. Lipofection . calcium chloride mediated gene trransfer

1. Aakifah Amreen H.E
MSc (BT)

2. Introduction
Transfection
Chemical methods
oLipofection
oCalcium chloride
oReference
Content

3. The cell membrane consists of a phospholipid bilayer with embedded proteins
and carries a net negative charge. Thus, It presents an impenetrable barrier to
large molecules that, like the phosphate backbones of DNA and RNA, are also
negatively charged. To sneak nucleic acids through the cell membrane,
researchers have developed a number of techniques each using a different
approach—from using chemicals and carrier molecules that coat the nucleic
acids to neutralize them to physical methods that create transient pores in the
membrane to introduce the DNA directly into the cell.
Introduction

4. Transfection
Is the process of artificially
introducing nucleic acids (DNA or
RNA) into cells, utilizing means
other than viral infection. Such
introductions of foreign nucleic acid
using various chemical, biological,
or physical methods can result in a
change of the properties of the cell.

5. Transfection technologies available today can be broadly classified into three groups:
chemical, biological, and physical.
Chemical methods that use carrier molecules to neutralize or impart a positive
charge to the negatively charged nucleic acids
Biological methods that rely on genetically engineered viruses to transfer non-viral
genes into cells
Physical methods directly deliver nucleic acids into the cytoplasm or the nucleus
of the cell

6. Chemical transfection is a popular technique due
to the ease, cost, and wide variety of transfection
reagents available.
There are different methods included :
DNA transfer by calcium chloride
Transfer of DNA by polyethelene glycol
Use of DEAE- Dextran for DNA transfer
Liposome mediated transfer
Chemical methods

7. Lipofection is the transfection using liposomes, small molecules which can
fuse with cell membranes and are able to release their contents. Liposomes
can be lipid-based (more common), or non-lipid based. Cationic polymers,
carrying a positive charge, bind well to negatively charged nucleic acids, and
are another method of transfection.
Lipofection

8. Principle
Lipofection is to associate nucleic acids with
cationic lipid formulation. The resulting molecular
complexes, known as lipoplexes, are then taken up by
the cells.
The main advantages of lipofection are its high
efficiency, its ability to transfect all types of nucleic
acids in a wide range of cell types, its ease of use,
reproducibility and low toxicity. In addition this
method is suitable for all transfection applications
Working
The lipid-based reagents used for lipofection are
generally composed of synthetic cationic lipids that
are often mixed with helper lipids such as DOPE or
cholesterol.
These lipids mixture assembles in liposomes or
micelles with an overall positive charge at
physiological pH and are able to form complexes
(lipoplexes) with negatively charged nucleic acids
The association of the lipid-based transfection
reagent with nucleic acids results in a tight
compaction and protection of the nucleic acids and
these cationic complexes are mainly internalized by
endocytosis.

9. Once inside the cells two mechanisms
leading to the nucleic acids release into the
cytoplasm:
1. The endosomes buffering capacity of
the polycationic residues.
2. the ability of cellular negatively
charged lipids to neutralize the
cationic residues of the transfection
reagent leading to destabilization of
endosomal membranes.
Finally, the cellular and molecular events
leading to the nuclear uptake of DNA
following by gene expression remain
highly speculative. However, the
significance of cell division on transfection
efficiency favours the assumption that
nuclear membrane disruption during the
mitosis process promote DNA nuclear
uptake.
Continued…..

11. Competent cells are bacterial cells that can accept extra-chromosomal DNA
or plasmids (naked DNA) from the environment.
The generation of competent cells may occur by two methods: natural
competence and artificial competence.
oNatural competence is the genetic ability of a bacterium to receive
environmental DNA under natural or in vitro conditions.
oArtificial competence Bacteria can also be made competent artificially by
chemical treatment and heat shock to make them transiently permeable to
DNA.
Calcium chloride mediated gene transfer

12. Calcium chloride transformation technique is the most efficient technique among
the competent cell preparation protocols. It increases the bacterial cell’s ability to
incorporate plasmid DNA, facilitating genetic transformation. Addition of calcium
chloride to the cell suspension allows the binding of plasmid DNA to LPS. Thus,
both the negatively charged DNA backbone and LPS come together and when heat
shock is provided, plasmid DNA passes into the bacterial cell.
Principle

14. Prepare a overnight culture of the bacteria in LB broth. Grow at 37°C without
shaking.
About 2 h before to begin the main procedure, use 1.0 mL of the overnight culture to
inoculate 100 mL of fresh LB broth. This culture is grown with rapid shaking at 37°C
until it reaches roughly 5 x 107 cells/ml. Thus corresponds to an OD650 for culture
Take a 5 mL aliquot of each transformation reaction and transfer to sterile plastic
centrifuge tubes. Cool on ice for 10 mm.
Pellet the cells by spinning for 5 min at 5000g. It is necessary for the centrifugation
to be performed at 4°C..
Pour off the supernatant and resuspend cells in 25 mL of cold 0.1M CaCl2. Leave on
ice for at least 20 min.
Centrifuge as in Step 3.
Procedure

15. Resuspend the cells in 0.2 mL of cold 0.1M CaCl2.
Transfer the suspensions to sterile, thin-walled glass bottles or tubes. The use of
glass makes the subsequent heat shocks more effective.
To each tube add up to 0.1 mg of DNA, made up in a standard DNA storage buffer
such as TE to a volume of 100 mL. Leave on ice for 30 min.
Transfer to a 42°C water bath for 2 min and return briefly to ice.
Transfer the contents of each tube to 2 mL of LB broth in a small flask. Incubate
with shaking at 37°C for 60-90 min.
Plate 0.1 mL aliquots of undiluted, 10-1 and 10-2 dilutions onto LB plates to which
the antibiotics to be used for selection have been added.
Incubate overnight at 37°C.

17. https://www.ozbiosciences.com/content/14-Lipofection-liposomal-
transfection-delivery-system
https://www.mybiosource.com/learn/testing-procedures/cacl2-transformation-
technique/
Refernce