Biomedical Engineering Reference
In-Depth Information
Table 10.3
Common markers used for
in situ
gene amplification. Many amplifiable markers can also be used as
endogenous or dominant selectable markers, but, in some cases, the drug used for amplification may not be the same as
that used for standard selection.
Marker
Product
Amplifying selective drug
References
Ada
as
Cad
Dhfr
gpt
GS
Hprt
Adenosine deaminase
Asparagine synthase
Aspartate transcarbamylase
Dihydrofolate reductase
Xanthine-guanine phosphoribosyltransferase
Glutamine synthase
Hypoxanthine-guanine
phosphoribosyltransferase
Inosine monophosphate dehydrogenase
Metallothionein 1
Multidrug resistance: P-glycoprotein 170 gene
Ornithine decarboxylase
Thymidine kinase
Uridine monophosphate synthases
Deoxycoformycin
b
-Aspartylhydroxamate
N
-Phosphonacetyl-l-aspartate
Methotrexate
Mycophenolic acid
Methionine sulphoxamine
Aminopterin
Kaufman
et al
. 1986
Cartier
et al
. 1987
Wahl
et al
. 1984
Kaufman
et al
. 1985
Chapman
et al
. 1983
Cockett
et al
. 1990
Kanalas & Suttle 1984
Impdh
Mt-1
M
res
Odc
Tk
Umps
Mycophenolic acid
Cd
2
+
Adriamycin, colchicine, others
Difluoromethylornithine
Aminopterin
Pyrazofurin
Collart & Huberman 1987
Beach & Palmiter 1981
Kane
et al
. 1988
Chiang & McConlogue 1988
Roberts & Axel 1982
Kanalas & Suttle 1984
subcloning,
in vitro
manipulation and purification of
recombinant proteins (p. 76).
2 More importantly, modular elements can be
included to drive transgene expression, and these
can be used with any transgene of interest. The pSV
and pRSV plasmids are examples of early expression
vectors for use in animal cells. As discussed in
Box 10.2, transcriptional control sequences from
SV40 and Rous sarcoma virus are functional in a
wide range of cell types. The incorporation of
these sequences into pBR322 generated convenient
expression vectors in which any transgene could be
controlled by these promoters when integrated into
the genome of a transfected cell (Fig. 10.2).
3 The inclusion of a selectable marker gene
obviates the need for cotransformation, since the
transgene and marker remain linked when they
cointegrate into the recipient cell's genome. A range
of pSV and pRSV vectors were developed containing
alternative selectable marker genes, e.g. pSV2-neo
(Southern & Berg 1982), pSV2-gpt (Mulligan & Berg
1980) and pSV2-dhfr (Subramani
et al
. 1981).
Plasmid vectors for DNA-mediated gene transfer
Stable transformation by integration can be achieved
using any source of DNA. The early gene-transfer
experiments discussed above were carried out using
complex DNA mixtures, e.g. genomic DNA, bacterial
plasmids and phage. Calcium phosphate trans-
fection was used in most of these experiments, and
the specific donor DNA was often bulked up with a
non-specific carrier, such as cleaved salmon-sperm
DNA. However, it is generally more beneficial to use
a purified source of the donor transgene. This prin-
ciple was originally demonstrated by Wigler
et al
.
(1977), who transfected cultured mouse cells with
a homogeneous preparation of the HSV
Tk
gene.
Later, this gene was cloned in
E. coli
plasmids to pro-
vide a more convenient source. The use of plasmid
vectors for transfection provides numerous other
advantages, depending on the modular elements
included on the plasmid backbone.
1 The convenience of bacterial plasmid vectors can
be extended to animal cells, in terms of the ease of
