Complex recombination constructs and fusion proteins.

<p>A) A BBW1.0-like construct is assembled into the middle entry vector via a Golden Gate reaction 8 entry vectors are used that contain either recombination elements (LoxP, Lox2272 and FRT sites) or fluorophores. Additionally, the ORF that encodes for the tamoxifen-inducible Cre recombinase was generated with the tamoxifen-sensitive estrogen receptor (ERT2) and flanking Flag tags. The Golden Gate assemblies are made in the Gateway^TM middle entry vector. Subsequently, these are combined with the ubiquituous beta actin 2 promoter and the globin intron-SV40 polyA. B-E) Both vectors were coinjected with Tol2 mRNA. 57% (n=43) of the fish were transiently eGFP-expressing. Those were split in two groups and treated with either tamoxifen dissolved in DMSO or DMSO alone. All fish transiently injected with the BBW1.0 construct and CreERT2 retained green fluorescence after addition of DMSO. Addition of tamoxifen induced cell-specific recombination in all embryos.</p

Basic nomenclature rules.

<p>The described nomenclature contains all necessary information to use the entry vectors for an assembly without the need to analyze the exact sequence. These rules are especially important for the generation of fusion proteins.</p

Golden Gate-based multisite mutagenesis.

<p>A) The FlpO ORF contains two internal BsaI sites. These are mutated by amplifying fragments of the FlpO ORF via PCR. The primers contain flanking BsaI sites that lead to compatible overhangs after restriction digest. The site-directed mutagenesis vector (pGG-sdm) contains BsaI sites for the mutagenesis assembly. Additionally, BamHI and KpnI sites allow the transfer of the mutated DNA assembly to Golden Gate entry vectors. From there, mRNA can be generated using SP6 RNA polymerase. Note that the pGG-sdm vector represents a standard Gateway^TM middle entry vector. B) A Golden Gate reaction is used to prepare a dual FRT-based recombination construct in the Gateway^TM middle entry vector. An LR reaction was prepared to generate a final expression construct. ISceI sites flank the expression cassette. The dual FRT element is driven by the zebrafish beta actin 2 promoter and a human globin intron with SV40 polyA is used in the 3’ entry vector. C) Zebrafish embryos were injected with the expression construct alone or in combination with FlpO mRNA. Without FlpO mRNA 64% (n=98) of the fish were positively injected and showed eGFP expression and the absence of mCherry. In combination with FlpO mRNA 60% (n=79) of the fish were positively injected and all of them showed the complete absence of green fluorescence and the appearance of red fluorescence, which is indicative for proper FlpO activity.</p

Generation of recombination templates using Golden Gate cloning.

<p>A) Schematic depiction of the generated recombination templates. The two recombination templates are based either on FRT or Lox elements in defined orientations. We included specific multiple cloning sites in the entry vectors -1, 3, 6 and 8’. B) Vector map of a subcloning destination vector with the restriction sites for the most common restriction endonucleases. The NcoI site (highlighted in red) is not present in the vector that lacks the ATG; otherwise restriction sites in all the vectors are identical.</p

Summary of overhangs and amino acid linkers.

<p>The entry vectors differ only in the overhangs that are created by BsaI restriction digest. These overhangs define the position of the fragment in the final assembly. Defined amino acid linker sequences are retained, since the overhangs as well as parts of the subcloning toolbox are retained in the final assembly. GS/x/GT are linkers introduced by the BamHI, KpnI restriction sites. TA-Cloning via the XcmI sites introduces the SGTA linker. Note compatible overhangs in consecutive entry vectors.</p

Golden Gate entry vector design and cloning.

<p>Cloning cassette used to fill Golden Gate entry vectors. Inserts can be introduced in two ways. XcmI restriction digest generates overhangs that can be used for TA cloning. Second, BamHI and KpnI sites can be used to clone inserts with different length either via standard ligation or an oligo annealing and cloning procedure. This cassette is flanked with BsaI sites used for the Golden Gate assembly. Each entry vector contains an SP6 promoter and a globin intron and SV40 polyA site flanking the insert for the generation of mRNA.</p