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.
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 Entry and RAS Pathway Clone Collections
The cost is U.S. $38.55 for each collection, plus each requestor must pay shipping expenses.
For additional information on the clone collections, to get more information on the Combinatorial Cloning Platform, or to order, please contact Dominic Esposito, Director, Protein Expression Laboratory, FNLCR at 301-846-7376 or firstname.lastname@example.org.
For technical questions about the system, please contact Carissa Grose, Protein Expression Laboratory, FNLCR at 301-360-3427 or email@example.com.
Overview of Clone Collections
The R3 KRAS Entry Clone Collection #1 is a set of vectors for use with the Gateway Cloning Platform (Life Technologies, Carlsbad, CA) to permit construction of KRAS expression vectors for in vitro and in vivo research. While these clones can be used for standard Gateway cloning, their utility is greatly enhanced with the use of the FNLCR Combinatorial Cloning Platform (CCP), which allows simplified construction of vectors with different elements. Some uses of this system might be:
- Construction of protein expression constructs with various fusion tags
- Generation of expression constructs with different promoters for in vivo expression
- Production of clones with fluorescent tags for localization experiments
- Generation of constructs for making mutant cell lines or transgenic animals
- Production of vectors for shRNA or miRNA delivery
The advantage of the CCP is in the exquisite specificity of the Multisite Gateway reactions, which permit linkage of multiple elements in a directional fashion and which involve no additional DNA amplification, thus ensuring the accuracy of the DNA sequence of the final products without the need for additional sequencing. There is also no need for restriction-based cloning processes which have a high rate of failure and may require optimization depending on the sites available in a given clone.
The KRAS Entry clone collection contains wild-type and mutant KRAS genes in two separate formats. The “closed” format contains a Kozak translational initiation sequence and ATG start site at the 5’ end, and a stop codon at the 3’ end. These constructs can be used to make native or aminoterminally tagged constructs using Gateway vectors or the CCP. The “open” format has the same 5’ sequence, but lacks a stop codon and is in frame with the Gateway attB2 site at the 3’ end. This allows production of C-terminal fusions using Gateway vectors or the CCP. These clones have been completely sequence validated and functionally tested in Gateway reactions to ensure proper performance.
A large collection of Gateway Multisite vectors tailored for mammalian expression experiments is available through Dr. Esposito. These plasmids combined with the KRAS entry clones allow mix–react–transform construction of plasmids with a variety of promoters (weak, strong, inducible, silencing-resistant) and N- and C-terminal fusions with fluorescent proteins, epitopes, and expression motifs.
Find more information at the PEL Combinatorial Cloning Platform
Gateway Recombinational Cloning
For more information on the Gateway system, please see Hartley JL, Temple GF, and Brasch MA. (2000) “DNA cloning using in vitro site-specific recombination.” Genome Res. 10, 1788-1795.
For more information on the FNLCR Combinatorial Cloning Platform (CCP), please see Wall VA, et. al. (2014). “Combinatorial assembly of clone libraries using site-specific recombination.” Meth. Mol. Biol. 1116, 193-208.
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.
Expression Plasmids for 15 RAS Pathway Genes
Researchers within the RAS Initiative have constructed a set of Gateway-compatible entry clones for the following 15 RAS pathway genes. All clones are available with and without stop codons (the latter configuration allows for C-terminal fusions). 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.
Both DNA and frozen E. coli cells are provided. The genes include:
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. Genbank RefSeq identifiers are listed below to identify the specific isoforms of the genes in these clones.
|Clone Identifier||Gene||GenBank RefSeq Identifier||C-terminus|