Golden GATEway cloning--a combinatorial approach to generate fusion and recombination constructs

Abstract

The design and generation of DNA constructs is among the necessary but generally tedious tasks for molecular biologists and, typically, the cloning strategy is restricted by available restriction sites. However, increasingly sophisticated experiments require increasingly complex DNA constructs, with an intricacy that exceeds what is achievable using standard cloning procedures. Many transgenes such as inducible gene cassettes or recombination elements consist of multiple components that often require precise in-frame fusions. Here, we present an efficient protocol that facilitates the generation of these complex constructs. The golden GATEway cloning approach presented here combines two established cloning methods, namely golden Gate cloning and Multisite Gateway(TM) cloning. This allows efficient and seamless assembly as well as reuse of predefined DNA elements. The golden Gate cloning procedure follows clear and simple design rules and allows the assembly of multiple fragments with different sizes into one open reading frame. The final product can be directly integrated into the widely used Multisite Gateway(TM) cloning system, granting more flexibility when using a transgene in the context of multiple species. This adaptable and streamlined cloning procedure overcomes restrictions of "classical construct generation" and allows focusing on construct design.

Figure 1. Summary of the Golden GATEway cloning kit.

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).

Figure 2. Golden Gate entry vector design and cloning.

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.

Figure 3. Generation of recombination templates using Golden Gate cloning.

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.

Figure 4. Golden Gate-based multisite mutagenesis.

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.

Figure 5. Complex recombination constructs and fusion proteins.

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.