Abstract
The Cre/lox recombinase system has been widely used for spatiotemporal control of gene expression in animal model systems, however, efficient methods to isolate zebrafish Cre drivers that reliably recapitulate endogenous expression patterns of known genes are in need. Here, we apply CRISPR/Cas9 double-strand breaks to direct targeted integration using short homology of a 2A-Cre recombinase transgene into the early coding region of proneural genes ascl1b, olig2 and neurod1. The pPRISM-2A-Cre targeting vector contains a linked lens-specific fluorescent secondary marker cassette for identification of transgenic embryos, and we observed high rates of germline transmission ranging from 10%-100% (2/20 olig2; 1/5 neurod1; 3/3 ascl1b). The established transgenic lines Tg(ascl1b-2A-Cre)is75, Tg(olig2-2A-Cre)is76, and Tg(neurod1-2A-Cre)is77 expressed functional Cre recombinase. in situ hybridization showed Cre expression reflected the pattern of the endogenous targeted proneural gene. In combination with Tg(ubi:loxP-EGFP-loxP-mCherry), each driver induced a switch from EGFP to mCherry expression in the expected proneural cell populations and their descendants, indicating efficient Cre-mediated recombination and excision of the floxed EGFP cassette. The results demonstrate Cre recombinase expression is driven by the native promoter and regulatory elements of the targeted genes. This approach provides a straightforward, efficient, and cost-effective method to generate cell type specific zebrafish Cre drivers whose spatial and temporal restricted expression mimics endogenous genes, surmounting the challenges associated with promoter BAC cloning and transposon mediated transgenesis.
Competing Interest Statement
SCE has a financial conflict of interest with LifEngine, and LifEngine Animal Health. Karl J Clark has a financial conflict of interest with Recombinetics, Inc, LifEngine and LifEngine Animal Health. JJE has a financial conflict of interest with Recombinetics, Inc; Immusoft, Inc; LifEngine and LifEngine Animal Health.
Footnotes
NIH R24OD020166 (MM, JJE, SCE, KJC), CNPq Brazilian National Council for Scientific and Technological Development (MPA).