Biomedical Engineering Reference
In-Depth Information
Table 10.2
Commonly used dominant selectable marker genes in animals (see Box 12.2 for selectable markers
used in plants).
Marker
Product (and source)
Principles of selection
References
as
Asparagine synthase
(
Escherichia coli
)
Glycopeptide-binding protein
(
Streptoalloteichus hindustantus
)
Blasticidin deaminase
(
Aspergillus terreus
)
Guanine-xanthine
phosphoribosyltransferase
(
E. coli
)
Toxic glutamine analog
albizziin
Andrulis &
Siminovitch 1981
Genilloud
et al
. 1984
ble
Confers resistance to glycopeptide antibiotics
bleomycin
,
pheomycin, Zeocin
TM
Confers resistance to basticidin S
bsd
Izumi
et al
. 1991
gpt
Analogous to
Hprt
in mammals, but possesses
additional xanthine phosphoribosyltransferase
activity, allowing survival in medium containing
aminopterin
and
mycophenolic acid
(Fig 10.1)
Confers resistance to
histidinol
Mulligan & Berg
1981b
hisD
Histidinol dehydrogenase
(
Salmonella typhimurium
)
Hygromycin phosphotransferase
(
E. coli
)
Neomycin phosphotransferase
(
E. coli
)
Puromycin N-acetyltransferase
(
Streptomyces alboniger
)
Tryptophan synthesis (
E. coli
)
Mantei
et al
. 1979
hpt
Confers resistance to
hygromycin-B
Blochlinger &
Diggelmann 1984
Colbère-Garapin
et al
.
1981
Vara
et al
. 1986
neo (nptII)
Confers resistance to
aminoglycoside
antibiotics
(e.g. neomycin, kanamycin, G418)
Confers resistance to
puromycin
pac
trpB
Confers resistance to
indole
Hartman & Mulligan
1988
(1980) demonstrated this principle by transfecting
methotrexate-sensitive mouse cells with genomic
DNA from the methotrexate-resistant cell line A29,
which contains multiple copies of an altered
Dhfr
gene. They linearized the A29 genomic DNA with
the restriction enzyme
Sal
I, and ligated it to
Sal
I-
linearized pBR322 DNA prior to transfection. Fol-
lowing stepwise drug selection of the transformed
cells, Southern-blot hybridization showed that the
amount of pBR322 DNA had increased more than
50-fold.
Methotrexate selection has been used for the
large-scale expression of many recombinant proteins,
including tissue plasminogen activator (Kaufman
et al
. 1985), hepatitis B surface antigen (HBSAg)
(Patzer
et al
. 1986) and human factor VIII (Kaufman
et al
. 1988). CHO cells are preferred as hosts for this
expression system because of the availability of a
number of
dhfr
−
mutants (Urlaub
et al
. 1983). In
non-mutant cell lines, non-transformed cells can
survive selection by amplifying the endogenous
Dhfr
genes, generating a background of 'false positives'.
Alternative strategies have been developed that
allow DHFR selection to be used in wild-type cells.
Expressing the
Dhfr
marker using a strong con-
stitutive promoter (Murray
et al
. 1983) or using a
methotrexate-resistant allele of the mouse gene
(Simonsen & Levinson 1983) allows selection at
methotrexate concentrations much higher than
wild-type cells can tolerate. The
E. coli dhfr
gene is
also naturally resistant to methotrexate, although
for this reason it cannot be used for amplifiable selec-
tion (O'Hare
et al
. 1981). Another useful strategy is
to employ a second marker gene, such as
neo
, allow-
ing non-transformed cells to be eliminated using
G418 (Kim & Wold 1985). Although
dhfr
is the most
widely used amplifiable marker, many others have
been evaluated, as shown in Table 10.3.
