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10:08 min
December 21st, 2021
DOI :
10.3791/63177-v
Chapters
0:05
Introduction
0:56
Synthesis of Guide RNAs
2:03
Microinjection of CRISPR-Cas9 Cocktail into a One-Cell Zebrafish Embryo to Generate Maternal Crispants
3:07
Screening for Somatic INDELs in F0 Injected Embryos
4:54
Identification of Maternal-Effect Phenotypes in Maternal Crispant Embryos
6:21
Sequencing Alleles in Maternal Crispant Haploids
8:18
Results: Identification of Maternal Effect Phenotypes in a Rapid and Resource Efficient Manner
9:08
Conclusion
Transcript
许多母系表达基因在早期发育中的作用目前尚不清楚。这种母体CRISPR技术允许快速鉴定母体效应基因及其在发育中的作用。将RNA复用引导到单个基因,使研究人员能够在一代中鉴定出新的母体效应表型。
这项研究有利于了解配子中mRNA转录物的功能,这是早期胚胎发生所必需的。该技术的关键是在单细胞阶段早期对胚胎进行微注射,并检查大多数引导RNA是否可以在注射的胚胎中产生体细胞突变。要为每个基因特异性寡核苷酸创建向导RNA模板,请分析恒定寡核苷酸并用T4 DNA聚合酶填充突出部分。
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Summary
早期发育依赖于母系遗传产品,其中许多产品的作用目前尚不清楚。在本文中,我们描述了一种使用CRISPR-Cas9鉴定单代母系效应表型的方案。
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