Concerted Assembly and Cloning of Multiple DNA Segments Using In Vitro Site-Specific Recombination: Functional Analysis of Multi-Segment Expression Clones

Abstract

The ability to clone and manipulate DNA segments is central to molecular methods that enable expression, screening, and functional characterization of genes, proteins, and regulatory elements. We previously described the development of a novel technology that utilizes in vitro site-specific recombination to provide a robust and flexible platform for high-throughput cloning and transfer of DNA segments. By using an expanded repertoire of recombination sites with unique specificities, we have extended the technology to enable the high-efficiency in vitro assembly and concerted cloning of multiple DNA segments into a vector backbone in a predefined order, orientation, and reading frame. The efficiency and flexibility of this approach enables collections of functional elements to be generated and mixed in a combinatorial fashion for the parallel assembly of numerous multi-segment constructs. The assembled constructs can be further manipulated by directing exchange of defined segments with alternate DNA segments. In this report, we demonstrate feasibility of the technology and application to the generation of fusion proteins, the linkage of promoters to genes, and the assembly of multiple protein domains. The technology has broad implications for cell and protein engineering, the expression of multidomain proteins, and gene function analysis.[Supplemental material is available online at www.genome.org.]The cloning and manipulation of DNA segments, typically encoding functional elements such as promoters, genes, protein domains, or fusion tags, are central to methods of cell engineering, protein production, and gene-function analysis. The large number of available genome sequences now makes it possible to create and apply repositories of defined functional elements to conduct high-throughput, genome-wide analyses. The Gateway Cloning Technology (Hartley et al. 2000) uses in vitro sitespecific recombination to clone and subsequently transfer DNA segments between vector backbones. This approach has been used to generate several large clone collections (Entry Clones), in some cases comprising the entire or nearly entire coding capacity of model genomes as open reading frames (ORFs). These ORFeomes include Caenorhabditis elegans (Walhout et al. 2000b;Reboul et al. 2001Reboul et al. , 2003, Pseudomonas aeruginosa (LaBaer et al. 2004), and Saccharomyces cerevisiae (G. Marsischky, pers. comm.), Arabidopsis (Yamada et al. 2003; also see Atome project http:// genoplante-info.infobiogen.fr/Databases/CT_Nouveaux_Outils/ NO2001054/), human (clones available from several commercial sources), and an incipient collection of Drosophila ORFs (http:// www.fruitfly.org/EST/gateway.shtml). A collection of sequenced, full-length Arabidopsis cDNAs in the Gateway Vector pCMV-SPORT6 will shortly be made available through INRA-Genoscope (Castelli et al. 2004). Repositories of full-length clones, some of which are in the Gateway format, are available for Xenopus (http://xgc.nci.nih.gov/), zebrafish (http://zgc.nci.nih.gov/), as well as many hu...

A versatile nonviral vector system for tetracycline-dependent one-step conditional induction of transgene expression

“…[8][9][10][11][12] Construction of multiple functional DNA elements in tandem on a single plasmid was performed by stepwise LR and BP reactions as indicated in Figure 1b. All functional elements required for Tet regulation were placed on one contiguous DNA fragment of 12 340 bp between att1 and att2 Gateway recombination signals.…”

Zebrafish enhancer detection (ZED) vector: A new tool to facilitate transgenesis and the functional analysis of cis‐regulatory regions in zebrafish

“…This promoter drives strong expression in somites and heart (Mohun et al, 1986;Ryffel et al, 2003). To facilitate the transference of putative insulator sequences to the reporter vector, we introduced a Gateway entry site between the promoter and the enhancer (Hartley et al, 2000;Cheo et al, 2004;Fig. 2).…”

“…First, to drive the expression of the reporter gene encoding the enhanced green fluorescent protein (EGFP), we have used a gata2a minimal promoter (Ellingsen et al, 2005), which has been selected from a test set of several TATA-containing and TATA-less promoters because of its high efficiency of enhancer activity detection. Second, we have placed a Gateway entry site 5Ј of the gata2a minimal promoter to facilitate the insertion of candidate enhancers in the reporter construct (Hartley et al, 2000;Cheo et al, 2004). Third, the enhancer reporter cassette in ZED is flanked by insulator sequences that we show decrease position effects, thereby increasing the specificity of the signal.…”

Zinc Finger Nuclease and Homing Endonuclease-Mediated Assembly of Multigene Plant Transformation Vectors

“…Only a handful of dedicated vector assembly systems have been developed in the past several years for the assembly of multigene transformation vectors (Cheo et al, 2004;Sasaki et al, 2004;Karimi et al, 2005;Chen et al, 2006Chen et al, , 2010Wakasa et al, 2006). Most of these vector assembly systems have a rather limited capacity and hence have been utilized for the delivery of only a small number (i.e.…”

“…The system was used to construct several multitransgene binary vectors and led to the production of transgenic plants with eight transgenes (Chen et al, 2010). A number of other recombination-based cloning strategies have also been developed for the construction of multigene plant transformation vectors (Cheo et al, 2004;Sasaki et al, 2004;Karimi et al, 2005;Chen et al, 2006Chen et al, , 2010Wakasa et al, 2006). However, here too, the irreversible nature of the recombination-based reactions does not enable the modification of existing binary vectors, and users of such systems may be required to completely rebuild their transformation vectors to give different gene combinations.…”