pFB-Neo-LacZ plasmid vector基于MMLV的逆病毒系统对照质粒载体 BioVector NTCC质粒载体菌种细胞基因保藏中心

pFB-Neo-LacZ plasmid vector基于MMLV的逆病毒系统对照质粒载体

Efficient delivery of markers, genes, or whole libraries into cultured cells is
a necessity for many areas of research. The development of tissue culture
systems for the production of high titer recombinant retrovirus that are
capable of infecting a virtually limitless range of cell types has had a
tremendous impact on these fields. With Moloney Murine Leukemia Virus
(MMLV)- based vectors, transduction efficiencies of >90% are achievable
for most mitotic cell types, and the copy number per cell can be easily
controlled by varying the multiplicity of infection. Standard transfection
methods typically result in only a small population of transfected cells
capable of the uptake and stable integration of vector DNA. Additionally,
copy number is unpredictable and often prohibitively high for many
applications, such as cDNA library expression screening.
The pVPack vector system comprises a set of 5 vectors that can be used
with any MMLV-based retroviral vector to produce viral supernatants that
have titers that are consistently >107
colony forming units (cfu)/ml in
transient triple-transfection experiments. The vectors include a gag-polexpressing vector that is cotransfected with the retroviral expression vector
together with one of a choice of 4 envelope (env)-expressing vectors. The
choice of the env-expressing vector is based on the range of cell types the
user wishes to transduce. With the pVPack vector system, all of the cis and
trans elements required to produce infectious virus are separated onto three
plasmids, with minimal or no sequence overlap between the plasmids. This
makes the pVPack system much safer than the majority of stable producer
cell lines or vector-based systems for which there is a large degree of
homology between the packaging vector(s) and the retroviral expression
vector. In these latter systems there is a relatively high probability of
production of replication-competent retrovirus (RCR) due to homologous
recombination between the vectors.
Although the relative speed and simplicity of the transient high titer virus
production protocol described in this manual will be attractive for most
applications, the gag-pol and env open reading frames (ORFs) are all
followed by an IRES linked to a downstream drug-resistance gene so that
these vectors may also be used to produce stable producer lines if desired.
The antibiotic-resistance genes used in the gag-pol and env vectors are
different from each other, and from those used in most of the more popular
retroviral vectors, so that any env vector may be used with the gag-pol
vector and with a wide range of antibiotic-resistant retroviral vectors to
produce triple-stable viral producer lines. The position of the antibioticresistant gene as the second ORF in a bicistronic expression cassette, as
opposed to its expression from a second cassette on the same plasmid,
ensures that expression of the viral packaging proteins is comaintained with
the antibiotic-resistant genes by the inclusion of antibiotics in the media.

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