PUBLICATION
Generation and application of endogenously floxed alleles for cell-specific knockout in zebrafish
- Authors
- Shin, M., Yin, H.M., Shih, Y.H., Nozaki, T., Portman, D., Toles, B., Kolb, A., Luk, K., Isogai, S., Ishida, K., Hanasaka, T., Parsons, M.J., Wolfe, S.A., Burns, C.E., Burns, C.G., Lawson, N.D.
- ID
- ZDB-PUB-230827-66
- Date
- 2023
- Source
- Developmental Cell 58(22): 2614-2626.e7 (Journal)
- Registered Authors
- Burns (Erter), Caroline, Burns, Geoff, Lawson, Nathan, Parsons, Michael, Shin, Masahiro, Wolfe, Scot A.
- Keywords
- Cre/lox, conditional genetics, foxc1a, gata2a, rasa1a, ruvbl1, zebrafish
- MeSH Terms
-
- Alleles
- Animals
- Gene Knockout Techniques
- Integrases*/genetics
- Mice
- Mice, Knockout
- Zebrafish*/genetics
- PubMed
- 37633272 Full text @ Dev. Cell
Citation
Shin, M., Yin, H.M., Shih, Y.H., Nozaki, T., Portman, D., Toles, B., Kolb, A., Luk, K., Isogai, S., Ishida, K., Hanasaka, T., Parsons, M.J., Wolfe, S.A., Burns, C.E., Burns, C.G., Lawson, N.D. (2023) Generation and application of endogenously floxed alleles for cell-specific knockout in zebrafish. Developmental Cell. 58(22):2614-2626.e7.
Abstract
The zebrafish is amenable to a variety of genetic approaches. However, lack of conditional deletion alleles limits stage- or cell-specific gene knockout. Here, we applied an existing protocol to establish a floxed allele for gata2a but failed to do so due to off-target integration and incomplete knockin. To address these problems, we applied simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counterscreening and comprehensive molecular analysis, to identify off-target insertions and confirm targeted knockins. We subsequently used our approach to establish endogenously floxed alleles of foxc1a, rasa1a, and ruvbl1, each in a single generation. We demonstrate the utility of these 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 routine generation and application of endogenously floxed alleles in zebrafish.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping