Transfection – basics & optimization tips

Transfection
Basics & Optimization Tips
Bio-Resource
www.technologyinscience.blogspot.com

What is Transfection??
Transfection means :
• Transfer of exogenous DNA into a cell.
Transfectants means:
• The cells which has incorporated the
exogenous (foreign) DNA

Types of Transfection
• There are two types of transfection
• Transient &
• Stable Transfection

Transient Transfection Protocol
Dilute Plasmid in a reduced serum media.
Add Transfection reagent and mix.
Incubate (15-30) minutes.
Add DNA complexes directly to cells in
complete medium.
Incubate 24 – 72 hours.
Harvest cells and perform assay.

Transfection Methodologies
• Reagent Based
• Instrument Based
• Viral Mediated

Transfection – Reagent Based
• Calcium Phosphate
• Lipids
• Cationic Polymers

Reagent Based Method
• Simple Method.
• The reagent used, neutralizes the negative
charge and condenses the DNA for effective
uptake

DNA Uptake Mechanism
• Clathrin dependent
• Caveolin dependent or
• Other pathways.

Cationic Lipids
• Polar lipids formed of highly positive charge
head groups attached to hydrophobic tails.

Lipoplexes

Helper Lipids in Transfection Reagent
• Reagents for Transfection are also made of
helper lipids.
• Helper lipids in conjunction with cationic lipids
forms structures called liposomes, they can
effectively encapsulate DNA.

DNA Uptake by Cell

• The DNA/Complex enter by endocytic pathways
and reaches really low pH zone of endolyzosome.
• DNA / Transfection complex need to be released
once inside the cytoplasm.

Cationic Polymers
• PEI (Polyethyleneimine) – Very effective
nucleic acid condensing agents.

• Cationic Polymers like PEI forms complex with
DNA called the Polypelex (DNA, Polycation).
• This complex has net positive charge, are very
effective in transfection.

DEAE - Dextran
• DEAE dextran is a cationic polymer that tightly
associated with negatively charged nucleic acid.
• The Positively charge polymer:DNA complex
comes into close association with negatively
charged membrane.

Lipid & Polymer Combination -
Lipopolyplex

Ideal Features of Transfection
Complex
• Size – 40 – 1000 nm.
• Charge – Transfection complex should have a net
cationic (+) charge.
• Positive surface charge density – Zeta Potential
(+mV).
• Charge ratio.

Charge Ratio – N/P ratio
• N/P Ratio =
Reagent Concentration in Nitrogen residues (mM)
DNA Concentration in phosphate moeities (mM)
*N/P ratio is very important for optimization.

Transfection – Instrument Based
• Electroporation
• Biolistic Technology
• Microinjection
• Laserfection / Optoinjection

Electroporation

Biolistic Technology
Biolistic Transformation is the transfer of nucleic acid into cells via high
velocity nucleic acid coated microparticles

Laserfection/Optoinjection
• This method laser light to transiently
permeabilize cells in very short time.

Transfection – Viral Mediated
• Retrovirus
• Adeno-associated Viruses
• Lentivirus

Viral Vectors

Viral Vectors - Characteristics

Advantages and Disadvantages of
different Transfection Methods

Evaluating Transfection Performance
Reporter systems
• Green Fluorescent Protein
• Luciferase Reporter
• Beta – Galactosidase
• Secreted Alkaline Phosphatase

Direct Visualization of Nucleic acid
delivery
• Nucelic Acid Labelling
• Fluorescent labels – Cyanine Dyes, Fluorescein
• Epitope Tags
• Covalent linkage of dye to the DNA.

Factors affecting Transfection
• Media
• Nucleic Acid
• Complexing Time
• Cell Culture condition / Confluency
• Harvesting Time

Transfection - Optimization
Media
• Transfection complex should be made in
serum free media.
• Serum has nucleases, which will chew up the
DNA.
• Antibiotics should be avoided in the
transfection complex formation media, as the
antibiotics are charged, they interfere in
complex formation.

Nucleic Acid
• Level of Purity: Plasmid purity 260/280 ratio
should be >1.8.
• Endotoxin contaminated DNA inhibits
transfection or yield low transfection
efficiency.
• Endotoxin is toxic to the cells.

Complexing Time
• Complex time formation is also important.
• Too much time – too big complex.
• Recommended incubation time for complex
formation is 15-30 mins. (Not more than 30
mins).
• For mRNA 5 – 10 mins.(Not more than 10
mins).

Cell Culture Conditions
• Adherent cell confluency affects transfection.
• Low confluency – 25% - Not good.
• 50 – 70% confluency is good for transfection.
• At >90% confluency, cells will not take up DNA to nuclei
because of the dissociation of nuclear membrane.

Refernces
• Life Technologies , Technical Resources
• Biorad, Technical Resources
• Qiagen, Technical Resources

Thank You

Transfection – basics & optimization tips

More Related Content

What's hot

Viewers also liked

Similar to Transfection – basics & optimization tips

More from ajithnandanam

Recently uploaded

Transfection – basics & optimization tips