Delivery of exogenous proteins into eggs by injection into the mother's ovary (IMO) in zebrafish.

Genome editing has had profound effects on biological experimentation and can now be applied to many organisms, including non-conventional models. However, the introduction of genome editing components is time- and labor-consuming and sometimes requires special skills for microinjection. In this study, we developed a technique to deliver exogenous proteins into eggs by injection into the mother's ovary (IMO), which leads to the delivery of CRISPR/Cas9 into the eggs of oviparous animals, including fish. To test this technique, we examined whether exogenous proteins tagged with GFP or luciferase (Luc), and fluorescent-labeled RNP (Cas9 and sgRNA complex), can be delivered into eggs by IMO. When GFP-Luc or Cas9-Luc was delivered by IMO, their incorporation into fertilized eggs was confirmed by GFP fluorescence or luciferase activity; proteins were accumulated in the yolk. Cas9-RNP (targeting tyrosinase) was also incorporated into the eggs. However, genome editing of the target gene, tyrosinase, was not observed yet. This is presumably because the RNP delivered by IMO was packed in the yolk granules and did not reach into the embryonic nuclei. Thus, this report shows that exogenous molecules including Cas9-RNP were successfully delivered into fertilized eggs by IMO. Transferring the delivered RNP into nuclei will be critical for successful genome editing via the IMO delivery system.

Plasmid delivery by electroporation into fish skeletal muscle for recombinant protein secretion and uptake by oocytes.

We examined the efficiency of electroporation for the delivery of plasmid into the skeletal muscle and also examined the subsequent secretion of recombinant protein into the circulation system, using zebrafish, Japanese flounder, and bubble-eye goldfish. The expression area of GFP fluorescence was markedly expanded by electroporation. Introduced plasmid was retained in the muscle cells and continued to express GFP for at least 180 days in zebrafish, indicating the long lifespan of plasmid DNA in the muscle cell. Luciferase and a fusion of growth hormone (GH) and luciferase were secreted into the blood from muscle electroporated with CMV:secNluc and CMV:GH-Luc plasmids, respectively, indicating that recombinant proteins such as peptide hormones can be supplied to the blood by plasmid electroporation into muscle. Interestingly, luciferase activity was detected from fertilized eggs laid by zebrafish females that had been electroporated with CMV:secNluc, indicating that maturing oocytes incorporated recombinant protein from the blood stream that had been secreted from the muscle. The plasmid electroporation system reported here also may work for the delivery of recombinant proteins, such as Cas9, into the oocytes. Since the GH-Luc fusion protein was detected in the lymph of the eye-sac of bubble-eye goldfish, this fish may be useful for the production of recombinant protein.