Yamagata Masahito, Sanes Joshua R
Center for Brain Science and Department of Molecular and Cellular Biology, Harvard University, Cambridge MA, 02138, USA.
Bio Protoc. 2021 Aug 5;11(15):e4105. doi: 10.21769/BioProtoc.4105.
The abilities to mark and manipulate specific cell types are essential for an increasing number of functional, structural, molecular, and developmental analyses in model organisms. In a few species, this can be accomplished by germline transgenesis; in other species, other methods are needed to selectively label somatic cells based on the genes that they express. Here, we describe a method for CRISPR-based somatic integration of reporters or Cre recombinase into specific genes in the chick genome, followed by visualization of cells in the retina and midbrain. Loci are chosen based on an RNA-seq-based cell atlas. Reporters can be soluble to visualize the morphology of individual cells or appended to the encoded protein to assess subcellular localization. We call the method eCHIKIN for lectroporation- and RISPR-mediated omology-instructed nock-.
标记和操纵特定细胞类型的能力对于模式生物中越来越多的功能、结构、分子和发育分析至关重要。在少数物种中，这可以通过种系转基因来实现；在其他物种中，则需要基于细胞所表达的基因来选择性标记体细胞的其他方法。在这里，我们描述了一种基于CRISPR的方法，可将报告基因或Cre重组酶体整合到鸡基因组中的特定基因中，随后对视网膜和中脑的细胞进行可视化。根据基于RNA测序的细胞图谱选择位点。报告基因可以是可溶的，以可视化单个细胞的形态，或者附加到编码蛋白上以评估亚细胞定位。我们将该方法称为eCHIKIN，即电穿孔和RISPR介导的同源性指导敲入。
