| 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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| Transfection of Human Naive CD4+ T Cells with PHA Activation and Neon Electroporation
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Author:
Date:
2013-08-05
[Abstract] Transfection of primary T cells can be challenging. This protocol describes a method to transfect primary human naive CD4+ T cells with an AP-1 luciferase reporter using low-level activation by phytohemagglutinin (PHA) and electroporation, as published (Palin et al., 2013). This technique is a modification of one previously described by our group (Cron et al., 2013). Anyone wishing to transfect murine T cells should consult the publication by Cron et al., 2013. This technique may be adapted for other primary T cell types by optimizing the Neon electroporation conditions, as described in the text. Other luciferase or GFP reporters may be used, and will require optimization of the stimulation conditions for that particular reporter.
[摘要] 原代T细胞的转染可能是具有挑战性的。 该方案描述了使用植物凝集素（PHA）和电穿孔的低水平激活，用AP-1荧光素酶报道子转染初级人初始CD4 + T细胞的方法，如公开的（Palin等人 。，2013）。 这种技术是先前由我们组描述的技术的修改（Cron等人，2013）。 任何希望转染鼠T细胞的人都应参考Cron等人2013年的出版物。该技术可以通过优化氖电穿孔条件来适应其它原代T细胞类型，如文中所述。 可以使用其他荧光素酶或GFP报道分子，并且将需要优化该特定报道分子的刺激条件。
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