The Drosophila Gatewayª Vector CollectionA resource developed by the Murphy lab for the Drosophila research communityContentsDownload a PDF version of this page. The Drosophila Gateway Vector collection Contact informationTerence Murphy Figure 1. The Gateway LR in vitro recombination reaction OverviewThe Drosophila Gatewayª Vector collection is a set of 68 Gateway-based vectors designed to express epitope-tagged proteins in Drosophila culture cells or flies. At its core is Invitrogen's Gatewayª recombination cassette, which allows you to recombine an Open Reading Frame (ORF) of interest into any of the vectors using a simple and efficient in vitro reaction. The result is a fusion gene with your ORF placed in frame with one of 7 different epitope tags and expressed by one of 4 different promoters.
Gatewayª technology uses lambda integrase to recombine your ORF, flanked by attL1 and attL2 recombination sites, with the attR1 and attR2 recombination sites of a destination vector (figure 1). The result is a "swap" of your ORF with the cassette containing the ccdB gene in the destination vector. Successfully recombined expression clones can be selected based on their resistance to ampicillin and lack of toxicity to standard laboratory strains of E. coli (the ccdB gene product is toxic, which prevents the original destination vector from forming colonies).
Figure 2. N- and C-terminal Tag/Gateway fusion modules The Drosophila Gateway Vector collectionWe have constructed a set of 17 Gateway/Tag modules (Figure 2 and Table 1) for use in making different destination vectors. Each module contains one of seven epitope tags placed either 5' or 3' of the Gateway cassette, followed by stop codons in all three reading frames. These modules can be subcloned into any vector containing a promoter and terminator of interest as an EcoRV (blunt) - NheI (XbaI compatible) restriction fragment. This subcloning step is facilitated by the presence of a chlR gene within the Gateway cassette, allowing the desired clones to be selected based on their resistance to chloramphenicol.
Table 1. The 17 Gateway/Tag modules The 17 modules have two or three letter designations, using combinations of a one-letter abbreviation for each epitope and "W" to stand for the Gateway cassette. Thus, "GW" designates an EGFP tag placed 5' of the Gateway cassette, suitable for producing N-terminal fusions.
Table 2. The four vectors currently available with the 17 Gateway/Tag modules The 17 modules are currently available in four different vectors (Table 2). The Actin5C- and Hsp70-based vectors (Huynh and Zieler, 1999) are intended for high and moderate levels, respectively, of transient expression in tissue culture cells. The UASt vectors contain a GAL4-responsive promoter for expression in vivo in cells expressing GAL4 (Brand and Perrimon, 1993). The UASp vectors contain a GAL4-responsive promoter modified to allow expression in the female germline in addition to somatic cells (Rorth, 1998), although anecdotal evidence suggests that UASp vectors may have some basal expression in the absence of GAL4 and induce less strongly in response to GAL4 than UASt vectors. How to get the vectorsIt has been a tedious and time-consuming process to arrange for permission to distribute the vectors from the various companies that hold applicable patents (Invitrogen, Sigma, Amersham, Aurora Biosciences, Riken Brain Science Institute, and UCSD/HHMI). Most of the arrangements have been finalized, with the exception of Amersham, which has reneged on their initial approval. It has also been impossible to make any progress with AmershamÕs lawyers due to the pending sale of Amersham to GE. Unfortunately, Amersham has legal control over distribution of GFP-based vectors, precluding us from distributing 24 of the 68 vectors at this time. We are continuing to work on ways around this legal roadblock and are hopeful that this barrier can be resolved, but we have no idea how long these negotiations will take. We also do NOT have blanket permission to distribute the mRFP-based vectors; however, interested labs can contact Roger Tsien and sign an individual MTA with HHMI/UCSD after which we can provide the 8 mRFP-based vectors.
As a further complication, we had planned on providing the entire collection of vectors as a microtiter plate of glycerol stocks, which were prepared LONG ago and currently sit in our freezer. Given that many people have been awaiting these vectors for 6 months or more, we can ship DNA of a small number of FLAG-, myc-, HA-, or mRFP-based vectors to not-for-profit institutions upon request. Please contact me by e-mail (see contact information) for further information and a Material Transfer Agreement.
Note that, under the terms of the MTA, you are not allowed to further distribute these vectors to parties that have not also signed an MTA with the Carnegie Institute. New labs and departing post-docs and students that want to take copies of these vectors should fill out their own MTA so that we don't get sued. Organization of the vectors
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