RAS Pathways and Reagents

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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

KRAS Entry Clone Collection 1 (Wild-type KRAS and 10 point mutations / Open and close formats / Plasmid DNA and glycerol stocks of E. coli DH10B harboring each plasmid)

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:

  • G12C
  • G12D
  • G12V
  • G13D
  • Q61L
  • Q61R
  • S181A
  • S181D
  • S181E
  • C185S

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.

Clone IdentifierGeneC-terminus
R750-E01 Hs.KRAS4b closed
R750-E02 Hs.KRAS4b open
R750-E03 Hs.KRAS4b G12C closed
R750-E04 Hs.KRAS4b G12C open
R750-E05 Hs.KRAS4b G12D closed
R750-E06 Hs.KRAS4b G12D open
R750-E07 Hs.KRAS4b G12V closed
R750-E08 Hs.KRAS4b G12V open
R750-E09 Hs.KRAS4b G13D closed
R750-E10 Hs.KRAS4b G13D open
R750-E11 Hs.KRAS4b Q61L closed
R750-E12 Hs.KRAS4b Q61L open
R750-E13 Hs.KRAS4b Q61R closed
R750-E14 Hs.KRAS4b Q61R open
R750-E15 Hs.KRAS4b S181A closed
R750-E16 Hs.KRAS4b S181A open
R750-E17 Hs.KRAS4b S181D closed
R750-E18 Hs.KRAS4b S181D open
R750-E19 Hs.KRAS4b S181E closed
R750-E20 Hs.KRAS4b S181E open
R750-E21 Hs.KRAS4b C185S closed
R750-E22 Hs.KRAS4b C185S open
  • Updated: April 7, 2016