biotechnology

1. CHEMICAL AGENTS USED IN CELL
TRANSFORMATION
Aden Razaq

2. 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

3. 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.

4. 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 sllec :
.emoneg rieht
 Transient transfectants eht ni etargetni ton seod AND ngierof :
96-24(emti detimil a rof desserpxe era seneg tub emoneghours(.

5. FACTORS AFFECTING
TRANSFECTION

6.  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.

7. TRANSFECTION WORKFLOW

8. Tissue culture
count cells
Resuspend cells in
electroporation buffer
Add nucleic acid
Transfect cells
Plate cells
Protein Expression Gene Expression
Analysis
Flow
Microscopy Real time PCR
cytometry
Reporter gene
activity
Western blot
analysis

9. COMMON TRANSFECTION
METHODS

10. 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.

11. 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.

12. Method overview

13. Pros and cons
ADVANTAGES 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.

14. 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.

15. Method overview
 Solution A: DNA in calcium
solution
 Solution B: 2X Hanks buffered
saline solution

16. Pros and Cons
Advantages:
 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

17. 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.

18. Method overview

19. Pros and Cons
Advantages:
 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

20. 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.

21. Method overview

22. Pros and cons
Advantages
 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.

23. Thank you