Recombinational Cloning with Plant Gateway Vectors

Abstract

The study of biological systems relies to a large extent on DNA cloning technologies enabling the analysis of recombinant genes through transgenic research. In this context, the advent of recombinational cloning methods was a significant progress because DNA fragments can now be assembled regardless of their sequence. In particular, the Gateway system was designed to join fragments in a predefined order, orientation, and reading frame. The recent development of transformation vectors and large-scale clone resources amply demonstrate that plant researchers have adopted the Gateway platform and that it will remain an important asset in projects requiring systematic cloning, modular assembly, and expression in various contexts. Agrobacterium tumefaciens binary vectors are widely used for plant transformation. They vary in size, origin of replication, bacterial selectable markers, T-DNA borders, and overall structure. Binary vectors are cumbersome to handle in conventional cloning schemes involving DNA restriction and ligation reactions, and substantial efforts have been invested in the creation of smaller vectors with a choice of unique restriction sites within the T-DNA region (Hajdukiewicz et al., 1994; Hellens et al., 2000a; Goderis et al., 2002; Tzfira et al., 2005; http://www.cambia.org/). But the recent introduction of robust site-specific recombinational cloning methods has greatly facilitated the construction of expression units in a large variety of in vivo and in vitro systems (Marsischky and LaBaer, 2004). In particular, the Gateway technology developed originally by researchers at Life Technologies, Inc. (Hartley et al., 2000), and now commercialized by Invitrogen, has been endorsed by a large community and compatible vectors have been created for most applications requiring the creation of recombinant DNA molecules. This review gives a summary of the site-specific Gateway recombinational cloning system and presents related vectors generated by different plant research laboratories. Schematic representation of att sites and Gateway recombination reactions. A, In a BP clonase reaction, attB sites (in a PCR product or plasmid) recombine with the matching attP sites of a donor vector (pDONR) to yield attL sites in a novel entry vector (pENTR) and attR sites in a byproduct. B, In a LR clonase reaction, attL sites in an entry vector (pENTR) recombine with the matching attR sites of a destination vector (pDEST) to yield attB in a novel expression vector (pEXPR) and attP in a byproduct. C, In a single MultiSite LR clonase reaction, the compatible att sites carried by entry clones originating from independent BP clonase reactions (in this example pENTR L4-fragment1-R1, pENTR L1-fragment2-L2, and pENTR R2-fragment3-L3) and by a MultiSite destination vector (for example pDEST R4-R3) recombine to yield a single contig in which the DNA fragments of interest are separated by short attB sites. Blue boxes, DNA fragments of interest assembled in BP and LR clonase reactions; black box with vertical white stripe, attB sites, also at the core of the attP, attL, and attR sites; yellow box, portion of the attP and attL sites; red box, portion of the attP and attR sites. B1 to B4, attB1 to attB4 sites; L1 to L4, attL1 to attL4; R1 to R4, attR1 to attR4. To enable directional cloning, the inventors of the Gateway system engineered variants of the original attB, attP, attL, and attR sites so that attB1 will react specifically with attP1, but not with attP2, attP3, etc. (Cheo et al., 2004; Sasaki et al., 2004). The standard oriented BP and LR reactions involving the att1 and att2 series (Fig. 1, A and B) always maintain the frame register that is necessary to build translational fusions. The availability of multiple recombination site series is also useful in alternative protocols to assemble simultaneously multiple DNA fragments in a single LR clonase reaction yielding an expression clone with two or more contiguous DNA fragments in predefined order and orientation. This specific technology with more than two att series has been dubbed MultiSite Gateway and has already been adapted for the construction of plant binary destination vectors (Fig. 1C; Karimi et al., 2005). For additional information about the basics of the Gateway technology, the reader is referred to the manuals accessible online (http://www.invitrogen.com/). A given destination vector intended for a particular functional assay can be recombined with any sequence captured in a compatible entry clone. Conversely, the same entry clone (such as an open reading frame [ORF] entry clone) can be recombined with many different destination vectors. The resulting expression clones are the constructs used to test gene functions, for example after transformation into plants. Typically, the desired plasmids are created by in vitro recombination, transformed in Escherichia coli strains, and segregated from other reaction by-products and input vectors through appropriate a