PUBLICATION
Efficient knock-in method enabling lineage tracing in zebrafish
- Authors
- Mi, J., Andersson, O.
- ID
- ZDB-PUB-230307-45
- Date
- 2023
- Source
- Life science alliance 6(5): (Other)
- Registered Authors
- Andersson, Olov
- Keywords
- none
- MeSH Terms
-
- Animals
- DNA Primers
- Genetic Loci
- Hematopoietic Stem Cells
- Liver*
- Zebrafish*/genetics
- PubMed
- 36878640 Full text @ Life Sci Alliance
Citation
Mi, J., Andersson, O. (2023) Efficient knock-in method enabling lineage tracing in zebrafish. Life science alliance. 6(5).
Abstract
Here, we devised a cloning-free 3' knock-in strategy for zebrafish using PCR amplified dsDNA donors that avoids disrupting the targeted genes. The dsDNA donors carry genetic cassettes coding for fluorescent proteins and Cre recombinase in frame with the endogenous gene but separated from it by self-cleavable peptides. Primers with 5' AmC6 end-protections generated PCR amplicons with increased integration efficiency that were coinjected with preassembled Cas9/gRNA ribonucleoprotein complexes for early integration. We targeted four genetic loci (krt92, nkx6.1, krt4, and id2a) and generated 10 knock-in lines, which function as reporters for the endogenous gene expression. The knocked-in iCre or CreERT2 lines were used for lineage tracing, which suggested that nkx6.1+ cells are multipotent pancreatic progenitors that gradually restrict to the bipotent duct, whereas id2a+ cells are multipotent in both liver and pancreas and gradually restrict to ductal cells. In addition, the hepatic id2a+ duct show progenitor properties upon extreme hepatocyte loss. Thus, we present an efficient and straightforward knock-in technique with widespread use for cellular labelling and lineage tracing.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping