Parameters and Efficiency of Direct Gene Disruption by Zinc Finger Nucleases in Medaka Embryos

摘要: Zinc finger nucleases (ZFNs) can generate targeted gene disruption (GD) directly in developing embryos of zebrafish, mouse and human. In the fish medaka, ZFNs have been attempted on a transgene. Here, we developed procedures parameters for ZFN-mediated direct GD gonad-specifically expressed gsdf locus medaka. A pair was designed to target first exon their synthetic mRNAs were microinjected into 1-cell stage embryos. We reveal dose-dependent survival rate efficiency. fry, ZFN mRNA injection at 10 ng/μl led efficiency 30 %. This value increased up nearly 100 % when dose enhanced 40 ng/μl. typical series experiments ng/μl, 420 injected 94 adults, 4 which had altered alleles. leads efficacy ∼4 adulthood. Sequencing revealed wide variety subtle allelic alterations including additions deletions 1∼18 bp length ZFN-injected samples. Most importantly, one adults examined capable germline transmission 15.2 its F1 progeny. Interestingly, ontogenic analyses profile that commenced early development, continued during subsequent stages development primordia different adult organs three germ layers. These results demonstrate feasibility and—for time our knowledge—the chromosomal medaka