Gene transfer to animal cells - Principles of Gene Manipulation - page 195

Only the cis -acting sites required for replication and
packaging are left behind. These include the LTRs
(necessary for transcription and polyadenylation of
the RNA genome as well as integration), the packag-
ing site

thus inactivating the LTR promoter while leaving
internal promoters intact. This strategy also helps to
alleviate additional problems associated with the
LTR promoter: (i) that adjacent endogenous genes
may be activated following integration; and (ii) that
the entire expression cassette may be inactivated by
DNA methylation after a variable period of expres-
sion in the target cell (Naviaux & Verma 1992).
Since retroviral vectors are used for the produc-
tion of stably transformed cell lines, it is necessary
to co-introduce a selectable marker gene along
with the transgene of interest. The expression of two
genes can be achieved by arranging the transgene
and marker gene in tandem, each under the control
of a separate promoter, one of which may be the LTR
promoter. This leads to the production of full-length
and subgenomic RNAs from the integrated provirus.
Alternatively, if the first gene is flanked by splice
sites, only a single promoter is necessary, because
the RNA is spliced in a manner reminiscent of the
typical retroviral life cycle, allowing translation of
the downstream gene (Cepko et al . 1984). Vectors in
which the downstream gene is controlled by an
internal ribosome entry site have also been used
(e.g. Dirks et al . 1993, Sugimoto et al . 1994; see
Box 13.3). Since the viral replication cycle involves
transcription and splicing, an important considera-
tion for vector design is that the foreign DNA must
not contain sequences that interfere with these
processes. For example, polyadenylation sites down-
stream of the transgene should be avoided, as these
will cause truncation of the RNA, blocking the rep-
lication cycle (Miller et al . 1983). Retroviruses also
remove any introns contained within the transgene
during replication (Shimotohno & Temin 1982).

, which is upstream of the gag gene, and
'primer-binding sites', which are used during the
complex replication process. The inclusion of a small
portion of the gag coding region improves packaging
efficiency by up to 10-fold (Bender et al . 1987).
Deleted vectors can be propagated only in the pres-
ence of a replication-competent helper virus or a
packaging cell line. The former strategy leads to the
contamination of the recombinant vector stock with
non-defective helper virus. Conversely, packaging
lines can be developed where an integrated provirus
provides the helper functions but lacks the cis -acting
sequences required for packaging (Mann et al . 1983).
Many different retroviruses have been used to
develop packaging lines and, since these determine
the type of envelope protein inserted into the virion
envelope, they govern the host range of the vector
(they are said to pseudotype the vector). Packaging
lines based on amphotropic MLVs allow retroviral
gene transfer to a wide range of species and cell
types, including human cells (e.g. see Cone &
Mulligan 1984, Danos & Mulligan 1988). It is still
possible for recombination to occur between the
vector and the integrated helper provirus, resulting
in the production of wild-type contaminants. The
most advanced 'third-generation' packaging lines
limit the extent of homologous sequence between
the helper virus and the vector and split up the
coding regions so that up to three independent
crossover events are required to form a replication-
competent virus (e.g. see Markowitz et al . 1988).
The simplest strategy for the high-level constitut-
ive expression of single genes in retroviral vectors is
to delete all coding sequences and place the for-
eign gene between the LTR promoter and the viral
polyadenylation site. Alternatively, an internal
heterologous promoter can be used to drive trans-
gene expression. However, many investigators have
reported interference between the heterologous pro-
moter and the LTR promoter (e.g. see Emmerman
& Temin 1984, Wu et al . 1996). Yu et al . (1986)
addressed this problem by devising self-inactivating
vectors , containing deletions in the 3

ψ

Sindbis virus and Semliki forest virus
(alphaviruses)

The alphaviruses are a family of enveloped viruses
with a single-strand positive-sense RNA genome.
One of the advantages of using such RNA viruses
for gene transfer is that integration into the host
genome is guaranteed never to occur. Alphavirus
replication takes place in the cytoplasm, and pro-
duces a large number of daughter genomes, allow-
ing very high-level expression of any transgene
(reviewed by Berglung et al . 1996). To date, these

′ LTR, which

are copied to the 5 ′ LTR during vector replication,