Biomedical Engineering Reference
In-Depth Information
does not need to integrate in order to be stably
maintained, thus stable transformation occurs with
an efficiency equal to that normally achieved with
transient transformation. Furthermore, while the
expression of integrated transgenes is often affected
by the surrounding DNA, a problem known as the
position effect (see Box 11.1), these episomal vectors
are not subject to such influences.
The human BK polyomavirus infects many cell
types and is maintained with a copy number of about
500 genomes per cell. Plasmid vectors containing
the BK origin replicate in the same manner as the
virus when the BK T antigen is provided
in trans
.
However, by incorporating a selectable marker such
as
neo
into the vector, it is possible to progressively
increase the concentration of antibiotic in the medium
and select for cells with higher vector copy number.
Cell lines have been propagated for over a year under
such conditions (De Benedetti & Rhoads 1991) and
stable lines with up to 9000 copies of the genome
have been produced. Any transgene incorporated
into the vector is expressed at very high levels, lead-
ing to the recovery of large amounts of recombinant
protein (Sabbioni
et al
. 1995). SV40 itself can also
be used as a stable episomal vector if the availability
of T antigen is rationed to prevent runaway replica-
tion. This has been achieved using a conditional
promoter and by mutating the coding region to
render the protein temperature-sensitive (Gerard &
Gluzman 1985, Rio
et al
. 1985).
The first virus to be developed as an episomal
replicon was bovine papillomavirus (BPV). The
papillomaviruses are distantly related to the poly-
omaviruses and cause papillomas (warts) in a range
of mammals. BPV has been exploited as a stable
expression vector because it can infect mouse cells
without yielding progeny virions. Instead, the viral
genome is maintained as an episomal replicon, with
a copy number of about 100. The molecular biology
of BPV is considerably more complex that that of
SV40, but the early part of the infection cycle is sim-
ilar, involving the production of a T antigen that:
(i) is required for viral replication; and (ii) causes
oncogenic transformation of the host cell. The early
functions of BPV are carried on a 5.5 kb section of
the genome, which is called the 69% transforming
fragment (BPV
69T
). This was cloned in the
E. coli
plasmid pBR322 and was shown to be sufficient to
establish and maintain episomal replication, as well
as inducing cell proliferation (Sarver
et al
. 1981a).
Initially, the ability of the virus to cause uncon-
trolled cell proliferation was used to identify trans-
formed cells, but this limited the range of cell types
that could be used. The incorporation of selectable
markers, such as
neo
, allows transformants to be
selected for resistance to G418, permitting the use of
a wider range of cell types (Law
et al
. 1983). BPV
69T
replicons have been used to express numerous pro-
teins, including rat preproinsulin (Sarver
et al
. 1981b)
and human
-globin (Di Maio
et al
. 1982). Gener-
ally, the plasmids are maintained episomally, but
the copy number varies from 10 to over 200 vector
molecules per cell. Some investigators have reported
the long-term maintenance of such episomal trans-
formants (Fukunaga
et al
. 1984), while others found
a tendency for the construct to integrate into the
genome (Ostrowski
et al
. 1983, Sambrook
et al
.
1985). Recombination within the vector or between
vectors is also a fairly common observation, resulting
in unpredictable spontaneous deletions and plasmid
oligomerization. Recombination events and copy
number appear to be affected by multiple factors,
including the host cell type, the incorporated trans-
gene and the structure of the vector itself (see Mecsas
& Sugden 1987).
β
Stable transformation with EBV replicons
Unlike BPV replicons, vectors based on EBV replicate
very stably in mammalian cells. EBV is a herpesvirus
(see also discussion of herpes simplex virus later in
the chapter), with a large double-stranded DNA
genome (approximately 170 kb), which predomin-
antly infects primate and canine cells. It is also
naturally lymphotrophic, infecting B cells in humans
and causing infectious mononucleosis. In cultured
lymphocytes, the virus becomes established as an
episomal replicon with about 1000 copies per cell
(Miller 1985). Although the virus itself only infects
lymphocytes, the genome is maintained in a wide
range of primate cells if introduced by transfection.
Only two relatively small regions of the genome are
required for episomal maintenance - the latent
origin (
oriP
) and a gene encoding a
trans
-acting
regulator called Epstein-Barr nuclear antigen 1
(EBNA1) (Yates
et al
. 1984, Reisman
et al
. 1985).
