Transfection is the process of introduction of foreign DNA into the nucleus of eukaryotic cell. The cells which has incorporated exogenous DNA are called transfectants.

Transfection Methodologies:

Following are the methodologies that are used for transfection, each of the below strategies has its own advantages and disadvantages, so one need to choose the suitable method based on the application.

1. Transfection  – Reagent Based

Reagent based methods are simple, The reagent used, neutralizes the negative charge and condenses the DNA for effective uptake.

As the cell membrane is negatively charged, -vely charged DNA molecules will be repelled from the cells, for the cells to take up DNA the net charge need to be positive, that is the reason why reagents are used. The main principle of using reagents is to give the DNA a net positive charge either by reagents binding to the DNA or by forming complexes with the DNA.

• Calcium Phosphate
• Lipids
• Cationic Polymers

Overview of Reagent Based Transfection

The mechanism of DNA uptake by calcium phosphate mediated method is not well understood. In this method, the DNA to transfected is mixed with calcium chloride, then this is added slowly to a buffered saline or phosphate solution. Incubating this mixture with the cells to be transfected. The cells will take up the DNA by endocytic pathways,

Lipid Mediated Transfection – Lipofection

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

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

Cationic Polymers like PEI forms complex with DNA called the Polypelex (DNA, Polycation).

The Positively charge polymer:DNA complex comes into close association with negatively charged membrane.

Charge ratio:

Transfection – Instrument Based

Nucleic acids are transported mechanically into the cells, following are the commonly used methods for instrument based transfection.

• Electroporation
• Biolistic Technology
• Microinjection
• Laserfection / Optoinjection

transferred into plastic or glass cuvette.The cuvettes used here are specially meant for electroporation, they have aluminium electrodes at the sides. The voltage and capacitance is set and the cuvette inserted into the electroporator. 

Once after electroporation, few ml medium is added , and is incubated at the the culture’s optimal temperature for an hour or more to allow recovery of the cells.The transformed cells can be screened out using selectable markers in the plasmid (usaually antibiotic resisitance genes are used).

Advantages and Disadvantages of Electroporation

Electroporation has the following advantages and disadvantages

Advantages:

Versatility: Electroporation is effective with nearly all cell and species types.

Efficiency: A large majority of cells take in the target DNA or molecule. 

Small Scale: The amount of DNA required is smaller as compared with other methods.

Disadvantages:

Cell Damage: If the electric pulses are given for long time or with more intensity, some pores may become too large or fail to close after membrane discharge causing cell damage or rupture
Nonspecific Transport: The transport of material into and out of the cell during the time of electropermeability is relatively nonspecific. This may result in an ion imbalance that could later lead to improper cell function and cell death.

 Characteristics of Viral Vectors

• Green Fluorescent Protein 
• Luciferase Reporter 
• Beta – Galactosidase 
• Secreted Alkaline Phosphatase

Nucelic Acid Labelling

Fluorescent labels – Cyanine Dyes, Fluorescein
Epitope Tags

Factors affecting Transfection

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

Transfection – Optimization 

Media

Culture media plays a major role in transfection efficiency. Cell culture media mostly supplemented with serum. For efficient transfection transfection complex should be made in serum free media. The reason for serum free media usage for transfection complex formation is that, the serum has nucleases in it which can chew up the nucleic acids,

Antibiotics also should be avoided in the transfection complex formation media, as the antibiotics are charged, they can interfere in the transfection complex formation and there by reducing the transfection efficiency.

Other charged compounds like polyanions (eg: heparin sulfate) should be avoided and also surfactants like pluronic F68, mostly used as antifoaming agent, should be avoided.