Gateway cloning strategy. A: Schematic of the 3-part LR recombination reaction used to generate expression constructs, using three entry clones and the pDestTol2pA/pA2 destination vector. B: Schematic of pDestTol2CG/CG2, destination vectors that include the cmlc2: EGFP-pA expression cassette. C: Strategy for generating a middle entry clone using pDONR221. Generation of 5′ and 3′ entry clones is similar, but uses different att sequences and donor vectors. D: Transformation of TOP10 cells with an LR recombination reaction yields two classes of colonies: clear (arrowheads) and opaque (arrow). Clear colonies yield the correct recombination product >99% of the time, whereas opaque colonies never do.

Validation of reporters and internal ribosome entry sequence (IRES) constructs. Embryos shown were injected at the one-cell stage with expression constructs made with the pISce-Dest destination vector, then mounted at 24 hours postfertilization (hpf) for confocal microscopy of the trunk. All plasmids tested generate functional bicistronic messages, as demonstrated by the presence of both mCherry and EGFP fluorescence. A-A″:bactin2: nlsmCherry-IRES-EGFPCAAXpA. B-B″:h2afx: nlsmCherry-IRES-EGFPCAAXpA. C-C″:bactin2: mCherry-IRES-EGFPCAAXpA. D-D″:bactin2: nlsmCherry-IRES-EGFPpA. E-E″:bactin2: mCherryCAAX-IRES-nlsEGFPpA. F:bactin2: nlsEGFP-IRES-EGFPCAAXpA. G:bactin2: EGFPCAAX-IRES-nlsEGFPpA. Scale bar = 50 μm.

Test of pDestTol2pA destination vector. Transposase greatly increases frequency and level of enhanced green fluorescent protein (EGFP) expression. A,B: DNA (30 pg) for pDestTol2pA; bactin2: EGFPCAAX-polyA was injected at the one-cell stage either without (A) or with (B) 25 pg of transposase RNA. Images were taken at 24 hours postfertilization.

Test of pDestTol2CG destination vector. Embryos were injected at the one-cell stage with 25 pg of DNA for pDestTol2CG; hsp70: mCherryCAAX-polyA, either with or without 25 pg of transposase RNA. A: Schematic of expression construct and graph showing fraction of embryos with a green heart at 30 hours postfertilization (hpf). Arrows in schematic indicate direction of transcription. Coinjection of transposase RNA greatly increased the fraction of embryos with green hearts. B-E: Further analysis of embryos selected at 30 hpf for green hearts. B-B′″: Embryos at 48 hpf, after heat shock for 1 hr at 30 hpf. C-C<′″: Embryos at 48 hpf, without heat shock. No red fluorescence is detectable at this magnification (C″).D-D′″: Close up of two heat-shocked embryos, shown at 48 hpf. Note bright red fluorescence (D″) and lack of ectopic green fluorescence (D′).E-E′″: Close up of two non-heat-shocked embryos, shown at 48 hpf. Note that there is no mCherry fluorescence in the heart. Dim, diffuse red fluorescence appears in one embryo (E″, arrowhead), indicating leaky expression from the hsp70 promoter.