INTRODUCTION
A broad variety of methods to introduce both RNA and DNA into cells have been
developed so far, including calcium phophate, cationic polymer/lipid, electroporation,
nucleofection, microinjection and virus mediated apporaches. A thorough review of
several transfection methods can be read at [5].
The development of such techniques has primarily been focused on the transfection of
DNA in contrast to RNA due to applicability in many di_erent research areas. A few
ideas have been suggested for RNA transfection by using cationic liposome transfer
which protects RNA from hydrolysis and by using self-replicating recombinant mRNA
derived by the Sinbis virus [6] to implement replication in the cell.
The mechanism of cationic lipid-mediated transfection
Specially designed cationic lipids, such as Lipofectamine® Transfection Reagents,
facilitate DNA and siRNA delivery into cells (1–3). The basic structure of cationic lipids
consists of a positively charged head group and one or two hydrocarbon chains. The
charged head group governs the interaction between the lipid and the phosphate backbone
of the nucleic acid, and facilitates DNA condensation. Often cationic lipids are
formulated with a neutral co-lipid or helper lipid, followed by extrusion or
microfluidization, which results in a unilamellar liposomal structure with a positive
surface
charge
when
formulated
in
water.
The positive surface charge of the liposomes also mediates the interaction of the nucleic
acid and the cell membrane, allowing for fusion of the liposome/nucleic acid
(“transfection complex”) with the negatively charged cell membrane. The transfection
complex is thought to enter the cell through endocytosis. Endocytosis is the process
where a localized region of the cellular membrane uptakes the DNA: liposome complex
by forming a membrane bound/intracellular vesicle. Once inside the cell, the complex
must escape the endosomal pathway, diffuse through the cytoplasm, and enter the nucleus
for gene expression. Cationic lipids are thought to facilitate transfection during the early
steps of the process by mediating DNA condensation and DNA/cellular interactions.
The principle of delivery using cationic lipid reagents thus differs from prior attempts to
use neutral liposomes for transfections. With cationic lipid reagents, the DNA solution is
not deliberately encapsulated within the liposomes, rather, the negatively charged DNA
binds spontaneously to the positively charged liposomes, forming DNA-cationic lipid
reagent complexes (Figure 1).
Some of the problems associated with traditional transfection methods like calcium
phosphate coprecipitation, DEAE-dextran, polybrene and electroporation, include low
efficiency of DNA delivery, poor reproducibility, cell toxicity, and inconvenience.
However, cationic lipid reagent-mediated transfection yields high and previously
unattainable transfection efficiencies in a wide variety of eukaryotic cells. It is simple to
perform, and ensures consistently reproducible results. Moreover, a number of cell lines
normally resistant to transfection by other methods transfect successfully using cationic
lipid reagents.
Day 1
» Prepare the mRNA transfection complex (in 6well format)
1. Thaw one 12ul aliquot of the eGFP mRNA on ice
2. Using RNase-free, aerosol-barrier pipette tips, add 48 ul of Opti-MEM to the tube
containing the mRNA and pipet gently to mix (Tube A)
3. Add 54 ul of Opti-MEM to a second sterile 1.5 ml RNase-free microfuge tube
4. Add 6 ul f RNAiMAX to the Opti-MEM in the second tube and pipet gently to
ensure none of the transfection reagent remains in the pipet tip Tube B)
5. Using a sterile tip, transfer the entire contents of the RNAiMAX+Opti-MEM mix
(60ul Tube B) to the tube containing the mRNA mix (Tube A)
6. Gently pipet to mix.
7. Incubate the tube at room temperature for 15 minutes to allow the mRNA to
complex with the transfection reagent.
Tube A
Tube B
48 ul Opti-MEM
54 ul Opti-MEM
12 ul eGFP mRNA
6 ul RNAiMAX
60 ul Total
60 ul Total
» Transfect cells
1. In a drop-wise fashion, add 120 ul of the mRNA transfection complex to each
well.
a. Note: Uniformly distribute the mRNA transfection complex across the 2
ml of culture medium
2. Gently rock the 6-well plate from side to side and front to back to distribute the
mRNA transfection complex across the well.
3. Incubate the cells for 4 hours at 37C and 5% CO2.
4. Next day examine the transfection efficiency using a fluorescence microscope for
GFP detection
Day 2
» Examine the expression of GFP post transfection
1. Using a fluorescence microscope try to detect the GFP signal
2. Take pictures of different magnifications (10x, 20x, 40x) of the transfected cells
3. Take notes of the transfection efficiency (estimate how many cells did get
transfected), right location of the GFP signal (where in the cell is the GFP
detected), fluorescence intensity (every cell the same?)
References
1. Chesnoy, S. and Huang, L. (2000) Annual Review of Biophysical Biomolecular
Structure 29: 27-47.
2. Hirko, A. et al. (2003) Current Medicinal Chemistry 10: 1185-1193.
3. Liu, D. et al. (2003) Current Medicinal Chemistry 10: 1307-1315.
4. http://www.invitrogen.com/site/us/en/home/Products-andServices/Applications/Cell-Culture/Transfection/transfection-methods/LipidTransfection.html
5. Tae Kyung Kim and James H Eberwine. Mammalian cell transfection: the present
and the future. Anal Bioanal Chem, 397(8):3173{8, Aug 2010.
6. T W Dubensky, Jr, D A Driver, J M Polo, B A Belli, E M Latham, C E Ibanez, S
Chada, D Brumm, T A Banks, S J Mento, D J Jolly, and S M Chang. Sindbis virus
dna-based expression vectors: utility for in vitro and in vivo gene transfer. J Virol,
70(1):508{19, Jan 1996.