Biomedical Engineering Reference
In-Depth Information
CHAPTER 10
Gene transfer to animal cells
Introduction
Overview of gene-transfer strategies
Gene transfer to animal cells has been practised now
for over 40 years. Techniques are available for the
introduction of DNA into many different cell types in
culture, either to study gene function and regulation
or to produce large amounts of recombinant protein.
Animal cells are advantageous for the production of
recombinant animal proteins because they perform
authentic post-translational modifications that are
not carried out by bacterial cells and fungi. Cell cultures
have therefore been used on a commercial scale to
synthesize products such as antibodies, hormones,
growth factors, cytokines and viral coat proteins for
immunization. There has been intense research into
the development of efficient vector systems and
transformation methods for animal cells. Although
this research has focused mainly on the use of mam-
malian cell lines, other systems have also become
popular, such as the baculovirus expression system,
which is used in insects. More recently, research has
focused on the introduction of DNA into animal cells
in vivo
. The most important application of this tech-
nology is
in vivo
gene therapy, i.e. the introduction of
DNA into the cells of live animals in order to treat
disease. Viral gene-delivery vectors are favoured for
therapeutic applications because of their efficiency,
but safety concerns have prompted research into
alternative DNA-mediated transfer procedures.
This chapter focuses on the introduction of DNA
into somatic cells. Unlike the situation in plants,
most animal cells are restricted in terms of their
developmental potential and cannot be used to
generate transgenic animals. Mouse embryonic stem
cells (ES cells) are exceptional in this respect because
they are derived from the early embryo prior to the
formation of the germ line. Gene transfer to ES cells is
therefore discussed in the next chapter, along with
the introduction of DNA into oocytes, eggs and cells
of the early embryo.
Gene transfer to animal cells can be achieved essen-
tially via three routes. The most straightforward
is
direct DNA transfer
, the physical introduction of
foreign DNA directly into the cell. For example, in
cultured cells this can be done by microinjection,
whereas for cells
in vivo
direct transfer is often
achieved by bombardment with tiny DNA-coated
metal particles. The second route is termed
transfec-
tion
, and this encompasses a number of techniques,
some chemical and some physical, which can be
used to persuade cells to take up DNA from their sur-
roundings. The third is to package the DNA inside an
animal virus, since viruses have evolved mechan-
isms to naturally infect cells and introduce their
own nucleic acid. The transfer of foreign DNA into a
cell by this route is termed
transduction
. Whichever
route is chosen, the result is
transformation
, i.e. a
change of the recipient cell's genotype caused by the
acquired foreign DNA, the
transgene
. Transforma-
tion can be transient or stable, depending on how
long the foreign DNA persists in the cell.
DNA-mediated transformation
Transformation techniques
DNA /calcium phosphate coprecipitate method
The ability of mammalian cells to take up exogen-
ously supplied DNA from their culture medium was
first reported by Szybalska and Szybalski (1962).
They used total uncloned genomic DNA to transfect
human cells deficient for the enzyme hypoxanthine-
guanine phosphoribosyltransferase (HPRT). Rare
HPRT-positive cells, which had presumably taken
up fragments of DNA containing the functional
gene, were identified by selection on HAT medium
(p. 177). At this time, the actual mechanism of DNA
