Transgenic manipulation in zebra fish by combination of Cre-loxP recombinant system, Tol2 transposon system, and RNAi technique

The overall goal of this research is to make precisely controlled transgene constructs to target genes in zebrafish and facilitate their functional studies. Many of the previous transgenic techniques (e.g. morpholino) can only produce transient gene expression or inhibition, which is not good for long-term studies. Also, transgenes that randomly integrate into the chromosomes are typically difficult to control and are poorly regulated. Thus, it would be of great benefit to develop a reversibly controlled transgenic tool to help study gene function. Here, we propose to combine three distinct techniques to generate a stable transgenic tool to facilitate gene functional study in zebrafish, including Cre-loxP recombinant system, Tol2 transposon system, and RNAi technique. The first objective is to combine both Cre-loxP and Tol2 systems to make a convertible and movable transgene construct for insertional inactivation assays in zebrafish. The generated transheterozygous rfp/gfp fragments can be translocated to other loci in the fish genome upon the introduction of Tol2 transposase, which may result in insertional mutations and/or new patterns of interest. The second objective is to make a precisely controlled shRNA transgene construct to inhibit the gfp and/or fli1 expression in zebrafish. Once the target gene is silenced, the zebrafish should show reduced green fluorescence, or some altered phenotypes (in the case of an endogenous blood cell specific target) that can be easily observed.;The results showed that we successfully established a stable homozygous pBa/RFP/loxP2/GFP/SBIR transgenic zebrafish line. The conversion from rfp to gfp expression was performed efficiently by heatshock-activated Cre recombinase in vivo. And the subsequent germline transmission of the converted gfp expression was observed in heat-shocked pTol-EF1alpha-RFP-loxP2-GFP transgenic fish outcrosses. The newly generated red/green transheterozygous fish from the cross of non-converted red fish and converted green fish are ready for further insertional mutation assay using Tol2 transgenic fish (Fuji70) or Tol2 mRNA. The construction of the shRNA plasmid was completed and the F0 microinjected zebrafish showed mosaic rfp expression in a few blood vessel cells as well as muscle cells, which indicated the potential success of fli1 driven shGFP transcription in vivo. Future goals include an examination of the efficiency of shGFP anti-gfp expression in pTol-Fli1-EGFP transgenic fish, and Cre recombinase mediated shGFP deletion and target gene expression recovery in vivo.