Transfections Flashcards
Transfection definition
the transfer, by artificial means, of genetic material from one cell to another. implies transfer of less than the whole nucleus of the donor cell and is usually achieved by using isolated chromosomes, DNA, or cloned genes
Way to make purified protein
put a cleavable tag on
put on end of protein (either 5’ or 3’ depending how the protein reacts)
Transient transfection
the plasmid gets lost or diluted out
lasts up to a week
Stable transfection
have to continuously apply pressure
occurs 1/10000
Anatomy of transfection plasmids
promoter
multiple cloning site
polyadenylation site
selectable marker
Ras farnesylation signal
20 amino acid farnesylation signal from c-ha-Ras fused to the C-terminus of EGFP
binds EGFP to the inner face of the plasma membrane in living and fixed cells
Endotoxin contamination
columns are needed to purify plasmids that brings endotoxins along
causes cytotoxicity problems
Cationic lipids structure
polar head connected to linker that connects to hydrophobic tails
Types of lipoplex structures
vesicle stacks stacks of flattened bilayers (most common) multilamellar vesicles bilayer tubes bunches of inverted lipid tubes
Rate-limiting step
getting the DNA released from the lipoplexes
PLUS reagent
enhances transfection
reduces the amount of DNA and LipofectAMINE required
allows for greater variation in DNA concentration
results in a higher percentage of cells expressing the transgene
expands the cell types that can be transfected
Lipofectamine
mixture of DOSPA (cationic) and DOPE (neutral)
Optimal transfection requirements
appropriate cell line correct transfection method cells healthy cell density DNA quality and quantity DNA:Lipo optimization (under 200 nm) lipoples exposure controls (checking for cytotoxicity)
Low efficiency transfections
cell type specific improper cell density poor DNA quality suboptimal lipid:DNA ratio transfection complexes on cells too briefly transfection period too short wrong transfection reagent
Cell death
too much DNA used transfection complexes on cell too long too much transfection complexes used stressed cells DNA introduced is toxic/lethal antibiotics used during transfection
