Generation and Application of Endogenously Floxed Alleles for Cell-Specific Knockout in Zebrafish
Author Type(s)
Student
Document Type
Article
Publication Date
11-20-2023
DOI
10.1016/j.devcel.2023.07.022
Journal Title
Developmental Cell
Keywords
Mice, Animals, Mice, Knockout, Zebrafish, Alleles, Integrases, Gene Knockout Techniques
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.
Recommended Citation
Shin, M., Yin, H., Shih, Y., 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., & Lawson, N. D. (2023). Generation and Application of Endogenously Floxed Alleles for Cell-Specific Knockout in Zebrafish. Developmental Cell, 58 (22), 2614-2626. https://doi.org/10.1016/j.devcel.2023.07.022
