RAS Pathways and Reagents

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Frederick National Laboratory for Cancer Research (FNLCR) scientists have developed unique reagents to study RAS biology. As many of these as possible will be made available to the community of RAS researchers at nominal cost. Below are reagents now available for purchase.

KRAS Entry and RAS Pathway Clone Collections

Cost: US $38.55 (each requestor must pay shipping expenses) for each collection.

Contact: 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 dom.esposito@nih.gov.

For technical questions about the system, please contact Carissa Grose, Protein Expression Laboratory, FNLCR at 301-360-3427 or grosecl@mail.nih.gov.

Overview

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

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 Identifier Gene C-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

Expression Plasmids for 15 RAS Pathway Genes

RAS Entry Clone Collection 1: 15 RAS pathway genes; Open and closed formats; Plasmid DNA and glycerol stocks of E. coli DH10B harboring each plasmid

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:

  • SOS1
  • SOS2
  • RASGRP1
  • RASGRP2
  • RASGRP3
  • RASGEF1A
  • KNDC1
  • RASA1
  • SYNGAP1
  • RASAL1
  • RASA4
  • BRAF
  • RAF1
  • ARAF
  • PDE6D

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
R702-E01 Hs.SOS1 NM_005633.3 closed
R702-E02 Hs.SOS1 NM_005633.3 open
R702-E03 Hs.SOS2 NM_006939.2 closed
R702-E04 Hs.SOS2 NM_006939.2 open
R702-E05 Hs.RASGRP1 NM_005739.3 closed
R702-E06 Hs.RASGRP1 NM_005739.3 open
R702-E07 Hs.RASGRP2 NM_153819.1 closed
R702-E08 Hs.RASGRP2 NM_153819.1 open
R702-E09 Hs.RASGRP3 NM_001139488.1 closed
R702-E10 Hs.RASGRP3 NM_001139488.1 open
R702-E11 Hs.RASGEF1A NM_145313.2 closed
R702-E12 Hs.RASGEF1A NM_145313.2 open
R702-E13 Hs.KNDC1 NM_152643.6 closed
R702-E14 Hs.KNDC1 NM_152643.6 open
R702-E15 Hs.RASA1 NM_002890.2 closed
R702-E16 Hs.RASA1 NM_002890.2 open
R702-E17 Hs.SYNGAP1 NM_006772.2 closed
R702-E18 Hs.SYNGAP1 NM_006772.2 open
R702-E19 Hs.RASAL1 NM_001193520.1 closed
R702-E20 Hs.RASAL1 NM_001193520.1 open
R702-E21 Hs.RASA4 NM_006989.5 closed
R702-E22 Hs.RASA4 NM_006989.5 open
R702-E23 Hs.BRAF NM_004333.4 closed
R702-E24 Hs.BRAF NM_004333.4 open
R702-E25 Hs.RAF1 NM_002880.3 closed
R702-E26 Hs.RAF1 NM_002880.3 open
R702-E27 Hs.ARAF NM_001654.4 closed
R702-E28 Hs.ARAF NM_001654.4 open
R702-E29 Hs.PDE6D NM_002601.3 closed
R702-E30 Hs.PDE6D NM_002601.3 open
  • Posted: September 22, 2014