Summary of the Golden GATEway cloning kit.

<p>A) Outline of the entire cloning procedure. Eight different entry vectors (pGG-EVx) contain different inserts (colored bars). These inserts are assembled in a predefined order using a Golden Gate reaction into Gateway^TM entry vectors at any position (Threeway Gateway^TM cloning). These can then be assembled to establish a final expression vector in an LR reaction. Compatible overhangs are indicated on each Golden Gate entry vector. B) The principle of Golden Gate cloning is illustrated in the top scheme; the principle of Gateway cloning is illustrated in the bottom scheme. Golden Gate cloning utilizes type II restriction endonucleases to generate compatible overhangs that can be ligated with T4 ligase. The ligation of the two compatible inserts from the entry vectors one and two is illustrated. Gateway cloning relies on recombination of specific att sites using a commercially available enzyme mix (LR Clonase II, Life Technologies).</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

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

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

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

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

The most suitable fluorescence intensity in zebrafish varies during development.

(A) Schematic overview of the mean of the tested fluorescent proteins. The lookup table is indicated at the top of the panel, the green fluorescent proteins are shown on the left hand side and the red fluorescent proteins are shown on the right hand side (embryos modified from [22]). (B) Green fluorescent protein fluorescence comparison over time. Plotted is the fluorescence normalized to mCherry per well against the hours post fertilization. Analysis was conducted as outlined in Fig 1A. (C) Red fluorescent protein fluorescence comparison over time. Plotted is the fluorescence normalized to eGFP per well against the hours post fertilization. Analysis was conducted as outlined in Fig 1A.</p

Codon adaptation of <i>eGFP</i> for medaka is not improving fluorescence.

(A) Relative comparison of in vivo fluorescence in medaka with in vivo fluorescence in E. coli. Indicated are red, green and yellow fluorescent proteins and a diagonal line for orientation. The red, green and yellow fluorescent proteins have been normalized to the same relative values published of mRFP1*, eGFP and Venus, respectively [18]. While many of the fluorescent proteins have similar properties, some are significantly different, reinforcing the need of a systematic review of fluorescent proteins in medaka. (B) Codon adaptation indices (CAIs) of all prior used sequences. Codon adaptation index of each sequence is plotted for medaka and human along with a diagonal line for orientation. Labelled are the two normalizing fluorescent proteins eGFP and mCherry, and the outlier mRuby2. For a plot with full labelling, refer to S2 Fig. (C) Pure codon usage table driven codon averaging does not improve fluorescence intensity of fluorescent proteins. OleGFP (medaka codon-averaged eGFP) fluorescence intensity is overall lower than SceGFP (yeast codon-averaged) and 25 to 30-fold lower than eGFP. (D) Codon adaptation indices of sequences used in order to score for the effect of codon averaging. Additional to the prior used sequences are an eGFP codon optimized for medaka (OleGFP) and an eGFP codon optimized for yeast (SceGFP).</p

The acute double pigment knockout utilizing the CRISPR/Cas9-system can be deployed in the injected generation.

(A) Medaka zygotes were microinjected with Cas9 mRNA and an injection mix according to Table 1. The injected embryos were scored for suitability for imaging either in the brightfield or in the green fluorescent spectrum. The ratio of the embryos suitable for imaging was determined and noted in panel B. (B) Transient targeting efficiency depends on the number of sgRNAs per gene. Compare op_1 and op_2. (C) By visual inspection no difference in pigmentation is observed between Oca2-/- or Tyr-/-, as well as Pax7a-/- Slc2a15b-/- embryos. (D) Medaka injected with the mix op_1 were raised, screened and imaged in F1 to compare the effects of sgRNA injection to wild-type. Wild-type embryos exhibit a dense, black pigment in the eyes, whereas adults additionally exhibit a non-translucent peritoneum. Fish injected with sgRNAs against oca2 and pnp4a show dramatic loss of pigmentation surrounding the eye and the peritoneum, rendering the fish more accessible for microscopy.</p