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Krasg12v

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The KRASG12V is a recombinant protein that contains the KRAS protein with the G12V mutation. It can be used for various research purposes, such as studying signal transduction pathways and cellular processes involving the KRAS protein.

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Lab products found in correlation

Sw1116, by American Type Culture Collection (1 mentions) Hct116, by American Type Culture Collection (1 mentions) Dld 1, by American Type Culture Collection (1 mentions) N ras q61k, by Addgene (1 mentions) Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions) Prl sv40 renilla luciferase reporter vector, by Promega (1 mentions) Plxin, by Takara Bio (1 mentions) Fopflash, by Addgene (1 mentions) Topflash, by Addgene (1 mentions) Retroviral vector, by Addgene (1 mentions) Lentiviral vector, by Addgene (1 mentions) S5130, by Merck Group (1 mentions) Torin1, by R&D Systems (1 mentions) Pp242, by Bio-Techne (1 mentions)

Spelling variants of this product

PBABE-KrasG12V (5 citations) PLenti6-KRASG12V (2 citations)

5 protocols using krasg12v

1

Genetic Manipulation of Colorectal Cancer Cells

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DLD1, HCT116, LOVO, and SW1116 cell lines were obtained from the American Type Culture Collection (Rockville, MD). DKS8 cells were from Prof. Senji Shirasawa, Fukuoka University. These cell were cultured in DMEM supplemented with 10% FBS and penicillin-streptomycin. 1CT normal colonic cells and 1CT cells expressing shAPC (1CT-A) was obtained from Prof. Jerry Shay (UT Southwestern Medical Center, TX)12 (link). Both of the cell lines were transduced with lentiviral-KRASG12V (Addgene: 35634) to provide 1CT-KRASG12V (1CT-K) and 1CT-shAPC-KRASG12V cells (1CT-AK).
Wong C.C., Wu J.L., Ji F., Kang W., Bian X., Chen H., Chan L.S., Luk S.T., Tong S., Xu J., Zhou Q., Liu D., Su H., Gou H., Cheung A.H., To K.F., Cai Z., Shay J.W, & Yu J. (2022). The cholesterol uptake regulator PCSK9 promotes and is a therapeutic target in APC/KRAS-mutant colorectal cancer. Nature Communications, 13, 3971.
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2

Transfection of cell lines

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5 × 103 SKOV3 and HT29 cells were seeded on the 96 well plate and transfected with siRNA against galectin-1 (200 nM) (Ambion) and pcDNA/gal-1 (100 ng) (GenScript) respectively. 1 × 105 MDCK cells were seeded on the 6 well plate and transfected with H-Ras(G12V), K-Ras(G12V) and N-Ras(Q61K) plasmids (2 ug) (Addgene). Transfection was performed using Lipofectamine 3000 transfection reagent (Life Technologies) according to the manufacturer’s instructions. MDCK transfected cells were selected using 2 μg/ml puromycin after additional 7 days.
Shih T.C., Liu R., Fung G., Bhardwaj G., Ghosh P.M, & Lam K.S. (2017). A Novel Galectin-1 Inhibitor Discovered through One-Bead-Two-Compounds Library Potentiates the Anti-tumor Effects of Paclitaxel in vivo. Molecular cancer therapeutics, 16(7), 1212-1223.
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3

Retroviral Expression Vectors for Kinases

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Expression vectors for hemagglutinin-tagged wild-type MEK1 (wtMEK) and caMEK, with a conversion of S218 and S222 RAF1-dependent regulatory phosphorylation sites to aspartic residues, were provided by Dr J. Pouysségur (Nice, France). The HA-wtMEK and HA-caMEK constructs were subcloned into the retroviral expression vector pLXIN (Clontech, Mountain View, CA, USA), as previously described.23 (link), 24 (link) The pRL-SV40 Renilla luciferase reporter vector was from Promega (Nepean, ON, Canada). Expression vectors encoding for human ΔNTCF4, wt LRP6, LRP6-5A and KRASG12V were all provided by Addgene (Cambridge, MA, USA). The TCF reporter constructs TOPFLASH and its negative control FOPFLASH as well as the c-myc promoter reporter (4 × TBE2) and its control (4 × TBE2-mutated) were also purchased from Addgene.
Lemieux E., Cagnol S., Beaudry K., Carrier J, & Rivard N. (2014). Oncogenic KRAS signalling promotes the Wnt/β-catenin pathway through LRP6 in colorectal cancer. Oncogene, 34(38), 4914-4927.
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4

Genetic Manipulation via Viral Vectors

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Lentiviral vector expressing CRE, shCTR and shLRF, and retroviral vectors containing c-myc, and K-RasG12V were obtained from Addgene. Viral vectors of control as well as containing shRNA against Lrf, Pml, Pten, Dlk1 and Sox9 were obtained from open biosystem. Vectors containing IDH2R172K were cloned in our lab. All the viral particles were produced in transfecting 293T cells with packaging vectors of second generation.
Guarnerio J., Riccardi L., Taulli R., Maeda T., Wang G., Hobbs R.M., Song M.S., Sportoletti P., Bernardi R., Bronson R.T., Castillo-Martin M., Cordon-Cardo C., Lunardi A, & Pandolfi P.P. (2015). A genetic platform to model sarcomagenesis from primary adult mesenchymal stem cells. Cancer discovery, 5(4), 396-409.
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5

Modeling PI3K and KRAS Oncogenes in Breast Cells

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All cell lines were obtained from ATCC (Manassas, VA, USA) and maintained in RPMI-1640 with 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Lipid-reduced FBS was made by mixing with fumed silica (20 mg/ml) (S5130, Sigma) for 3 hours. Pools of MCF10A cells stably expressing pBabe-Puro-vector, -PI3KCAH1047R (Addgene #12524)63 (link), or K-RasG12V (Addgene #9052), via retroviral transduction, were selected and cultured in DMEM-F12 with 5% horse serum, EGF (20 ng/ml), hydrocortisone (0.5 mg/ml), cholera toxin (100 ng/ml), Insulin (10 μg/ml), and puromycin (1 μg/ml) at 37°C and 5% CO2. Rapamycin (553210, Calbiochem, San Diego, CA, USA), PP242 (4257, Tocris, Bristol, UK), and Torin1 (4247, R&D Systems, Minneapolis, MN, USA) were used to inhibit mTOR.
Ricoult S.J., Yecies J.L., Ben-Sahra I, & Manning B.D. (2015). Oncogenic PI3K and K-Ras stimulate de novo lipid synthesis through mTORC1 and SREBP. Oncogene, 35(10), 1250-1260.
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