Research Skills

Ras Oncogene + NMR | Trypanosomes

Alamar Blue Assays

Resazurin-based metabolic indicator used to evaluate cell proliferation, viability and cytotoxicity

Nuclear Magnetic Resonance (NMR)

Spectroscopic technique used to evaluate changes in glucose metabolism

CRISPR-based Restriction Fragment Length Polymorphism

Molecular technique used to identify CRISPR Cas9 induced gene edits and detects gene mutations.

Confocal Studies

High-res optical imaging technique using laser point source and pinhole aperture to eliminate out of focus light and 3D images of thick samples.

HRAS (Harvey rat sarcoma)

encodes proteins that act as molecular switches - regulate cell growth division. MAPK/ERK pathway - translate external cellular signals to nucleus

mutated HRAS - bladder cancer, follicular thyroid cancer, oral squamous cell carcinoma

Costello Syndrome: intellectual disability

KRAS

mutations: error in gene cause protein to get stuck in active state

• pancreatic cancer

• colorectal

• NS cell lung cancer

NRAS (neuroblastoma)

• tumor development

• melanoma

• colorectal

• acute myleloid leukemia

• thyroid cancer

Noonan syndrome

gremlin gain of function mutation in RAS-MAPK pathway

• PTPN11 - encodes SHP-2 protein = increase activation of RASMAPK

• higher risk of leukemia and solid tumor

RAS in Cancer

Ras protein harbor single mutation at codon 12, 13, or 61

• favor GTP binding + constitutive activation of Ras

• downstream signaling changes cell metabolism, growth, proliferation, and endocytosis

FTase (farnesyltransferase)

enzyme that catalyses in addition to 15-carbon farnesyl group to ras protein on C-terminus

• allows ras to associate with plasma membrane = interact with RTK

Sotorasib/Adagrasib

anti-Ras drugs: target codon 12 mutants of RAS

Inactivation of FTase

When blocked = related GGTase can still add prenyl group which can bring Ras to membrane

Rigosertib ON-01910

“Ras mimetic”/inhibitor: binds to Ras-binding domain of Pi3K and Raf = block signaling

• binds to Ras-BD of multiple RAS effector proteins

BAY-293

inhibitor of KRAS-SOS1: prevents SOS1 from loading GTP onto KRAS = inhibition of RAS activation in cancer cells

• selective inhibitor of K-RAS

Son of Sevenless 1 (SOS1)

a GEF that stimulates conversion of Ras from inactive GDP-bound form to the active GTP-bound form

MDA-MB-231

Breast cancer cell line - BAY and Rigo for effect on metabolism, endocytosis, and cellular architecture of line

• known mutation in gene coding K-Ras at codon 13

  • results in normal glycine being replaces with aspartic acid

MCF-7

second breast cancer cell line - normal, non-mutated K-Ras gene

Objective of Practical

Use CRISPR to distinguish non-mutated and mutated K-ras genes in the two cell lines using mod version of Restriction Fragment Length Polymorphism

Restriction Fragment Length Polymorphism (RFLP)

Technique commonly used in clinic to identify cancer mutations and monogenic diseases (e.g. sickle cell, CF, Huntingtons)

• PCR amplification of short region of gene mutation

• region of KRAS gene = amplified in practical