pig embryo. Our results demonstrated that mutations caused by these CRISPR/Cas9 plasmids occurred before and at the 2-cell stage of pig embryos, indicating that besides the cytoplasmic microinjection of in vitro transcribed RNA, the pronuclear microinjection of CRISPR/Cas9 DNA vectors provided an efficient solution to ...
[摘要] 构建了一套Cas9和单引导CRISPR RNA表达载体。 需要一个非常简单的程序来制备具有高目标精度的特异性单向RNA表达载体。 由于在1细胞期的小鼠胚胎中已经检测到了合并的合子转录，所以将编码Cas9和GGTA1基因特异性单向RNA的质粒DNA载体微注射到合子原核中以确认 1细胞猪胚胎现象。 我们的研究结果表明，这些CRISPR / Cas9质粒引起的突变发生在猪胚胎的2细胞阶段之前和之后，表明除了体外转录的RNA的细胞质显微注射外，CRISPR / Cas9 DNA载体为产生基因敲除猪提供了有效的解决方案。
背景 由于最初发现了大肠杆菌基因组（Ishino）中的大肠杆菌基因组下游的32bp间隔的串联重复的高度保守的29个碱基对（bp）序列，等等，1987; Nakata等人，1989），在约50％至50bp的大小变化的短定期间隔重复序列的家族中发现约50％细菌和90％的古细菌（Makarova等人，2015）。根据它们的特征结构，Mojica（Mojica等人，2009）和Jansen（Jansen等人）引入了定期交织的短回文重复序列（CRISPR）的名称， ，2002），目前普遍使用。首先通过核苷酸序列比对（Jansen等人，2002）在CRISPR基因座的侧翼首先鉴定了一组CRISPR相关基因 cas1 ...
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| Ex utero Electroporation into Mouse Embryonic Neocortex
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Author:
Date:
2013-12-20
[Abstract] This technique allows highly efficient and reproducible transfer of DNA/RNA into the embryonic neocortex of rodents across multiple ages. Ex utero electroporation compliments the more technically difficult in utero electroporation technique by maximizing the number of embryos for available for a given experiment, as well as increasing the variety of constructs used in each experiment, thereby helping to reduce their overall numbers. Ex utero electroporation followed by short term organotypic slice culture of embryonic brain sections allows immediate access to multiple slices for choosing optimal ones for live-cell imaging experiments, and characterization of various NSC manipulations in the intact stem cell niche. (see also “In utero Electroporation of Mouse ...
[摘要] 这种技术允许高效和可重复地将DNA / RNA转移到多个年龄的啮齿动物的胚胎新皮质中。通过最大化可用于给定实验的胚胎数量，以及增加每个实验中使用的构建体的多样性，从而有助于减少它们的总体数量，子宫内电穿孔在技术上更难以满足子宫电穿孔技术。胚胎电穿孔之后进行短期器官切片培养，可以立即进入多个切片，选择用于活细胞成像实验的最佳切片，以及在完整干细胞生态位中对各种NSC操作的表征。 （参见“子宫内电穿孔小鼠胚胎脑”（Ge，2012）;“胚胎脑切片的器官切片培养”（Calderon de Anda，2013）。此外，子宫内电穿孔新生儿可用于体外原代细胞培养进一步解剖，分离成单个细胞，并根据标准技术在盖玻片或多孔培养皿中铺板：注意，该程序可以在电穿孔后立即进行，在异位基因表达发生之前，或在隔膜培养至仅收集电穿孔细胞的区域。
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| In utero Electroporation of Mouse Embryo Brains
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Author:
Date:
2012-07-20
[Abstract] This is a non-invasive technique to introduce transgenes into developing brains. In this technique, DNA is injected into the lateral ventricle of the embryonic brains, and is incorporated into the cells through electroporation. Embryos then continue their development in normal conditions in vivo. The effects of genes of interest can be evaluated at certain time points after in utero electroporation. This technique allows acute knockdown or over expression of genes of interest. Compensatory effects from other genes are less likely to happen; it also circumvents possible chronic detrimental effects.
[摘要] 这是将转基因引入发育中的大脑的非侵入性技术。 在该技术中，将DNA注入胚胎脑的侧脑室，并通过电穿孔掺入细胞中。 胚胎然后在正常条件下在体内继续其发育。 可以在子宫内电穿孔后的某些时间点评估感兴趣的基因的效应。 这种技术允许急性敲减或过表达感兴趣的基因。 其他基因的补偿效应不太可能发生; 它也绕过可能的慢性有害影响。
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