From Gateway to MultiSite Gateway in one recombination event
© Magnani et al; licensee BioMed Central Ltd. 2006
Received: 30 September 2006
Accepted: 06 December 2006
Published: 06 December 2006
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.
We created a construct, pDONR-R4-R3, to easily convert any available Gateway destination vector to a MultiSite Gateway vector in a single recombination reaction. In addition, we designed pDONR-R4-R3 so that DNA fragments already cloned upstream or downstream of the Gateway cassette in the original destination vectors can still be utilized for promoter-gene or translational fusions after the conversion.
Our tool makes MultiSite Gateway a more widely accessible technology and expands its applications by exploiting all the features of the Gateway vectors already available.
Recombinase-based cloning technologies are becoming increasingly popular because of their easy use and high efficiency. These tools exploit bacterial or viral site-specific recombinases like the bacteriophage P1 Cre, the Saccharomyces cerevisiae FLP or the bacteriophage lambda integrase [1–3]. These enzymes catalyze a reciprocal double-stranded DNA exchange between two specific DNA sites .
Here we present a method to convert any Gateway vector to a MultiSite Gateway vector via a single recombination event.
Results and discussion
Last, we performed an LR Plus reaction with pDEST22-R4-R3 and pDEST32-R4-R3 to test the functionality of the att R4-att R3 cassette flanked by att B1-att B2 sites (Fig 4B). We used three entry clones bearing a 3434 bp, 3116 bp and 561 bp DNA fragment cloned into pDONR-P4-P1R (Invitrogen), pDONR221 and pDONR-P2R-P3 (Invitrogen) respectively . Two LR Plus reactions were performed with the entry clones and either pDEST22-R4-R3 or pDEST32-R4-R3. Both reactions led to the creation of pDEST22 and pDEST32 expression clones containing the inserts in the expected order (Fig 4B). Correct functioning of the technology was further tested by cloning a different set of three entry clones into pDEST22-R4-R3 (data not shown). When the converted MultiSite Gateway destination vector bears a kanamycin resistance gene, like pMDC163-R4-R3, the entry clones need to be linearized in order to specifically recover the expression clone.
Figure 4C shows the external att B1-att B4 and att B3-att B2 sites of a converted MultiSite Gateway expression clone which maintain the Gateway frame and lack start and stop codons to allow promoter-gene or translational fusions. Thus, converted MultiSite Gateway vectors can take advantage of all reporter genes, tags or promoter sequences already present in the original Gateway destination vectors.
We have developed a new method to convert virtually all Gateway vectors to MultiSite Gateway. A careful combination of compatible att sites allowed us to use the Gateway recombination technology to deliver a MultiSite Gateway cassette. This strategy opens a new way of thinking about recombination sites as cloning and clonable elements at the same time and could apply to other recombination cassettes. Our method makes the Invitrogen technology available to many biological systems for translational fusion of different proteins or tags, expression of genes or reporter genes under the control of specific promoters and 3'-UTRs, and combinatorial analysis of promoters, genes or other DNA fragments. Our improvement also expands the potential use of this recombination system by utilizing all the features of the large number of Gateway vectors already created.
Construction of pDONR-R4-R3
We PCR amplified the att R4-att R3 cassette from the pDEST-R4-R3 vector (Invitrogen) using the primers att B1-att R4-F (5'GGGGACAAGTTTGTACAAAAAAGCAGGCTCAACTTTGTATAGAAAAGTTGAAC3') and att B2-att R3-R (5'GGGGACCACTTTGTACAAGAAAGCTGGGTCAACTATGTATAATAAAGTTGAAC3'). We digested the pDONR221 vector (Invitrogen) with EcoRI and used it in a BP recombination reaction (Invitrogen) according to the manufacturer with the att R4-att R3 cassette PCR fragment. We used the reaction to transform DB3.1 E. coli cells (Invitrogen) and selected them for kanamycin resistance. We named the resultant vector pDONR-R4-R3.
Construction of pDEST22-R4-R3, pDEST32-R4-R3 and pMDC163-R4-R3
We digested the pDEST22 (Invitrogen), pDEST32 (Invitrogen) and pMDC163  vectors with EcoRI, XbaI and NcoI respectively. We conducted two independent LR reactions (Invitrogen) according to the manufacturer combining pDONR-R4-R3 with digested pDEST22 and pDEST32. An additional LR reaction was performed combining EcoNI linearized pDONR-R4-R3 with digested pMDC163. We used the three reactions to transform DB3.1 E. coli cells (Invitrogen) and selected them for ampicillin (pDEST22/pDONR-R4-R3 reaction), gentamicin (pDEST32/pDONR-R4-R3 reaction) or kanamycin (pMDC163/pDONR-R4-R3 reaction) resistance. We named the resultant vectors pDEST22-R4-R3, pDEST32-R4-R3 and pMDC163-R4-R3 respectively.
Construction of pDONR-P4-P1R-pPNY, pDONR221-PNY and pDONR-P2R-P3-YC
We PCR amplified 3434 bp upstream the start codon of the Arabidopsis thaliana PENNYWISE (PNY) gene  using pny-att B4F (GGGGACAACTTTGTATAGAAAAGTTGTTGGCACGATTCTGAAACACG) and pny-att B1R (GGGGACTGCTTTTTTGTACAAACTTGGGGAAAGGATGATGTCGATGAG) primers, the PNY gene (3116 bp) using pny-att B1F (GGGACAAGTTTGTACAAAAAAGCAGGCTTGATGGCTGATGCATACGAGCC) and pny-att B2R (GGGGACCACTTTGTACAAGAAAGCTGGGTAACCTACAAAATCATGTAGAA) primers and a 561 bp fragment of the pUC-SPYCE vector  using att B2-SPYCE-F (GGGGACAGCTTTCTTGTACAAAGTGGGGATGTACCCATACGATGTTCCA) and att B3-SPY-R (GGGGACAACTTTGTATAATAAAGTTGGAATTCCCGATCTAGTAACATAGATG) primers. The PNY promoter region, the PNY gene and pUC-SPYCE PCR fragments were cloned into the pDONR-P4-P1R (Invitrogen), pDONR221 and pDONR-P2R-P3 (Invitrogen) vectors respectively via three independent BP reactions according to the manufacturer. We used the reactions to transform TOP10 E. coli cells (Invitrogen) and selected them for kanamycin resistance. We named the resultant vectors pDONR-P4-P1R-pPNY, pDONR221-PNY and pDONR-P2R-P3-YC respectively.
Construction of pDEST22-pPNY-PNY-YC and pDEST32-pPNY-PNY-YC
We performed an LR Plus reaction (Invitrogen) according to the manufacturer combining pDONR-P4-P1R-pPNY, pDONR221-PNY, pDONR-P2R-P3-YC, and pDEST22-R4-R3. We used the reaction to transform TOP10 E. coli cells and selected for ampicillin resistance. We named the resultant vectors pDEST22-pPNY-PNY-YC. We performed another LR Plus reaction according to the manufacturer combining pDONR-P4-P1R-pPNY, pDONR221-PNY, pDONR-P2R-P3-YC, and pDEST32-R4-R3. We used the reaction to transform TOP10 E. coli cells and selected for gentamicin resistance. We named the resultant vectors pDEST32-pPNY-PNY-YC.
List of abbreviations
We thank Hector Candela, Elisa Fiume, Jennifer Fletcher, Elise Kikis, China Lunde, and Peter Quail for critically reading the article. EM and LB were supported by NRI 04-03387.
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