Biomedical Engineering Reference
In-Depth Information
uptake was not understood. Much later, it was
appreciated that successful DNA transfer in such
experiments was dependent on the formation of a
fine DNA/calcium phosphate coprecipitate, which
first settles on to the cells and is then internalized.
The precipitate must be formed freshly at the time of
transfection. It is thought that small granules of
calcium phosphate associated with DNA are taken
up by endocytosis and transported to the nucleus,
where some DNA escapes and can be expressed
(Orrantia & Chang, 1990). The technique became
generally accepted after its application, by Graham
and Van der Erb (1973), to the analysis of the infect-
ivity of adenoviral DNA. It is now established as a
general method for the introduction of DNA into a
wide range of cell types in culture. However, since
the precipitate must coat the cells, this method is
suitable only for cells growing in monolayers, not
those growing in suspension or as clumps.
As originally described, calcium phosphate trans-
fection was limited by the variable and rather low
proportion of cells that took up DNA (1-2%). Only a
small number of these would be stably transformed.
Improvements to the method have increased the
transfection frequency to 20% for some cell lines
(Chu & Sharp 1981). A variant of the technique,
using a different buffer system, allows the precipitate
to form slowly over a number of hours, and this
can increase stable transformation efficiency by up
to 100-fold when using high-quality plasmid DNA
(Chen & Okayama 1987, 1988).
(1984) and Sussman and Milman (1984) by adding
after-treatments, such as osmotic shock or expos-
ure to chloroquine, the latter having been shown
to inhibit the acidification of endosomal vesicles
(Luthmann & Magnusson 1983). Although efficient
for the transient transfection of many cell types,
DEAE-dextran cannot be used to generate stably
transformed cell lines. Another polycationic chem-
ical, the detergent Polybrene, has been used for
the transfection of Chinese hamster ovary (CHO)
cells, which are not amenable to calcium phosphate
transfection (Chaney
et al
. 1986).
Phospholipids as gene-delivery vehicles
An alternative to chemical transfection procedures
is to package DNA inside a phosopholipid vesicle,
which interacts with the target cell membrane and
facilitates DNA uptake. The first example of this
approach was provided by Schaffner (1980), who
used bacterial protoplasts containing plasmids to
transfer DNA into mammalian cells. Briefly, bac-
terial cells were transformed with a suitable plasmid
vector and then treated with chloramphenicol to
amplify the plasmid copy number. Lysozyme was
used to remove the cell walls, and the resulting pro-
toplasts were gently centrifuged on to a monolayer
of mammalian cells and induced to fuse with them,
using polyethylene glycol. A similar strategy was
employed by Wiberg
et al
. (1987), who used the
haemoglobin-free ghosts of erythrocytes as delivery
vehicles. The procedures are very efficient in terms of
the number of transformants obtained, but they are
also labour-intensive and so have not been widely
adopted as a general transfection method. However,
an important advantage is that they are gentle,
allowing the transfer of large DNA fragments with-
out shearing. Yeast cells with the cell wall removed
(sphaeroplasts) have therefore been used to intro-
duce yeast artificial chromosome (YAC) DNA into
mouse ES cells by this method, for the production of
YAC transgenic mice (see Chapter 11).
More widespread use has been made of artificial
phospholipid vesicles, which are called
liposomes
(Schaefer-Ridder
et al
. 1982). Initial liposome-based
procedures were hampered by the difficulty encoun-
tered in encapsulating the DNA, and provided a
transfection efficiency no better than the calcium
Other chemical transfection methods
The calcium phosphate method is applicable to many
cell types, but some cell lines are adversely affected
by the coprecipitate due to its toxicity and are hence
difficult to transfect. Alternative chemical transfec-
tion methods have been developed to address this
problem. One such method utilizes diethylamino-
ethyl dextran (DEAE-dextran), a soluble polycationic
carbohydrate that promotes interactions between
DNA and the endocytotic machinery of the cell. This
technique was initially devised to introduce viral
DNA into cells (McCutchan & Pango 1968) but was
later adapted as a method for plasmid DNA transfer
(Milman & Herzberg 1981). The efficiency of the
original procedure was improved by Lopata
et al
.
