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Bhattacharya Group Molecular Biology Resources

dna-2.jfif

The Bhattacharya Group has several plasmids that are useful in designing high-throughput cell-based screens. These vectors were developed by Matt Benson, Anuja Neve, Sheny Chen and Shoumo Bhattacharya. Tools are available on a collaborative basis if unpublished, and freely if published, but are not to be redistributed. You will need to download the free editor APE (please make the author a donation) to visualise the sequence and maps for these plasmids. APE allows you to perform in silico GATEWAY recombination to confirm frame and structure. Note that this resource is only visible if you are logged in through the Oxford University network.

Plasmid reporters

Luciferase reporters

Luciferase reporters consist of either a natural or a concatemerised transcription factor binding site upstream of firefly luciferase. They report on the final step in many signalling pathways. Concatemerisation enhances specificity and signal/noise ratio. Limitations are that they are subject to cross talk from common pathway components, and specific reporters for many pathways are yet to be identified. A good example of a pathway based high-throughput siRNA screen using a WNT-reporter is here. When a DNA binding element for a transcription factor is not available it is possible to develop a screen by fusing your factor to the yeast GAL4-DNA binding domain, and using a GAL4-luc reporter. A good example of such a screen is here. Many pathway reporters are available commercially - e.g. from Qiagen or from Addgene.

We have the following plasmids for creating new luciferase or GFP reporters:

  • 521 pGL3-Basic (Promega)
  • 2015 pGL4.13 (luc2/SV40) (Promega)
  • 238 pCMX-GAL4N (Ron Evans)
  • 553 3XGAL4-Luc (Ron Evans)
  • 399 pGAL4x5-GFP (Andy Kung)

MicroRNA reporters

A microRNA reporter typically consists of miRNA binding sites fused to the luciferase 3'-UTR. Luciferase expression is under the control of a strong promoter e.g. CMV. Several are available commercially - e.g. from Signosis. We have the pMIRGlo plasmid from Promega that allows you to clone in known or potential miRNA binding sites.

GATEWAY and INFUSION System Plasmids

Many reporter plasmids work by fusion of a gene of interest to another protein e.g. GFP or luciferase. Typically you will need to test both N and C-terminal fusion orientations. Creating such constructs using restriction enzymes and ligation can be difficult. The GATEWAY Cloning System (Invitrogen) allows ligation independent transfer of inserts from ENTRY plasmids containing a gene of interest to DESTINATION plasmids. The INFUSION System (Clontech) allows ligation independent transfer of appropriate PCR products to any plasmid cut at a single appropriate restriction site.

Gateway ENTRY clones

Over 16000 ENTRY clones cane be obtained from commercial suppliers (e.g. Invitrogen ULTIMATE ORF Clones) or from the ORFEOME consortium. They are also easy to create by PCR and INFUSION (Clontech) cloning, using a GATEWAY vector (LabID 1922 pENTR-SfoI).

Fluorescent protein fusions

We have created several DESTINATION plasmids that allow you to fuse various fluorescent proteins to your protein of interest in either orientation. Some have a IRES - Puro / Neo selection cassette that allows rapid creation of pooled stable cells or cloned cell lines. Such fusion proteins can be used to monitor protein localisation or stability. A good example of such a screen is published here.

Relevant plasmids (all Gateway compatible - DEST is the GATEWAY destination site) are:

2131 pcDNA3-DEST-mVenusC

2132 pcDNA3-mCherryN-DEST

2133 pcDNA3-DEST-mCherryC

2134 pcDNA3-mCeruleanN-DEST

2135 pcDNA3-DEST-mCeruleanC

For making stable pools and clones using puro selection

2439 CMV-DEST-mVenusC-IRESPuro

2440 CMV-mCherryN-DEST-IRESPuro

2441 CMV-DEST-mCherryC-IRESPuro

2442 CMV-mCerN-DEST-IRESPuro

2443 CMV-DEST-mCerC-IRESPuro

Protein Complementation Assay (PCA) Reporters

The concept is that splitting certain proteins into two fragments result in loss of function. Fragment proximity in a cell (e.g. as a result of protein interaction between proteins fused to either fragment) results in complementation and restoration of function. This allows sensitive detection of protein interactions. If the protein interaction is responsive to a change in cell environment it then functions as a reporter of the changed environment.

We have modified the split-renilla PCA system of Michnick to make it compatible with the GATEWAY system. This allows fusion of renilla fragments to your protein of choice in either orientation. Several PCA reporters were also made using RLuc8 fragments, a stable form of RLuc made in the Gambhir lab. Several were also made with weak promoters (mutant CMV or PGK) to minimise artifactual interactions due to high level expression.

Plasmids need to be used in pairs; rLuc1 or rluc8.1 need to be partnered with rLuc2 or rLuc8.2. For each PCA we usually test 4 combinations (rluc8.1N + rluc8.2N, rluc8.1N + rluc8.2C, rluc8.1C + rluc8.2N, rluc8.1C + rluc8.2C). IRESNeo/Puro are used to create double stable cell lines.

1816 pcDNA3-rLuc1N-DEST

1822 pcDNA3-rLuc2N-DEST

1825 pcDNA3-rLuc8.1N-DEST

1828 pcDNA3-rLuc8.2N-DEST

1831 pcDNA3-DEST-rLuc8.1C

1834 pcDNA3-DEST-rLuc8.2C

2342 CMV-Rluc8.2N-DEST-IRESNeo

2343 CMV-DEST-RLuc8.2C-IRESNeo

2344 CMV-RLuc8.1N-DEST-IRES-Puro

2345 CMV-DEST-RLuc8.1C-IRES-Puro

Luciferase fusions

Fusion to renilla or firefly luciferase can also allow monitoring of protein stability. A good example of a high-throughput screen using this approach is published here. We have created DESTINATION plasmids that allow you to fuse firefly or renilla luciferase to your protein of interest.

  • 1845 pcDNA3-DEST-rLuc8C