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利用同源 CRISPR 对小鼠进行谱系条形码标记。

Lineage barcoding in mice with homing CRISPR.

机构信息

Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Center for Epigenetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

出版信息

Nat Protoc. 2021 Apr;16(4):2088-2108. doi: 10.1038/s41596-020-00485-y. Epub 2021 Mar 10.

Abstract

Classic approaches to mapping the developmental history of cells in vivo have relied on techniques that require complex interventions and often capture only a single trajectory or moment in time. We have previously described a developmental barcoding system to address these issues using synthetically induced mutations to record information about each cell's lineage in its genome. This system uses MARC1 mouse lines, which have multiple homing guide RNAs that each generate hundreds of mutant alleles and combine to produce an exponential diversity of barcodes. Here, we detail two MARC1 lines that are available from a public repository. We describe strategies for using MARC1 mice and experimental design considerations. We provide a protocol for barcode retrieval and sequencing as well as the analysis of the sequencing data. This protocol generates barcodes based on synthetically induced mutations in mice to enable lineage analysis.

摘要

经典的体内细胞发育史绘图方法依赖于需要复杂干预的技术,而且通常只能捕获单个轨迹或时间点。我们之前描述了一种发育条形码系统,通过合成诱导突变来解决这些问题,以记录基因组中每个细胞谱系的信息。该系统使用 MARC1 小鼠品系,其具有多个同源指导 RNA,每个 RNA 产生数百个突变等位基因,并组合产生指数多样性的条形码。在这里,我们详细介绍了两个可从公共库中获得的 MARC1 品系。我们描述了使用 MARC1 小鼠和实验设计注意事项的策略。我们提供了一种用于条形码检索和测序以及测序数据分析的方案。该方案基于小鼠中的合成诱导突变生成条形码,以实现谱系分析。

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