Gene transfer to animal cells - Principles of Gene Manipulation - page 183

Box 10.1 continued

gene is expressed in clearly distinct cell types: insulin
is synthesized in endocrine β cells and chymotrypsin
in exocrine cells. Plasmid DNA was introduced into
either pancreatic endocrine or pancreatic exocrine
cell lines in culture and, after a subsequent 44 h
incubation, cell extracts were assayed for CAT
activity. It was found that the constructs retained
their preferential expression in the appropriate cell
type. The insulin 5 ′ -flanking DNA conferred a high
level of CAT expression in the endocrine but not the
exocrine cell line, with the converse being the case for
the chymotrypsin 5 ′ -flanking DNA. The analysis was
extended by creating deletions in the 5 ′ -flanking
sequences and testing their effects on expression.
From such experiments it could be concluded that
sequences located 150-300 bp upstream of the
transcription start are essential for appropriate
cell-specific transcription.

An example of in vitro promoter analysis using
chloramphenicol acetyltransferase
The first reporter gene to be used in animal cells was
cat , derived from E. coli transposon Tn 9 (Gorman
et al . 1982b); it has also been used to a certain
extent in plants (Herrera-Estrella et al . 1983a,b).
This gene encodes the enzyme chloramphenicol
acetyltransferase (CAT), which confers resistance to
the antibiotic chloramphenicol by transferring acetyl
groups on to the chloramphenicol molecule from
acetyl-CoA. If 14 C-labelled chloramphenicol is used
as the substrate, CAT activity produces a mixture
of labelled monoacetylated and diacetylated
forms, which can be separated by thin-layer
chromatography and detected by autoradiography.
The higher the CAT activity, the more acetylated
forms of chloramphenicol are produced. These
can be quantified in a scintillation counter or using a
phosphorimager. Gorman et al . (1982a,b) placed the
cat gene downstream of the SV40 and Rous sarcoma
virus (RSV) promoters in the expression vectors pSV2
and pRSV2, to create the pSV-CAT and pRSV-CAT
constructs, respectively. These vectors, and derivatives
thereof, have been widely used to analyse transient-
transfection efficiency, because the promoters are
active in many animal cells and CAT activity can
be assayed rapidly in cell homogenates.
The cat gene has also been used to test regulatory
elements by attaching it to a 'minimal promoter',
typically a simple TATA box. This basic construct
generates only low-level background transcription
in transiently transfected cells. The activity of
other regulatory elements, such as promoters and
enhancers, and response elements , which activate
transcription in response to external signals, can be
tested by subcloning them upstream of the minimal
promoter and testing their activity in appropriate cell
types. In an early example of this type of experiment,
Walker et al . (1986) attached the promoter and
5 ′ -flanking sequences of human and rat insulin and
rat chymotrypsin genes, which are expressed at a
high level only in the pancreas, to the cat gene. Each

Other reporter genes
The cat reporter gene has been widely used for in
vitro assays but has a generally low sensitivity and is
dependent on a rather cumbersome isotopic assay
format. An alternative reporter gene, SeAP (secreted
alkaline phosphatase), has been useful in many cases
because various sensitive colourimetric, fluorometric
and chemiluminescent assay formats are available.
Also, since the reporter protein is secreted, it can be
assayed in the growth medium, so there is no need
to kill the cells (Berger et al . 1988, Cullen & Malim
1992). The bacterial genes lacZ and gusA have
been used as reporters for in vitro assays using
colourimetric and fluorometric substrates. An
important advantage of these markers is that they
can also be used for in situ assays, since histological
assay formats are available. More recently,
bioluminescent markers, such as luciferase and green
fluorescent protein, have become popular because
these can be assayed in live cells and whole animals
and plants. The lacZ , gusA , luciferase and green
fluorescent protein reporter genes are discussed in
more detail in Box 13.1.