From Gateway to MultiSite Gateway in one recombination event

Background  

Invitrogen Gateway technology exploits the integrase/att site-specific recombination system for directional cloning of PCR products and the subsequent subcloning into destination vectors. One or three DNA segments can be cloned using Gateway or MultiSite Gateway respectively. A vast number of single-site Gateway destination vectors have been created while MultiSite Gateway is limited to few destination vectors and therefore to few applications. The aim of this work was to make the MultiSite Gateway technology available for multiple biological purposes.     

Focusing transgene expression in Drosophila by coupling Gal4 with a novel split-LexA expression system

Here we report the development of a ternary version of the LexA::VP16/LexAop system in which the DNA-binding and trans-activating moieties are independently targeted using distinct promoters to achieve highly restricted, intersectional expression patterns. This Split LexA system can be concatenated with the Gal4/upstream activating sequence system to refine the expression patterns of existing Gal4 lines with minimal genetic manipulations.     

A Hormone Receptor-Based Transactivator Bridges Different Binary Systems to Precisely Control Spatial-Temporal Gene Expression in Drosophila

The GAL4/UAS gene expression system is a precise means of targeted gene expression employed to study biological phenomena in Drosophila. A modified GAL4/UAS system can be conditionally regulated using a temporal and regional gene expression targeting (TARGET) system that responds to heat shock induction. However heat shock-related temperature shifts sometimes cause unexpected physiological responses that confound behavioral analyses. We describe here the construction of a drug-inducible version of this system that takes advantage of tissue-specific GAL4 driver lines to yield either RU486-activated LexA-progesterone receptor chimeras (LexPR) or β-estradiol-activated LexA-estrogen receptor chimeras (XVE). Upon induction, these chimeras bind to a LexA operator (LexAop) and activate transgene expression. Using GFP expression as a marker for induction in fly brain cells, both approaches are capable of tightly and precisely modulating transgene expression in a temporal and dosage-dependent manner. Additionally, tissue-specific GAL4 drivers resulted in target gene expression that was restricted to those specific tissues. Constitutive expression of the active PKA catalytic subunit using these systems altered the sleep pattern of flies, demonstrating that both systems can regulate transgene expression that precisely mimics regulation that was previously engineered using the GeneSwitch/UAS system. Unlike the limited number of GeneSwitch drivers, this approach allows for the usage of the multitudinous, tissue-specific GAL4 lines for studying temporal gene regulation and tissue-specific gene expression. Together, these new inducible systems provide additional, highly valuable tools available to study gene function in Drosophila.     

Comparison of constitutive promoter activities and development of maize ubiquitin promoter- and Gateway-based binary vectors for rice

In transgenic experiments, we often face fundamental requirements such as overexpressing a certain gene, developing organelle markers, testing promoter activities, introducing large genomic fragments, and combinations of them. To fulfill these multiple requirements in rice, we developed simple binary vectors with or without maize ubiquitin (UBQ) promoter, Gateway cassette and fluorescent proteins. First, we compared stabilities of cauliflower mosaic virus 35S and maize UBQ promoters for constitutive gene expression in transgenic rice. We show that the 35S promoter was frequently silenced after shoot regeneration, whereas maize UBQ promoter achieved stable expression in various young tissues. Binary vectors with Gateway cassettes under the control of the UBQ promoter allowed us to develop stable organelle markers for nuclei, microtubules and P-bodies in rice. The maize UBQ promoter can be easily replaced with any promoters of interest as exemplified by reporters of mitotic cells and provascular bundles. Finally, by introducing two genomic fluorescent reporters, we showed utilities of the Gateway cassette and two selection markers in large DNA fragment transfer and sequential transformations, respectively. Thus, these binary vectors provide useful choices of transgenic experiments in rice.     

A Gateway MultiSite recombination cloning toolkit

The generation of DNA constructs is often a rate-limiting step in conducting biological experiments. Recombination cloning of single DNA fragments using the Gateway system provided an advance over traditional restriction enzyme cloning due to increases in efficiency and reliability. Here we introduce a series of entry clones and a destination vector for use in two, three, and four fragment Gateway MultiSite recombination cloning whose advantages include increased flexibility and versatility. In contrast to Gateway single-fragment cloning approaches where variations are typically incorporated into model system-specific destination vectors, our Gateway MultiSite cloning strategy incorporates variations in easily generated entry clones that are model system-independent. In particular, we present entry clones containing insertions of GAL4, QF, UAS, QUAS, eGFP, and mCherry, among others, and demonstrate their in vivo functionality in Drosophila by using them to generate expression clones including GAL4 and QF drivers for various trp ion channel family members, UAS and QUAS excitatory and inhibitory light-gated ion channels, and QUAS red and green fluorescent synaptic vesicle markers. We thus establish a starter toolkit of modular Gateway MultiSite entry clones potentially adaptable to any model system. An inventory of entry clones and destination vectors for Gateway MultiSite cloning has also been established (www.gatewaymultisite.org).     

MosSCI and gateway compatible plasmid toolkit for constitutive and inducible expression of transgenes in the C. elegans germline

Here we describe a toolkit for the production of fluorescently tagged proteins in the C. elegans germline and early embryo using Mos1-mediated single copy insertion (MosSCI) transformation. We have generated promoter and 3'UTR fusions to sequences of different fluorescent proteins yielding constructs for germline expression that are compatible with MosSCI MultiSite Gateway vectors. These vectors allow tagged transgene constructs to be inserted as single copies into known sites in the C. elegans genome using MosSCI. We also show that two C. elegans heat shock promoters (Phsp-16.2 and Phsp-16.41) can be used to induce transgene expression in the germline when inserted via MosSCI transformation. This flexible set of new vectors, available to the research community in a plasmid repository, should facilitate research focused on the C. elegans germline and early embryo.     

Building blocks for plant gene assembly

The MultiSite Gateway cloning system, based on site-specific recombination, enables the assembly of multiple DNA fragments in predefined order, orientation, and frame register. To streamline the construction of recombinant genes for functional analysis in plants, we have built a collection of 36 reference Gateway entry clones carrying promoters, terminators, and reporter genes, as well as elements of the LhG4/LhGR two-component system. This collection obeys simple engineering rules. The genetic elements (parts) are designed in a standard format. They are interchangeable, fully documented, and can be combined at will according to the desired output. We also took advantage of the MultiSite Gateway recombination sites to create vectors in which two or three genes can be cloned simultaneously in separate expression cassettes. To illustrate the flexibility of these core resources for the construction of a wide variety of plant transformation vectors, we generated various transgenes encoding fluorescent proteins and tested their activity in plant cells. The structure and sequence of all described plasmids are accessible online at http://www.psb.ugent.be/gateway/. All accessions can be requested via the same Web site.     

Efficient expressions of reporter genes in the industrial filamentous fungus Sclerotium rolfsii mediated by Agrobacterium tumefaciens

Sclerotium rolfsii (teleomorph Athelia rolfsii) is one of the plant pathogenic basidiomycetes, which causes severe stem-rot disease in hundreds of plants and produces important metabolites, such as scleroglucan and TF-specific lectin. However, further molecular biological research on this filamentous fungus is severely plateaued out due to the lack of genetic methods. In this study, the A. tumefaciens strain LBA4404 harboring a binary vector containing the basta resistance gene fused with three reporters (DsRed, tdTomato, and GUSPlus) respectively, driven by the SrGPD promoter, was used for genetic transformation of S. rolfsii. The results showed that the three reporter genes were all effectively expressed in S. rolfsii. This study also showed that the intron of the SrGPD promoter is not necessary for transgene expression in this fungus. Besides, we showed that these reporters’ signals could be observed easily but in a short time window. The efficient Agrobacterium-mediated transformation system and the three reporter gene plasmids for S. rolfsii developed in this study are of significance in overcoming current limitations of no available transformation and genetic manipulation techniques in S. rolfsii, facilitating further genetic manipulations and gene function exploration.     

A Gateway-Based System for Fast Evaluation of Protein-Protein Interactions in Bacteria

Protein-protein interactions are important layers of regulation in all kingdoms of life. Identification and characterization of these interactions is one challenging task of the post-genomic era and crucial for understanding of molecular processes within a cell. Several methods have been successfully employed during the past decades to identify protein-protein interactions in bacteria, but most of them include tedious and time-consuming manipulations of DNA. In contrast, the MultiSite Gateway system is a fast tool for transfer of multiple DNA fragments between plasmids enabling simultaneous and site directed cloning of up to four fragments into one construct. Here we developed a new set of Gateway vectors including custom made entry vectors and modular Destination vectors for studying protein-protein interactions via Fluorescence Resonance Energy Transfer (FRET), Bacterial two Hybrid (B2H) and split Gaussia luciferase (Gluc), as well as for fusions with SNAP-tag and HaloTag for dual-color super-resolution microscopy. As proof of principle, we characterized the interaction between the Salmonella effector SipA and its chaperone InvB via split Gluc and B2H approach. The suitability for FRET analysis as well as functionality of fusions with SNAP- and HaloTag could be demonstrated by studying the transient interaction between chemotaxis response regulator CheY and its phosphatase CheZ.     

A Modified MultiSite Gateway Cloning Strategy for Consolidation of Genes in Plants

The genome information is offering opportunities to manipulate genes, polygenic characters and multiple traits in plants. Although a number of approaches have been developed to manipulate traits in plants, technical hurdles make the process difficult. Gene cloning vectors that facilitate the fusion, overexpression or down regulation of genes in plant cells are being used with various degree of success. In this study, we modified gateway MultiSite cloning vectors and developed a hybrid cloning strategy which combines advantages of both traditional cloning and gateway recombination cloning. We developed Gateway entry (pGATE) vectors containing attL sites flanking multiple cloning sites and plant expression vector (pKM12GW) with specific recombination sites carrying different plant and bacterial selection markers. We constructed a plant expression vector carrying a reporter gene (GUS), two Bt cry genes in a predetermined pattern by a single round of LR recombination reaction after restriction endonuclease-mediated cloning of target genes into pGATE vectors. All the three transgenes were co-expressed in Arabidopsis as evidenced by gene expression, histochemical assay and insect bioassay. The pGATE vectors can be used as simple cloning vectors as there are rare restriction endonuclease sites inserted in the vector. The modified multisite vector system developed is ideal for stacking genes and pathway engineering in plants.     

Long-range regulation by shared retinoic acid response elements modulates dynamic expression of posterior Hoxb genes in CNS development

Retinoic acid (RA) signaling plays an important role in determining the anterior boundary of Hox gene expression in the neural tube during embryogenesis. In particular, RA signaling is implicated in a rostral expansion of the neural expression domain of 5? Hoxb genes (Hoxb9–Hoxb5) in mice. However, underlying mechanisms for this gene regulation have remained elusive due to the lack of RA responsive element (RARE) in the 5? half of the HoxB cluster. To identify cis-regulatory elements required for the rostral expansion, we developed a recombineering technology to serially label multiple genes with different reporters in a single bacterial artificial chromosome (BAC) vector containing the mouse HoxB cluster. This allowed us to simultaneously monitor the expression of multiple genes. In contrast to plasmid-based reporters, transgenic BAC reporters faithfully recapitulated endogenous gene expression patterns of the Hoxb genes including the rostral expansion. Combined inactivation of two RAREs, DE-RARE and ENE-RARE, in the BAC completely abolished the rostral expansion of the 5? Hoxb genes. Knock-out of endogenous DE-RARE lead to significantly reduced expression of multiple Hoxb genes and attenuated Hox gene response to exogenous RA treatment in utero. Regulatory potential of DE-RARE was further demonstrated by its ability to anteriorize 5? Hoxa gene expression in the neural tube when inserted into a HoxA BAC reporter. Our data demonstrate that multiple RAREs cooperate to remotely regulate 5? Hoxb genes during CNS development, providing a new insight into the mechanisms for gene regulation within the Hox clusters.     

Development of Gateway Binary Vector Series with Four Different Selection Markers for the Liverwort Marchantia polymorpha

We previously reported Agrobacterium-mediated transformation methods for the liverwort Marchantia polymorpha using the hygromycin phosphotransferase gene as a marker for selection with hygromycin. In this study, we developed three additional markers for M. polymorpha transformation: the gentamicin 3''-acetyltransferase gene for selection with gentamicin; a mutated acetolactate synthase gene for selection with chlorsulfuron; and the neomycin phosphotransferase II gene for selection with G418. Based on these four marker genes, we have constructed a series of Gateway binary vectors designed for transgenic experiments on M. polymorpha. The 35S promoter from cauliflower mosaic virus and endogenous promoters for constitutive and heat-inducible expression were used to create these vectors. The reporters and tags used were Citrine, 3×Citrine, Citrine-NLS, TagRFP, tdTomato, tdTomato-NLS, GR, SRDX, SRDX-GR, GUS, ELuc(PEST), and 3×FLAG. These vectors, designated as the pMpGWB series, will facilitate molecular genetic analyses of the emerging model plant M. polymorpha.     

LexA protein of cyanobacterium Anabaena sp. strain PCC7120 exhibits in vitro pH-dependent and RecA-independent autoproteolytic activity

The LexA protein of the nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120 exhibits a RecA-independent and alkaline pH-dependent autoproteolytic cleavage. The autoproteolytic cleavage of Anabaena LexA occurs at pH 8.5 and above, stimulated by the addition of Ca²⁺ and in the temperature range of 30–57 °C. Mutational analysis of Anabaena LexA protein indicated that the cleavage occurred at the peptide bond between Ala-84 and Gly-85, and optimal cleavage required the presence of Ser-118 and Lys-159, as also observed for LexA protein of Escherichia coli. Cleavage of Anabaena LexA was affected upon deletion of three amino acids, ⁸⁶GLI. These three amino acids are unique to all cyanobacterial LexA proteins predicted to be cleavable. The absence of RecA-dependent cleavage at physiological pH, which has not been reported for other bacterial LexA proteins, is possibly due to the absence of RecA interacting sites on Anabaena LexA protein, corresponding to the residues identified in E. coli LexA, and low cellular levels of RecA in Anabaena. Exposure to SOS-response inducing stresses, such as UV-B and mitomycin C neither affected the expression of LexA in Anabaena nor induced cleavage of LexA in either Anabaena 7120 or E. coli overexpressing Anabaena LexA protein. Though the LexA may be acting as a repressor by binding to the LexA box in the vicinity of the promoter region of specific gene, their derepression may not be via proteolytic cleavage during SOS-inducing stresses, unless the stress induces increase in cytoplasmic pH. This could account for the regulation of several carbon metabolism genes rather than DNA-repair genes under the regulation of LexA in cyanobacteria especially during high light induced oxidative stress.     

Marking embryonic stem cells with a 2A self-cleaving peptide: a NKX2-5 emerald GFP BAC reporter

Background

Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene''s native ATG start site has not been widely available.

Methodology

Here, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice.

Conclusions

Our results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library.