Generation of floxed alleles for cell-specific knockout in zebrafish
Shin, M.; Nozaki, T.; Toles, B.; Kolb, A.; Luk, K.; Isogai, S.; Ishida, K.; Hanasaka, T.; Parsons, M. J.; Wolfe, S. A.; Lawson, N. D.
Show abstract
A benefit of the zebrafish as a model is its amenability to genetic approaches. However, a lack of conditional deletion alleles makes it a challenge to perform stage- or cell-specific knockout. Here, we initially applied an existing protocol to establish a floxed allele for gata2a and encountered several issues, including off-target integration and incomplete knock-in. To address these problems, we developed a protocol incorporating simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counter-screening to identify off-target insertions. We applied this protocol to establish a floxed allele of foxc1a in a single generation. We demonstrate the utility of our floxed alleles by verifying Cre-dependent deletion, which yielded expected phenotypes in each case. Finally, we used the floxed gata2a allele to demonstrate an endothelial autonomous requirement in lymphatic valve development. Together, our results provide a framework for straightforward generation and application of floxed alleles in zebrafish.
Matching journals
The top 3 journals account for 50% of the predicted probability mass.
Similar papers in this journal
Similar papers in this journal
- Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development 95%
- Rapid and efficient adaptation of the dTAG system in mammalian development reveals stage specific requirements of NELF 95%
- The mesodermal source of fibronectin is required for heart morphogenesis and cardiac outflow tract elongation by regulating cell shape, polarity, and mechanotransduction in the second heart field 95%
Similar papers in this journal
- Genetic context of transgene insertion can influence neurodevelopment in zebrafish 96%
- Scarless engineering of the Drosophila genome near any site-specific integration site 94%
- Caenorhabditis elegans junctophilin has tissue-specific functions and regulates neurotransmission with extended-synaptotagmin 94%