Constructs and Injections

We have cloned the Tol2 transposon from medaka DNA by using PCR primers designed based on Tol2 sequence (GeneBank accession no. D84375). Cloning of Tol2 was performed in two steps using PCR primers. The first PCR product was cloned into pBK-CMV (Stratagene, USA) using ApaI and NotI restriction sites incorporated into the primers, followed by cloning the 3’-end product into the construct using NotI and XhoI restriction sites so that both PCR fragments were joined at the internal NotI site. The construct containing EGFP (Clontech Laboratories, USA) under 2.2-kb promoter of keratin4 (krt4) gene (GeneBank accession no. AF440690) was obtained from Dr Zhiyuan Gong, National University of Singapore. A fragment containing the EGFP gene and only 460 bp of the krt4 promoter was amplified using this plasmid as a template. The product was digested by NotI and cloned into the Tol2 construct between EcoRV and NotI sites. The final enhancer trap construct consists of 2,671 bp of Tol2 5’end, 529 bp Tol2 3’ end, and the mini-krt4 promoter-EGFP fragment (1,200 bp). Transposase cDNA was cloned from the medaka cDNA. We used mMESSAGE mMACHINE kit (Ambion) for synthesis of in vitro transcribed capped transposase RNA and RNeasy Mini Kit (QIAGEN, Germany) for RNA cleanup. A total of 5-10 pg of plasmid DNA with 25-50 pg of in vitro synthesized transposase mRNA were co-injected into zebrafish embryos at the one– to two-cell stage. The actual concentration of RNA was empirically adjusted to produce 50% embryo survival rate. When they became adult crosses with wild type fish were used to obtain embryos with characteristic expression pattern of a fluorescent protein. These embryos were analyzed by TAIL-PCR to map insertions by obtaining sequence of DNA adjacent to the insertion site (Parinov et al., 2004). For in vivo remobilization of a single-copy of Tol2 transposon-based ET construct, 50-100 pg of transposase mRNA was injected into zebrafish embryos from the donor lines at one- or two-cell stage and embryos with changes of expression pattern in somatic cells were grown to adulthood and used to establish families (Kondrychyn et al., 2009).

Similar approach was used to generate transgenics expressing the membrane-tethered red fluorescent protein KillerRed (Teh et al., 2010) and more recently the nuclear-localized KillerRed (unpublished).