RAS Pathways and Reagents
Scientists at the Frederick National Lab have developed unique reagents to study RAS biology. As many as possible of these will be made available to the community of RAS researchers.
Clones for All RAS Pathway Genes Now Available
The Reference Reagents Group of the NCI RAS Initiative has released a collection of Gateway entry clones comprising RAS pathway v2.0. Importantly, the 180 transcripts comprising the collection were chosen based on analysis of TCGA data to represent the most common variants in 38 types of human cancer. Many of the transcripts are not available from existing sources. For a listing of the clones and their RefSeqs, click here. All the plasmids (stop and no-stop versions) are available from Addgene.
Materials for Producing Fully-Processed KRAS 4b Protein
FNL scientists recently published a description of the cloning, expression, and characterization of fully-processed (farnesylated, carboxymethylated) KRAS 4b protein (Gillette WK, et al., Sci Rep. 2015, 5:15916. doi: 10.1038/srep15916). Both the baculovirus (expresses KRAS 4b and the human farnesyltransferase) and the baculovirus host strain (Hi5, Trichoplusia ni) used in the FNL procedure are available under a Material Transfer Agreement.
Contact Dominic Esposito for further information.
KRAS-4b wild-type and Mutant Expression Plasmids Clone Collection 1
The RAS Initiative collection of KRAS entry clones comprises 11 KRAS-4b genes, each in the closed (i.e., with a stop codon) and open (no stop codon, for C-terminal fusions) configuration. Both DNA and frozen E. coli cells are provided. Miniprep plasmid DNA for all Entry clones is provided, and concentrations are noted on the vials. In addition, glycerol stocks of E. coli DH10B cells containing the plasmids are provided. Gateway Entry clones are in the pDonr255 backbone and should be propagated with 50 μg/ml spectinomycin to ensure plasmid stability.
The NCI KRAS entry clone collection includes wild-type KRAS-4b and the following mutants:
All clones are in a standard attL1-attL2 Gateway Entry vector (pDonr255) and contain a 5’ Kozak translation initiation sequence prior to the ATG start codon.