ARIFPHD Scholar

What is Transfection?

Transfection vs. Transformation

Purpose of Transfection

How it Works

Experimental methods/processes of doing it.

Strengths and weaknesses of each method

Since the early 1980s, fruit flies, fish, sea urchins, frogs, laboratory mice and farm animals, such as cows, pigs, and sheep have been successfully produced.

The ability to manipulate the genome of the whole animal and the production of transgenic animals has influenced the science dramatically in the last 15 years.

The procedure for introducing exogenous donor DNA into a recipient cell is called Transfection. ( Non-viral methods)

Chromosomes are taken up inefficiently so that intact chromosomes rarely survived the procedure. Instead the recipient cell usually get a part of the DNA.

Transfection: Introduction of foreign DNA into the nucleous of eukaryotic cells. Cells that have incorporated the foreign DNA are called transfectants

Stable transfectants: Cells that have integrated foreign DNA in their genome

Transient transfectants: Foreign DNA does not integrate into the host genome but genes are expressed for a limited period of time (24-96 hours)

Which one should you use?

Protofection is the transfection of foreign mitochondrial DNA into the mitochondria of all cells in a tissue to supplement or replace the native mitochondrial DNA already present.

Transformation: genetically altering cells by changing their growth characteristics from finite to infinite by transporting in foreign genetic material.

Transfection: the process of transporting genetic material and/or macromolecules into eukaryotic cells through typically non-viral methods.

DNA transfer by natural methods

1. Conjugation

2. Bacterial transformation

3. Agrobacterium mediated transfer

Transduction

Viral vectors

Bactofection: bacterial delivery systems

Physical methods

1. Microinjection

2. Macroinjection

3. Biolistics transformation

4. Protoplast fusion

Chemical methods

1. DNA transfer by calcium phosphate method

2. Liposome mediated transfer

3. Transfer of DNA by use of polyethene glycol

4. Use of DEAE-Dextran for DNA transferElectrical methods

1. Electroporation 2. Electrofusion

Bactofection: The technique using bacteria for the direct gene transfer into the target organism, organ or tissue is called bactofection

Disadvantages of this method include: Low copy number integration.

Additional steps required to produce retroviruses.

Limitations on the size of the foreign DNA insert (usually 9 to 15 kb) transferred.

Potential for undesired genetic recombination that may alter the retrovirus.

High frequency of mosaicism.

Possible interference by integrated retroviral sequences in transgene expression.

Also the provirus attracts methylation which possibly in conjugation with other mechanisms disables its expression when it passes through the germ line.

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This transfection method has been verified

by Graham and Van in 1973.Principle: DNA is mixed with calcium

chloride. Addition to buffered

saline/phosphate solution and incubating at RT.

Formation of DNA-calcium phosphate co-precipitates which adhere to surface of cells.

Uptake presumably by endocytosis.

Frequency is very low.

Integrated genes undergo substantial modification.

Many cells do not like having the solid precipitate

adhering to them and the surface of their culture

vessel.

Due to above limitations transfection applied to

somatic gene therapy is limited.

Principle (liposome mediated gene transfer):

A cationic lipid is mixed with a neutral lipid/helper lipid (e.g. DOPE), unilamellar liposome vesicles are formed carrying a net positive charge.

Nucleic acids adsorb to these vesicles/packed structure.

Ionic absorption to the cellular membrane.

Uptake presumably by endocytosis.

Neutral »helper« lipids such as DOPE allow entrapped DNA to escape the endosomes by fusion of the lipsome with the membrane.

1. Simplicity.

2. Long term stability.

3. Low toxicity.

4.Protection of nucleic acid from degradation.

1. The ‘proton sponge’ phenomenon

Flip-flop mechanism

It is an efficient process to transfer DNA into cells.

Microscopic pores are induced in biological membrane by

the application of electric field. These pores are known as

electropores which allow the molecules, ions and water to

pass from one side of the membrane to another.

Electroporation has been reported to enhance the level of

gene expression and significantly improve immune

responses elicited to DNA vaccines in both large and small

animals

1. Method is fast.

2. Less costly.

3. Applied for a number of cell types.

4. Simultaneously a large number of cell can be treated.

5. High percentage of stable transformants can be produced.