Retroviral Delivery of ECM Genes to Cells - Methods in Molecular Biology: Extracellular Matrix Protocols - page 190

Fig. 2. Diagram of the recombinant retroviral vector. Retroviral vectors consist of
the 5' LTR containing viral promoter and enhancer, an extended sequence for effi-

cient RNA packaging, a drug-resistant gene, a multiple cloning site (MCS) where a
cDNA can be inserted, and the 3' LTR containing polyadenylation site. Upon transfec-
tion into packaging cells, a retroviral vector can transiently express (or integrate and
stably express) a transcript containing the

ψ

sequence, the puromycine resistance gene
and the inserted gene. In this vector the 5' LTR controls the drug resistance gene
( puro R ), whereas the CMV early promoter controls expression of inserted gene.

ψ

integrated systems are that virus could be produced in days rather than months.
The NX packaging cells contain integrated gag-pol and env coding sequences

driven by two different promoters. Separate introduction and integration of the
structural genes into the packaging cells minimize the chances of producing
replication-competent virus because of recombination events. In addition, the
CD8 surface marker cDNA sequence was placed downstream of the reading
frame of the gag-pol construct. Thus, monitoring of CD8 expression reflects
directly an intracellular gag-pol expression and the stability of the packaging
cell population's ability to produce gap-pol production. Two cell lines,

Φ

Φ NX-

eco and NX -ampho are used to infect rodent host range and all host range

target cells, respectively.
Most retroviral vectors are derived from Moloney murine leukemia virus
and consist of the 5' LTR containing viral promoter and enhancer, an extended
ψ

Φ

sequence for efficient RNA packaging, drug-resistant gene for selection and
a cDNA of a gene of interest, and the 3' LTR containing polyadenylation site
( Fig. 2 ). In addition, it contains the -lactamase gene and a plasmid origin of

replication for bacterial propagation. Two promoters of different strength are
incorporated to allow cloning and expression of the gene of interest and anti-
biotic resistant gene, neo R or puro R , for selection of transfected cells. The
cytomegalovirus (CMV) early promoter is typically a stronger promoter than
the viral LTR, but both promoters exhibit cell line-specific variation . A self-
inactivating (SIN) retroviral vector was originally developed as a safer alterna-
tive to be used in human gene therapy (6) and was recently shown to sustain a
prolonged gene expression in vivo (7) . Deletion of promoter and enhancer
sequences at the 3' LTR in SIN vector results in inactivation of transcription by

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