IGP Methodology Class

1
IGP Methodology Class

• Transfection Methods
• Jared Boustead
• Molecular Physiology and Biophysics
• Extension 6-1629

2
Purpose of Transfection

• Introduction of exogenous DNA into cells in
  culture
• Analysis of expression regulation
• Effects of gene on cellular growth
• Overexpression of gene for purification of product

3
Types of Transfection

• Transient
• DNA not integrated into host genome
• Expression usually peaks 24-72 hours after
  introduction of DNA
• Efficiency 1-90 of cells
• Stable
• DNA integrates into host genome
• Expression is constant or inducible
• Efficiency 100 after selection

4
Transient Transfection
5
Types of Transfection

• Transient
• DNA not integrated into host genome
• Expression usually peaks 24-72 hours after
  introduction of DNA
• Efficiency 1-90 of cells
• Stable
• DNA integrates into host genome
• Expression is constant or inducible
• Efficiency 100 after selection

6
Stable Transfection
7
Considerations for Transfection

• Methods
• Calcium Phosphate
• DEAE-Dextran
• Liposome-mediated
• Electroporation
• Optimization
• Preparation of Reagents
• DNA
• Amount
• Method of preparation
• Standing Time
• Culture conditions

8
Calcium Phosphate

• Advantages
• Relatively inexpensive
• Can accommodate high mass of DNA
• Representative sampling of DNA in precipitate
• Disadvantages
• Efficiency is often low
• Can be toxic to cells
• Does not work well with non-adherent cells
• Mechanism is unclear

9
Calcium Phosphate

• Method
• Mix DNA suspension with CaCl2 solution
• Alternatively, DNA can be precipitated and
  resuspended in CaCl2 solution
• Add HEPES buffered saline (contains sodium
  phosphate)
• Allow to stand in order to form precipitate
• Add cells to CaPO4 or add CaPO4 to dish
• Incubate with precipitate (time is variable).
  Some protocols call for DMSO or glycerol shock
• After appropriate time for expression, harvest
  and assay

10
Calcium Phosphate

• Variables
• Amount of DNA
• Time precipitate is left on cells
• To shock or not to shock?
• pH
• Controls
• Transfection of vector lacking cDNA of interest
• Internal control plasmid

11
DEAE-Dextran

• Advantages
• Relatively inexpensive
• Requires small amount of DNA
• Can be used with adherent and suspension cultures
• Disadvantages
• Efficiency is often low
• Can induce morphological changes in cells
• Does not work well for stable transfections
• Works best when added in presence of
  reduced-serum concentration
• Mechanism is by endocytosis

12
DEAE-Dextran

• Method
• Mix DNA suspension with DEAE-Dextran in DMEM and
  add chlorquine
• Add DEAE-Dextran-DNA containing medium to dish
• Incubate with medium (time is variable). Some
  protocols call for DMSO or glycerol shock
• After appropriate time for expression, harvest
  and assay

13
DEAE-Dextran

• Variables
• Concentration of DEAE-Dextran
• Ratio of DEAE-Dextran to DNA
• Duration of transfection
• Presence and timing of chloroquine
• Controls
• Transfection of vector lacking cDNA of interest
• Internal control plasmid

14
Liposome-Mediated Transfection

• Advantages
• Higher transfection efficiencies than other
  chemical methods
• Works with wide variety of cells
• Low cell toxicity
• Can be used to transfect RNA and oligonucelotides
• Disadvantages
• Higher cost than other chemical methods which
  often precludes large-scale use
• Mechanism was originally thought to be by
  membrane fusion but is now thought to be by
  endocytosis

15
Liposome-Mediated Transfection

• Method
• Mix DNA with lipid in serum-free medium
• Add to cells
• Incubate for several (3-5) hours
• Switch to normal growth medium
• After appropriate time for expression, harvest
  and assay

16
Liposome-Mediated Transfection

• Variables
• Type of lipid mixture
• Amount of DNA
• Amount of lipid
• Length of incubation
• Controls
• Transfection of vector lacking cDNA of interest
• Internal control plasmid

17
Electroporation

• Advantages
• Can be used with most cell types
• High efficiency
• Requires few steps
• Disadvantages
• Requires special equipment
• High fraction of cells die during electroporation
• Mechanism is by exposing cells to high-voltage
  impulse that is thought to induce transient
  breakdown of cell membrane

18
Electroporation

• Method
• Harvest cells and wash
• Resuspend in electroporation buffer (low ionic
  strength) and put in cuvette
• Add DNA
• Mix well
• Electroporate
• Transfer to dish and add medium
• After appropriate time for expression, harvest
  and assay

19
Electroporation

• Variables
• Electric field strength and pulse duration
• Temperature
• Ionic strength of buffer in which cells are
  suspended
• DNA concentration
• Controls
• Transfection of vector lacking cDNA of interest
  or mock transfected cells
• Internal control plasmid

20
Viral Transduction

• Advantages
• High efficiency delivery of gene of interest
  leading to high levels of expression
• Can often be used for delivery of gene in vivo
• Disadvantages
• Increased time and cost involved in preparation
  of virions
• Some viruses present special safety
  considerations. Careful selection of viral
  system can minimize safety concerns.

21
Electroporation Equipment
22
Viral Transduction

• Method
• Generate construct with gene of interest and part
  of viral genome
• Transfect into packaging cell line and collect
  supernatents or produce stable packaging cell
  lines
• Purify virus
• Infect cell line of interest with virus
• Allow time for expression and perform desired
  assay

23
Viral Transduction

• Variables
• Type of virus
• Retrovirus
• Adenovirus
• Baculovirus
• Amount of virus
• Controls
• Infection with virus not carrying cDNA of interest

24
Reporters and Selectable Markers

• Reporters
• Green fluorescent protein
• Luciferase
• ?-galactosidase
• Cell-surface antigen for FACS
• Selectable Markers
• Neor G418
• Hygromycin

25
Cotransfection of Internal Control
26
Use of Bicistronic Vector
IRES
Bicistronic vector
assay transfected cells
transfect
Allow reporter expression of your protein and
GFP protein in the same cell. (24-72 hrs)
27
Inducible Expression
(
-
)
T
E
T
tet
tAK
tAK
tAK
Ptet
tAK
Ptet
tAK
reporter
reporter
Ptet
Ptet
28
References

• Ravid K and Freshney RI, Eds. 1998. DNA
  transfer to cultured cells. New York
  Wiley-Liss.
• Ausubel F, et al. Eds. 2003. Current protocols
  in molecular biology. New York Wiley.
• Sambrook J and Russell DW, Eds. 2001. Molecular
  cloning a laboratory manual, 3rd ed. Cold
  Spring Harbor Cold Spring Harbor Laboratory
  Press
• Notes If you are transfecting a cell line with
  which you have no experience, look at the primary
  literature to see what others have done.
  Manufacturers of commercial reagents often have
  helpful literature available. There are also
  some other methods that have not been discussed
  here.