Poster for TAGC-Bing.pdf (143.9 MB)

Establishment and characterization of zebrafish mosaic analysis with double markers system for phenotypic analysis at the single-cell resolution

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posted on 20.04.2020 by Bing Xu, Sarah Kucenas, Hui Zong

Conditional knockout animal model is developed to study gene functions in a specific cell type. However, field of mutant cells prevent one from studying interactions between mutant and WT cells. To achieve gene inactivation in rare cells, genetic mosaic

animal models, including mosaic analysis with a repressible cellular marker (MARCM) in Drospholia, mosaic analysis with double markers (MADM) in mouse, were developed. Through Flp/FRT or Cre/loxP mediated inter-chromosomal mitotic recombination, sporadic

mutant cells can be generated and unequivocally labeled by fluorescent protein, allowing phenotypic analysis at single-cell resolution. Although these models led to many groundbreaking discoveries, dynamic analysis of mutant and wild type cells behavior in vivo is

still difficult due to the non-transparent nature of these model organisms. Here, we report the establishment of a zebrafish Mosaic Analysis with Double Markers (zMADM) system, to take advantage of the resolution provided by MADM and embryonic transparency of

zebrafish. The two zMADM cassettes were knocked into the pre-selected genomic region with CRISPR/Cas9, respectively, to create two stock lines. After breeding two lines, the injection of Cre mRNA or plasmid produced MADM-labeled cells, confirming the successful

establishment of the zMADM system. We quantified fluorescently labeled cells and determined that the labeling efficiency with Cre mRNA in zMADM was ~0.5%. To demonstrate the application of zMADM system, we showed the Cre controlled by cell type-specific promoters

can sparsely label those cell types faithfully. Next, we used live imaging to witness the birth of two sibling cells, and traced the development and migration of them in zMADM zebrafish. In particular, we discovered asymmetric cell division of neural progenitor

cells contributes to the formation of optic tectum neuronal column. In summary, we have successfully established and characterized zMADM system, which can be used to study genetic determinants of lineage development in vivo, and to model human diseases for

both basic and translational studies.


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