CHEMICAL AGENTS USED IN CELL TRANSFORMATION

Aden Razaq

Overview Termionology

Factors affecting transfection

• HOST CELL

• cell health

cell culture

• GENETIC MATERIAL

DNA quantity and quality

Transfection workflow

Common transfection methods

Reagent based

Instrument based

Virus based

What is cell transformation? The insertion of foreign DNA or exogenous genetic material into a

cell.

The cell can be a bacterium, a virus, plant or animal cell.

Transfection Introduction of foreign DNA into the nucleus of Eukaryotic cells.

Cells that have incorporated the foreign DNA are called transfectants.

Stable transfectants : ni AND ngierof detargetni evah taht sllecemoneg rieht.

Transient transfectants : eht ni etargetni ton seod AND ngierof(emti detimil a rof desserpxe era seneg tub emoneg24-96 hours(.

FACTORS AFFECTING TRANSFECTION

CELL HEALTH:

Cells should be grown in appropriate medium with all necessary factors.

Culture should be free of contamination and cells should be maintained in log phase growth.

CELL CULTURE:

Cells should be transfected at 40-80% confluency (cell type dependent).

The number of passages should be low (< 50)

DNA QUALITY AND QUANTITY:

Use of high quality plasmid DNA that is free of proteins , RNA and chemicals for transfection.

The optimal amount of DNA vary widely depending upon the type of DNA, transfection method , target cell line and No of cells.

TRANSFECTION WORKFLOW

Tissue culture

count cells

Resuspend cells in electroporation buffer

Add nucleic acid

Transfect cells

Plate cells

Analysis

Microscopy Real time PCRFlow

cytometryReporter gene

activityWestern blot

analysis

Gene ExpressionProtein Expression

COMMON TRANSFECTION METHODS

Cationic Lipids : Liposome/Lipoplexes

Cationic lipids are ampiphilic molecules that have a positively charged polar head group linked, via an anchor to an apolar hydrophobic domain generally comprising two alkyl chains.

Electrostatic interactions b/w the positive charges of cationic lipid head groups and the negatively charged phosphates of the DNA backbone are the main forces that allow DNA to spontaneously associate with cationic lipids.

Lipid-Mediated Gene Delivery

Transfer of genetic material into the cells takes place via liposomes , which are vesicles that can merge with cell membrane since they are both made of phospholipid bilayer.

Method overview

Pros and consADVANTAGES OF LIPIDS:

• Deliver nucleic acids to the cells in culture dish with high efficiency.

• Easy to use, minimal steps required.

DISADVANTAGES:

• Not applicable to all cell types.

Calcium phosphate

The protocol involves mixing DNA with calcium chloride, adding this in a controlled manner to a buffer saline/phosphate solution, and allowing the mixture to incubate at room temp.

This step generates a precipitate that is dispersed onto the cultured cells. The precipitate is taken up via endocytosis or phagocytosis.

Method overview Solution A: DNA in calcium

solution

Solution B: 2X Hanks buffered saline solution

Pros and ConsAdvantages:

Inexpensive

High efficiency

Can be applied to wide range of cell types

Disadvantages:

Reagent consistency is critical for reproducibility

Small pH changes can compromise transformation efficiency

Size and quality of the precipitate are crucial to the success of transfection

DEAE-Dextran DEAE-Dextran is a cationic polymer that tightly associates with the

negatively charges nucleic acids.

The positively charged DNA-polymer complex comes in close association with negatively charged cell membrane.

DNA-polymer complex uptake into the cell is presumed to occur via endocytosis.

Method overview

Pros and ConsAdvantages:

Inexpensive

Easy to perform and quick

Can be applied to a wide range of cell types.

Disadvantages:

High conc of DEAE-Dextran can be toxic to the cell

Transfection efficiency will vary with cell type

Can be used only for transient transfection

Magnet-mediated transfection Magnet-mediated method uses magnetic force to deliver DNA into

the target cells.

Nucleic acids are first associated with magnetic nanoparticles .Then ,application of magnetic force drives the nucleic acid-particle complex toward and into the target cells, where the cargo is released.

Method overview

Pros and consAdvantages

Rapid

Increased transfection efficiency by direct transport, esp for low amount of nucleic acid

High transfection rates for adherent mammalian cell lines and primary cell cultures

Mild treatment of cells

Disadvantages

Relatively new method

Require adherent cells; suspension cells need to be immobilized or centrifuged.

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