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MARTIN MARX
(\jf+, gag-, pol-, env-) Ieads to production of replication-incompetent retroviruses which contain an RNA copy of the plasmid-encoded provirus transfected into the packaging cellline.
This chapter outlines the components necessary to perform in vitro gene
transfer using animal or human cells in tissue culture as targets. A standard
and weil established protocol for the use of a Moloney Murine Leukemia
Virus (MoMuLV)-derived vector is described. Finally, potential biohazards
and related biological safety issues are discussed.
Outline
Standard transfection protocol
No single retroviral vector will be appropriate for allexperimental purposes.
However, similar methods are used when employing different vectors to
generate replication-incompetent viruses able to integrate a foreign
DNA in to the genome of target cells. In the following, a standard protocol
is described that was used frequently to insert a specific DNA into the retroviral vector pLXSN (Figure 1) which was developed by A.D. Miller (3) and to
generate ecotropic and amphotropic viral stocks suitable for the infection of
a broad range of mammalian cells. The vector contains lang terminal repeats derived from the Moloney Murine Leukemia Virus (MoMLV) and
from the Moloney Murine Sarcoma Virus (MoMSV). The EcoRI, Hpal,
Xhol or BamHi restriction sites in the cloning region of pLXSN can be
used for the insertion of foreign DNA. Further downstream the neomycin
resistance geneisdriven by the SV 40 promoter enhancer. The use of this and
related vectors in human and animal cells has previously been extensively
reported (4-12).
Materials
Components of a retroviral gene delivery system
The following components are required to set up a retroviral gene delivery
system:
• The plasmid containing specific MoMuL V sequences
• The DNA to be Iigated into the plasmid and inserted into the genome of
the target cell
MARTIN MARX
(\jf+, gag-, pol-, env-) Ieads to production of replication-incompetent retroviruses which contain an RNA copy of the plasmid-encoded provirus transfected into the packaging cellline.
This chapter outlines the components necessary to perform in vitro gene
transfer using animal or human cells in tissue culture as targets. A standard
and weil established protocol for the use of a Moloney Murine Leukemia
Virus (MoMuLV)-derived vector is described. Finally, potential biohazards
and related biological safety issues are discussed.
Outline
Standard transfection protocol
No single retroviral vector will be appropriate for allexperimental purposes.
However, similar methods are used when employing different vectors to
generate replication-incompetent viruses able to integrate a foreign
DNA in to the genome of target cells. In the following, a standard protocol
is described that was used frequently to insert a specific DNA into the retroviral vector pLXSN (Figure 1) which was developed by A.D. Miller (3) and to
generate ecotropic and amphotropic viral stocks suitable for the infection of
a broad range of mammalian cells. The vector contains lang terminal repeats derived from the Moloney Murine Leukemia Virus (MoMLV) and
from the Moloney Murine Sarcoma Virus (MoMSV). The EcoRI, Hpal,
Xhol or BamHi restriction sites in the cloning region of pLXSN can be
used for the insertion of foreign DNA. Further downstream the neomycin
resistance geneisdriven by the SV 40 promoter enhancer. The use of this and
related vectors in human and animal cells has previously been extensively
reported (4-12).
Materials
Components of a retroviral gene delivery system
The following components are required to set up a retroviral gene delivery
system:
• The plasmid containing specific MoMuL V sequences
• The DNA to be Iigated into the plasmid and inserted into the genome of
the target cell