Novel Approaches to Transgenesis in the Teleost Medaka (Oryzias latipes)

Abstract

The aim of thesis was to improve the generation of transgenic medaka fish. General transgenesis including transient expression of reporter genes and germ line integration of reporter genes was improved by application of two novel techniques. In addition, one of these methods allows for the first time efficient enhancer trapping in fish. First, a transposon-based approach using the artificially reconstructed Sleeping Beauty (SB) transposon was established. To address the potential of SB for transgenesis, microinjection experiments were performed. Transgenes (GFP) and promoter fragments were flanked with the SB recognition sequences (inverted direct repeats (IR/DR)) and injected into one-cell stage medaka embryos with or without SB10 mRNA. Upon injection of a control construct, that lacks SB recognition sequences and without transposase, only 13 % of surviving embryos expressed GFP uniformly in the entire body. Conversely, when SB IR/DRs were included, uniform, promoter-dependent expression was the predominant effect (45 %). The presence of IR/DRs alone strongly enhanced promoter-dependent transgene expression in G0, indicating that SB IR/DRs significantly enhance transient transgene expression. G0 expression was a reliable indicator for the efficient selection of transgenic founders. Embryos that exhibit a uniform GFP expression in G0 result in the highest yield of transgenic fish. This facilitates an easy selection of putative founder fish for medium- to large-scale approaches. The SB system enhanced total transgenesis frequencies to 32 % compared to 4 % resulting from control construct injections. Strikingly, 12 % (21/174) of the transgenics featured typical characteristics of enhancer trap lines, i.e. spatially and/or temporally restricted transgene expression due to regulation imposed by sequences adjacent to the insertion site. Thus, a set of transgenic lines expressing GFP in developmentally important structures/organs can be established and used without devoting a major effort on the isolation and characterization of promoter elements. Second, a meganuclease approach was applied. Transgenes of interest were flanked by two I-SceI meganuclease recognition sites, and co-injected together with the I-SceI meganuclease enzyme into medaka embryos at the one-cell stage. Upon injection, the promoter-dependent expression was strongly enhanced. Already in G0, 78 % of injected embryos exhibited uniform promoter dependent expression compared to 26 % when injections were performed without meganuclease. The transgenesis frequency was raised to 30.5 % compared to 5-18 % for naked DNA. Even more striking was the increase in germ line transmission rate. In standard protocols it does not exceed a few percent, the number of transgenic F1 offspring of an identified founder fish generated with I-SceI reached the optimum of 50 % in most lines, indicating genome insertion events already at the one-cell stage. Meganuclease co-injection thus provides a simple and highly efficient tool to improve transgenesis by microinjection.