(Curr Protoc Cell Biol.)
1: Curr Protoc Cell Biol. 2003 Aug;Chapter 20:Unit 20.7.
Optimization of transfection.
Yale University School of Medicine, New Haven, Connecticut, USA.
When embarking upon any transfection procedure, a critical first step is to
optimize conditions. Every mammalian cell type has a characteristic set of
requirements for optimal introduction of foreign DNA; there is a tremendous
degree of variability in the transfection conditions that work, even among cell
types that are very similar to one another. Often, an experimenter must screen a
wide variety of cell types for a desired regulatory trait, such as an appropriate
response to a particular effector molecule. It is thus helpful to have a
straightforward, systematic approach to optimizing transfection efficiency. This
unit provides guidelines to optimizing transfection experiments.
2: Curr Protoc Cell Biol. 2003 Aug;Chapter 20:Unit 20.4.
Transfection using DEAE-dextran.
Massachusetts General Hospital, Charlestown, Massachusetts, USA.
Transfection of cultured mammalian cells using diethylaminoethyl
(DEAE)-dextran/DNA can be an attractive alternative to other transfection methods
in many circumstances. The major advantages of the technique are its relative
simplicity and speed, limited expense, and remarkably reproducible
interexperimental and intraexperimental transfection efficiency. Disadvantages
include inhibition of cell growth and induction of heterogeneous morphological
changes in cells. Furthermore, the concentration of serum in the culture medium
must be transiently reduced during the transfection. In general, DEAE-dextran DNA
transfection is ideal for transient transfections with promoter/reporter plasmids
in analyses of promoter and enhancer functions, and is suitable for
overexpression of recombinant protein in transient transfections or for
generation of stable cell lines using vectors designed to exist in the cell as
episomes. This unit presents a general description of DEAE-dextran transfection,
as well as two more specific protocols for typical experimental applications. The
basic protocol is suitable for transfection of anchorage-dependent (attached)
cells. For cells that grow in suspension, electroporation or lipofection is
usually preferred, although DEAE-dextran-mediated transfection can be used.
3: Curr Protoc Cell Biol. 2003 Aug;Chapter 20:Unit 20.3.
Calcium phosphate transfection.
Kingston RE, Chen CA, Okayama H.
Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts,
This unit presents two methods of calcium phosphate-based eukaryotic cell
transfection that can be used for both transient and stable transfections. In
these protocols, plasmid DNA is introduced to monolayer cell cultures via a
precipitate that adheres to the cell surface. A HEPES-buffered solution is used
to form a calcium phosphate precipitate that is directly layered onto the cells.
For some cells, shocking the cells with glycerol or DMSO improves transfection
efficiency. In the alternate high-efficiency method, a BES-buffered system is
used that allows the precipitate to form gradually in the medium and then drop
onto the cells. While the alternate method is particularly efficient for stable
transformation of cells with circular plasmid DNA, both protocols yield similar
results for transformation with linear plasmid or genomic DNA, or for transient
4: Curr Protoc Cell Biol. 2003 Aug;Chapter 20:Unit 20.6.
Transfection of cultured eukaryotic cells using cationic lipid reagents.
Hawley-Nelson P, Ciccarone V.
Life Technologies, Inc., Rockville, Maryland, USA.
The development of high-efficiency methods for the introduction of functional
genetic material into eukaryotic cells using cationic lipids has accelerated
biological research in the studies of gene expression, control of cell growth,
and cell lineage. Transfection mediated by cationic lipids is commonly used in
industrial protein production as well as in some clinical gene therapy protocols.
Replacing our previous unit on this topic, this new version describes how to
perform transfection of adherent and suspension cells, insect cells, and RNA
transfection using the cationic lipid system.
siRNA transfection efficiency is dependent on a variety of factors. To chose the right siRNA transfection reagent is the key to achieve maximum silencing effect of siRNA. The most appropriate way to measure the silencing effect is through protein expression detection of the targeted gene. Several siRNA transfection reagents are available from different vendors to deliver siRNA duplex into cells. The general guideline is to chose siRNA transfection reagents with maximum transfection efficiency and minimum toxicity.
Recommended siRNA user guide:
Selection of siRNA duplexes from the target mRNA sequence
(Tuschl lab, Rockefeller University)