Download Transfecting Cells

Transfection of Cells
I. Transformation vs Transfection
A. transfection - similar to infection done by viruses but now a more general term
for introducing DNA into cells
B. Transformation- same meaning, but confused with cell transformation during
formation of cancer cells
C. Prokaryotes - natural event where bacteria undergo transformation (ex.
horizontal gene transfer ) - to create diversity
D. In eukaryotes- DNA transfer is not a natural phenomenon
II. Vectors
A. Transfection is relatively inefficient, so you need to find the cells that got the
DNA to separate them from cells that did not.
1. Fluorescence sorting
a. GFP expression allows you to find the fluorescent cells that have
incorporated the DNA
2. Drug selection
a. Various antibiotic resistance genes used
i. Geneticin/G418, blasticidin, puromycin, bleomycin, hygromycin
B. Extrachromosomal or integrating
1. If the vector has an origin of replication, it can be maintained
extrachromosomally as single copy or high copy
C. Promoters for driving expression
1. The promoter must match the cell type and species
a. Some promoters are fairly universal (viral promoters like CMV)
b. Some are tissue or cell type specific (only turned on in muscle or
lymphoid cells)
c. Some are inducible or repressible
i. ecdysone, dexamethasone, heat shock, metal response, UV response
2. We are using constitutive actin promoters
a. Actin 15 promoter - Actin (ubiquitous, 17 actin genes) - drives the
expression of a selectable marker.
b. Actin 8 promoter - drives the expression of GFP or GFP fused to filamin
II. Different Methods of Introducing DNA into cells
A. Chemical
1. CaPO4 co-precipitation- Cloudy precipitate at certain pH - co-precipitate
DNA out of the solution
2. PEG, others
3. Not used much anymore, but were the initial methods that showed it could
work
B. Microinjection
1. hard to do
2. only some cell types
3. often not stable
C. Lipofection
1. amphipathic molecule- polar/nonpolar regions creating micelles- presumed
to fuse with the plasma membrane to introduce DNA into cells.
2. somewhat expensive
3. works with many cell types
4. transient or stable
D. Electroporation
1. pass electric current through solution of cells and DNA. Current creates
transient pores in the membrane and DNA diffuses inside the cell. It then
somehow moves to the nucleus and is incorporated into chromatin
a. The amount of current (capacitor charge in µFarads), time it takes to
pass through the solution (time constant), voltage, resistance of the
solution all matter to efficiency
b. Electric circuit with cells in the middle of charge carrying ions (V=IR)
2. cheap once you have equipment
3. works with many cell types
4. stable or transient
5. Show cuvette and how it works
E. virus infection
1. Difficult to make virus
2. Easy to use once made
3. Some limits on cells type that can be transfected
4. Some viruses will transfect non-dividing cells
F. Gene gun [biolistic particle delivery]
1. Mostly used in for plant transfection to get DNA past cell wall
2. Coat a plastic particle with DNA and drive particles into cells with air gun
G. Liposomes
H. Sono-poration
I. Magnetofection
J. OPtical transfection
K. Impalefection
III.
Transient vs Stable transformation
A. Transient- 4-6 hours after DNA introduction, you can see gene expression (GFP).
1. THe DNA is not yet chromatin and not yet regulated, but the promoter works
2. Over the next few days, the DNA is lost from the cell and expression tails off
B. Stable - chromosomal or extrachromosomal
1. The DNA is randomly intergrated into a chromosomal site
2. The DNA is homologously intergrated into a chromosomal site
a. homologous recombination (single vs double crossovers) - gene targeting
3. Restriction Enzyme Mediated Integration (REMI)
a. Electroporate DNA with restriction enzyme into cells and the DNA
integrates at restriction sites randomly.
4. The DNA has an origin of replication and is maintained extrachromosomally