Chapter 22
Expression in Eukaryotic Cells: Gene Transfer Using
Retroviral Vectors
MARTIN MARX
lntroduction
Retroviral vectors are the most efficient gene delivery systems available for
in vitro and in vivo studies. Retroviral vectors are particularly useful for
achieving stable and highly efficient integration of a gene in the genome
of target cells. They have a wide host range, and infection does not lead
to cell death. Transfeeted or infected packaging celllines may produce replication-incompetent infectious virus over an indefinite time period. The
DNA introduced into the target cells is driven by a strong promoter, leading
to efficient expression of the foreign gene. In addition, inducible promoters
are available. Fora complete description ofthe retrovirallife cycle and construction ofvectors, see Weisset al. 1985 (1). Briefly, replication-incompetent retroviral vectors are constructed by deletion of the genes encoding the
structural proteins gag, env and pol. These deletions make the retrovirus
replication-incompetent and provide room for the insertion of foreign
genesthat can be expressed in the target cell or used as a selectable marker.
In the vector described in this chapter, expression of neomycin phosphotransferase encoded bythe bacterial transposon TnS (2) provides resistance
to the aminoglycoside antibiotics kanamycin, neomycin and geneticin. Despite the extensive deletions, the vectors retain the cis-acting viral sequences
that are necessary for transmission and the \Jf packaging sequence essential
for encapsidation into the viral particle. The deleted gag, pol and env genes
are provided by a helper or packaging cellline that contains mutant provirus with non-functional packaging signals due to a deletion of the \Jf
packaging sequence. When the plasmid form of the retroviral vector is
transfected into the packaging cellline, genetic complementation between
the primary provirus (\Jf-, gag+, env+, pol+) and the transfected provirus
Martin Marx, Universität Köln, Medizinische Klinik I, Ostmerheimerstrasse 200, Köln,
51109, Germany (phone +49-221-8907-2200; fax +49-221-8907-2335; e-mail martin.
marx@uni-koeln.de)
Expression in Eukaryotic Cells: Gene Transfer Using
Retroviral Vectors
MARTIN MARX
lntroduction
Retroviral vectors are the most efficient gene delivery systems available for
in vitro and in vivo studies. Retroviral vectors are particularly useful for
achieving stable and highly efficient integration of a gene in the genome
of target cells. They have a wide host range, and infection does not lead
to cell death. Transfeeted or infected packaging celllines may produce replication-incompetent infectious virus over an indefinite time period. The
DNA introduced into the target cells is driven by a strong promoter, leading
to efficient expression of the foreign gene. In addition, inducible promoters
are available. Fora complete description ofthe retrovirallife cycle and construction ofvectors, see Weisset al. 1985 (1). Briefly, replication-incompetent retroviral vectors are constructed by deletion of the genes encoding the
structural proteins gag, env and pol. These deletions make the retrovirus
replication-incompetent and provide room for the insertion of foreign
genesthat can be expressed in the target cell or used as a selectable marker.
In the vector described in this chapter, expression of neomycin phosphotransferase encoded bythe bacterial transposon TnS (2) provides resistance
to the aminoglycoside antibiotics kanamycin, neomycin and geneticin. Despite the extensive deletions, the vectors retain the cis-acting viral sequences
that are necessary for transmission and the \Jf packaging sequence essential
for encapsidation into the viral particle. The deleted gag, pol and env genes
are provided by a helper or packaging cellline that contains mutant provirus with non-functional packaging signals due to a deletion of the \Jf
packaging sequence. When the plasmid form of the retroviral vector is
transfected into the packaging cellline, genetic complementation between
the primary provirus (\Jf-, gag+, env+, pol+) and the transfected provirus
Martin Marx, Universität Köln, Medizinische Klinik I, Ostmerheimerstrasse 200, Köln,
51109, Germany (phone +49-221-8907-2200; fax +49-221-8907-2335; e-mail martin.
marx@uni-koeln.de)