Transfection of Cos7 cells with GFP

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Transfection of Cos-7 cells with GFP
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Cos-7 Cells

• Derived from the African Green Monkey
• Originally CV-1
• Transformed with a defective mutant of the simian
  virus 40 (SV-40)

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Transfection

• Delivery of foreign molecules (DNA) into
  eukaryotic cells
• Transient or stable
• Study and control gene expression
• Biochemical characterization
• Effects of gene expression on cell growth
• Production of specific protein for purification
• Gene therapy

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Methods of transfecting cells

• Create a complex of DNA plus some substance that
  will go into the cell
• Calcium phosphate (1973)
• Tricky but inexpensive
• Cationic liposomes (1987)
• Virus mediated
• Microinjection
• Electroporation
• Activated dendrimers
• Non-liposomal lipids

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Cationic liposome

• Cationic and neutral lipids form lipid bilayer
  structures
• Ionic interactions between liposome and DNA form
  a complex
• High efficiency
• FuGENE, Gene Juice, Lipofectamine

Liposomes are also be useful for drug and
cosmetic delivery
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• Liposome-DNA complex uptake may be mediated by
  endocytosis

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Phagocytosisthe uptake of particles larger than
250nm
Mmm...yummy bacteria!!
Help! Im to be broken down to mere
macro- molecules!!
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Pinocytosisthe uptake of particles less than
150nm
Lumen inside capillary
Capillary endothelial cell
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Factors that Decrease Transfection Efficiency

• Presence of antibiotics
• Lowers transfection rates
• Reagent may bring antibiotic into the cells
• Or, complex may interact with the antibiotic
• Presence of serum
• Conversely, lack of serum might make liposome
  more toxic to cells

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Serum Free Media
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Disadvantages of Serum

• Physiological Variability
• Shelf-life and consistency
• Quality control
• Availability
• Contamination
• Cost
• Standardization (batch to batch variability)

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Contaminants from Serum

• A big problem for industry if company wishes to
  create human-use products
• Also cant use phenol red
• media will not be pink/red, but will be yellowish

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Bush approved stem cells are contaminated

• January 2005
• Researchers face the possibility that their
  experiments have been ruined by contamination
  from animal cells.
• Antibodies created by body against the sugar
• N-glycolylneuraminic acid
• Could lead to rejection of implanted cells
• Either from feeder plates or from the serum

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Advantages of Serum Free Media

• Eliminates disadvantages of serum
• Can create a selective media
• Good for some primary cultures
• E.g. selecting for parenchymal cells (breast
  epithelia or keratinocytes) over fibroblasts
• Regulate proliferation and differentiation
• Can easily change growth and differentiation
  factors

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Disadvantages of Serum Free media

• Multiplicity of media
• Each cell line needs different media
• Selectivity
• May select a sub-lineage in cell line
• Reagent purity
• Serum removal also removes protective,
  detoxifying action of some serum proteins
• Cell proliferation
• Cell growth is slower
• Fewer generations produced

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Potential Changes to Subculture Procedure when
Serum is Removed

• May need to add adhesion factors to plate
• Poly-lysine or fibronectin to plate or well
• No protease inhibitors available
• Trypsin must be more dilute
• Cells are more fragile in presence of trypsin

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Factors needed when using serum free media

• Hormones
• Growth factors
• Nutrients
• Proteins
• Matrix proteins
• Lipids
• Vary by cell line

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GFP-tubulin plasmid
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GFP

• Green fluorescent protein
• Gives luminescent jelly fish their greenish glow
• A marker protein on a plasmid vector
• Wild type doesnt express in all cell lines
• Mutant variables available

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Plasmid Vector

• Relatively small circular DNA molecules that can
  replicate inside a cell
• Contains a replication origin
• Can replicate independently of chromosome
• Usually antibiotic resistance
• Cells taking up DNA are easily identified
• Can make multiple copies by growing in bacteria

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GFP Fusion Protein Strategy

• Can splice in protein of interest
• Our plasmid has tubulin spliced in
• Tubulin, a cytoskeletal protein, should be
  overexpressed
• Determine distribution of protein
• Track movement of protein
• Track movement of cells

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• View with fluorescent microscope
• Use blue or ultraviolet light

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eGFP-tubulin transfected into COS-7 cells using
fuGENE
Cos-7 cells are monkey fibroblasts that have
been transformed with SV-40 so the cell is
Easily transfected Cell is now Biosafety Level 2
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Handling FuGENE 6

• It is attracted to the sides of the tubes
• Immerse the pipet tip below the level of the
  serum free media rather than laying FuGENE on the
  top
• It is very volatile
• Close the caps on the FuGENE tube and your
  media/FuGENE mixture as soon as you are done
  retrieving each aliquot!

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Working with DNA

• Concentration of DNA this week is 1mg/ml
• Wear gloves
• DNA remains on ice!
• small volumes
• Use 0.5-10ml pipettor
• Specialized tips

Remember your aseptic technique!