Lab 3/4: GFP Flashcards
Cos-7 cells
Derived from African green monkey kidney cells transformed with SV-40 large T-antigen. Adherent fibroblast cell line. Grown in dulbeccos modified eagles medium DMEM+ 10% FBS+ antibiotics/antifungal agents.
Normal vs transformed cells
Normal cells have a limited number of cell divisions, are contact inhibited, and generally require attachment to a substrate to grow,
Transformed cells have unlimited growth potential, form foci, sometimes can be grown in suspension, have fewer growth factor requirements, and may be tumorigenic.
What does DMEM supply?
Minerals, amino acids, nucleaic acids, vitamins, glucose, bicarbonate buffer.
What does FBS supply?
Growth factors and hormones.
Culturing adherent cells
Cells attach to specially treated plastic. Glass surfaces may be treated with attachment factors: polylysine and/or matrix proteins (fibronectin, collagen) to enhance binding. Trips in is used to detach cells from substrate. Trypsin solution contains EDTA which chelates Calcium that is required for attachment of cells to each other and the growth surface.
Transient vs stable transfection
Transient: gene to be expressed is maintained on a plasmid. Plasmid is lost due to unequal partitioning as cells divide. Expression usually peaks at 48 hours.
Stable: plasmid incorporates into host DNA via recombination. Selectable gene e.g. gene encoding gentamyacin resistance protein is incorporated into genome along with gene of interest.
Chemical methods of transfection
Calcium phosphate, DEA-Dextran, artificial liposomes.
Fugene transfection reagent
Mixture of synthetic lipids in ethanol. When added to an aqueous solution containing plasmid DNA it will form lipid vesicles. Vesicles containing DNA are added to cells. Vesicles fuse to the cell membrane, releasing DNA into the cytoplasm. Plasmid enters the nucleus where it’s genes are expressed as mRNA and translated into proteins in the cytoplasm.
What are microtubules?
Structural complements of the cytoskeleton. Composed of heterodimers of alpha and beta Tunisian subunits; heterodimers polymerize into protofilaments; which then form sheets and sheets from a hollow tube (MT). MTs are polar, they have a plus and minus end.
Why are microtubules important?
Involved in: chromosomal segregation and orientation of the plane of cleavage during cell division. Position organelles such as mitochondria, Golgi apparatus, and endoplasmic reticulum. Provide network if intracellular roads over which vesicular transport occurs. Interact with actin filaments to facilitate cell migration. Serve as the principle structural element of flagella and cilia.
Nocodazole
Binds to microtubule dimers, at micro molar concentrations nocodazole inhibits microtubules polymerization and promotes depolymerization. Nanomolar concentrations are sufficient to arrest cells in mitosis.
MOA of MT targeting drugs
Loss of MT attachment and/or tension. The spindle assembly checkpoint remains active, sustained activation leads to apoptotic death.
Problem of fixation
Various agents such as methanol, formaldehyde, and glutaraldehyde are used to preserve protein-based structures in cells. Soluble tubulin in cells may also be fixed in place resulting in high background fluorescence. Solution is to detergent permeabilize cells in the presence of a MT stabilizing agent (glycerol) prior to fixation: extraction buffer.
What does extraction buffer have?
80mM PIPES, pH 6.7. 1mM EGTA 1mM MgCl2 10% glycerol 0.5% Triton X-100 detergent
