Generation and application of endogenously floxed alleles for cell-specific knockout in zebrafish.
Dev Cell
; 58(22): 2614-2626.e7, 2023 Nov 20.
Article
in En
| MEDLINE
| ID: mdl-37633272
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Zebrafish
/
Integrases
Limits:
Animals
Language:
En
Journal:
Dev Cell
Journal subject:
EMBRIOLOGIA
Year:
2023
Document type:
Article
Affiliation country:
United States
Publication country:
EEUU
/
ESTADOS UNIDOS
/
ESTADOS UNIDOS DA AMERICA
/
EUA
/
UNITED STATES
/
UNITED STATES OF AMERICA
/
US
/
USA