PUBLICATION
zMADM (zebrafish mosaic analysis with double markers) for single-cell gene knockout and dual-lineage tracing
- Authors
- Xu, B., Kucenas, S., Zong, H.
- ID
- ZDB-PUB-220225-3
- Date
- 2022
- Source
- Proceedings of the National Academy of Sciences of the United States of America 119(9): (Journal)
- Registered Authors
- Kucenas, Sarah
- Keywords
- single-cell gene knockout, lineage tracing, mosaic analysis with double markers, zebrafish
- MeSH Terms
-
- Animals
- CRISPR-Cas Systems
- Cell Lineage*
- Gene Knockdown Techniques
- Genetic Markers*
- Mosaicism*
- Single-Cell Analysis/methods*
- Zebrafish/genetics*
- PubMed
- 35197298 Full text @ Proc. Natl. Acad. Sci. USA
Citation
Xu, B., Kucenas, S., Zong, H. (2022) zMADM (zebrafish mosaic analysis with double markers) for single-cell gene knockout and dual-lineage tracing. Proceedings of the National Academy of Sciences of the United States of America. 119(9).
Abstract
As a vertebrate model organism, zebrafish has many unique advantages in developmental studies, regenerative biology, and disease modeling. However, tissue-specific gene knockout in zebrafish is challenging due to technical difficulties in making floxed alleles. Even when successful, tissue-level knockout can affect too many cells, making it difficult to distinguish cell autonomous from noncell autonomous gene function. Here, we present a genetic system termed zebrafish mosaic analysis with double markers (zMADM). Through Cre/loxP-mediated interchromosomal mitotic recombination of two reciprocally chimeric fluorescent genes, zMADM generates sporadic (<0.5%), GFP+ mutant cells along with RFP+ sibling wild-type cells, enabling phenotypic analysis at single-cell resolution. Using wild-type zMADM, we traced two sibling cells (GFP+ and RFP+) in real time during a dynamic developmental process. Using nf1 mutant zMADM, we demonstrated an overproliferation phenotype of nf1 mutant cells in comparison to wild-type sibling cells in the same zebrafish. The readiness of zMADM to produce sporadic mutant cells without the need to generate floxed alleles should fundamentally improve the throughput of genetic analysis in zebrafish; the lineage-tracing capability combined with phenotypic analysis at the single-cell level should lead to deep insights into developmental and disease mechanisms. Therefore, we are confident that zMADM will enable groundbreaking discoveries once broadly distributed in the field.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping