Gene Expression and Genome Editing Systems by Direct Delivery of Macromolecules Into Rice Egg Cells and Zygotes
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Author:
Date:
2020-07-20
[Abstract] Polyethylene glycol calcium (PEG-Ca2+)-mediated transfection allows rapid and efficient examination to analyze diverse cellular functions of genes of interest. In plant cells, macromolecules, such as DNA, RNA and protein, are delivered into protoplasts derived from somatic tissues or calli via PEG-Ca2+ transfection. To broaden and develop the scope of investigations using plant gametes and zygotes, a procedure for direct delivery of macromolecules into these cells has recently been established using PEG-Ca2+ transfection. This PEG-Ca2+-mediated delivery into rice egg cells/zygotes consists of four microtechniques, (i) isolation of gametes, (ii) production of zygotes by electrofusion of gametes, (iii) PEG-Ca2+-mediated delivery of ...
[摘要] [摘要 ] P olyethylene乙二醇钙(PEG-的Ca 2+ )介导的转染允许快速和有效的检查来分析感兴趣的基因的不同的细胞功能。在植物细胞中,大分子,例如DNA,RNA和蛋白质,通过PEG-Ca 2+ 转染被传递到源自体组织或愈伤组织的原生质体中。牛逼Ø拓宽和开发利用植物的配子和合子,直接递送大分子进入这些细胞最近已经采用PEG-钙建立过程调查范围2+ 转染。这种PEG-Ca 2+ 介导的传递至水稻卵细胞/受精卵的过程包括四个 微技术,(i )配子的分离,(ii)通过配子的电融合产生合子,(iii)PEG-Ca 2+ 介导的大分子向分离的卵细胞或产生的合子的传递,以及(iv)的培养和随后的分析转染的卵细胞/受精卵。因为对于完整的协议microtechniques (我)和(ii)已在户报告等人。,2016年,microtechniques (iii)和(iv)的主要是在第描述是协议。
[背景 ] 在被子植物中,受精和随后的发育事件,诸如胚胎发生和胚乳发育,发生在深深嵌入卵形组织胚囊(Nawaschin ,1898; Guignard ,1899;罗素,1992;拉加,2003) ...
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Robust Generation of Knock-in Cell Lines Using CRISPR-Cas9 and rAAV-assisted Repair Template Delivery
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Author:
Date:
2017-04-05
[Abstract] The programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease 9 (Cas9) technology revolutionized genome editing by providing an efficient way to cut the genome at a desired location (Ledford, 2015). In mammalian cells, DNA lesions trigger the error-prone non-homologous end joining (NHEJ) DNA repair mechanism. However, in presence of a DNA repair template, Homology-Directed Repair (HDR) can occur leading to precise repair of the lesion site. This last process can be exploited to enable precise knock-in changes by introducing the desired genomic alteration on the repair template. In this protocol we describe the delivery of long repair templates (> 200 nucleotides) using recombinant Adeno Associated Virus (rAAV) for CRISPR-Cas9-based knock-in of a ...
[摘要] 可编程集群定期间隔短回归度(CRISPR)相关核酸酶9(Cas9)技术通过提供在所需位置切割基因组的有效方式,彻底改变了基因组编辑(Ledford,2015)。 在哺乳动物细胞中,DNA损伤触发易发生非同源末端连接(NHEJ)DNA修复机制。 然而,在DNA修复模板的存在下,可以发生同源性定向修复(HDR),导致病变部位的精确修复。 可以利用最后的方法,通过在修复模板上引入所需的基因组改变来实现精确的敲入变化。 在本协议中,我们描述了使用重组腺相关病毒(rAAV)在人细胞系中进行基于CRISPR-Cas9的C-末端标签序列敲入的长修复模板(> 200个核苷酸)的递送。
尽管有关CRISPR-Cas9产生的敲门模型系统的大量报告,敲门砖报告仍然落后。由于许多应用,产生敲入细胞系仍然是基因组编辑的明显目标。敲入改变的引入通常依赖于修复模板DNA的存在,并且在位点特异性双链(ds)DNA断裂被引入接近改变位点的基因组中后,HDR修复机制的激活。不同的模板可以传送到修复机器,范围从含有广泛同源区域和可选选择盒的经典线性化载体到约200个核苷酸的单链(ss)DNA寡核苷酸(Chen等人, ...
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