Transfection
Transcript of Transfection
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(.
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.
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
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.
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.
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.
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.
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.