This is a lipid-mediated transfection technique used to inject genetic material into a cell by means of liposomes. Generally uses a positively charged (cationic) lipid or neutral lipids to form a structure with the negatively charged (anionic) genetic material.
1. Method of gene transfer:
Lipofection
Presented By:
Sonali Mohapatra
Roll No.: 201649
2. About:
● This is a lipid-mediated transfection technique used to inject genetic material into a cell by
means of liposomes.
● Liposomes are microscopic vesicles (diameter ranging from 20 nm to a few micrometers), that
can easily merge with the cell membrane since they are both made of a phospholipid bilayer.
● Depending on the size, liposomes can be categorized in the following classes:
● Small unilamellar vesicles (SUV), size range 20–100 nm
● Large unilamellar vesicles (LUV), size >100 nm
● Giant unilamellar vesicles (GULV), size >1000 nm
● Oligo Lamellar vesicles (OLV), size range 100–1000 nm
● Multilamellar large vesicles (MLV), with size >500 nm
● Multivesicular vesicles, size from 1000 nm to several thousand nanometers.
3. Liposomes:
Desired
components
Components:
● Phospholipids: Main component of the bilayer.
■ Natural: Phosphatidylcholine,
Phosphatidylethanolamine
■ Synthetic: Dioleoyl phosphatidylcholine (DOPC),
Dioleoylphosphatidylethanolamine (DOPE)
● Cholesterol:
■ Improves membrane fluidity.
■ Reduces its permeability to body fluids like
blood and plasma.
Cholesterol
Structure of Liposomes
Hydrophobic tail
nonpolar cation chains
Hydrophilic head (polar
region)
6. Liposome-mediated Transfection:
● Lipofection generally uses a positively charged (cationic) lipid or neutral
lipids to form a structure with the negatively charged (anionic) genetic
material.
● The basis for using cationic lipids as a delivery system for negatively charged
DNA is that the positively charged hydrophilic head group can condense with
the DNA while the hydrophobic tail can form bilayer structures around the
DNA. This complexation of lipids around the DNA has been termed a
lipoplex and yields DNA protection against nucleases.
● Fusion of the liposome-nucleic acid transfection complex with the negatively
charged cell membrane takes place. The transfection complex is thought to
enter the cell through endocytosis. Once inside the cell, the complex must
escape the endosomal pathway, diffuse through the cytoplasm, and enter the
nucleus for gene expression.
7. Cationic Lipid Mediated Transfection:
● Cationic lipid-mediated delivery is a fast, simple, and reproducible means for
easily introducing DNA, RNA, siRNA, or oligonucleotides into eukaryotic cells.
● Uses a helper lipid to increase the transfection efficiency.
● For example: Cationic lipid having an ethylenediamine cationic head group, denoted
by DA, associated with DOPE, achieved a greater efficiency than alone.
● Allows the highly efficient transfection of a broad range of cell types, including
adherent, suspension, and insect cells, as well as primary cultures.
● Example of cation reagent is Lipofectamine 2000, mainly used to increase
transfection efficiency of RNA and plasmid DNA.
9. Applications:
➔ Cationic liposomes are used in gene therapy
due to their ability to successfully encapsulate
nucleic acids.
➔ Used to induced mutation into zygotes during
in vitro fertilization and in embryos at the 2-
and 4-cell stages.
➔ Used to transfer plasmids into sensitive cells.
➔ The use of liposomes in drug delivery aims at
modifying the pharmacokinetics of drugs to
improve the therapeutic efficacy while
minimizing potential toxicity.
➔ Used in various diagnosis procedures.
10. Advantages:
● Yields high transfection efficiencies.
● Works in a wide variety of eukaryotic
cells.
● Low toxicity.
● Simple to perform.
● Highly stable.
● Moreover, a number of cell lines
normally resistant to transfection by
other methods transfect successfully
with cationic lipid reagents.
● Quite low transfection
efficiency in suspension cells.
● Dependence on cell division.
As well as on high rate of
endocytosis.
● Not Applicable to all cell types.
Disadvantages:
