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Achieve inducible gene expression in
difficult-to-transfect cells by high-efficiency retroviral
transduction
New Retroviral Vectors for Inducible, Tightly Controlled
Expression
Katherine Felts • Lisa Eldridge • Peter Vaillancourt
To overcome the barrier for gene
expression in hard-to-transfect cells, we designed the Complete
Control ® retroviral
inducible expression system ±, a novel retroviral version of
our successful plasmid-vector inducible mammalian expression system. The
new transduction- based method blends tightly controlled expression with
high-efficiency transduction using two retroviral vectors: one
replication-defective retroviral vector that encodes two insect
ecdysone-receptor proteins and another vector, which contains the
ecdysone-inducible expression cassette. Once these vectors are transfected
into packaging cell lines, the resulting infectious retroviral
supernatants achieve transduction efficiencies approaching 100%. To
demonstrate the performance of this system, we tested it in two different
cell lines.
Inducible expression systems most
accurately re-create biological processes in which the timing of gene
expression is intricately linked to gene function. Such processes include
development, disease, and aging, as well as research involved in the study
of toxic genes, the screening of activators and inhibitors of various
pathways, and the evaluation of gene therapies.
The most common approach for
transmission of the genetic material of interest into cells is
transfection with an inducible plasmid, such as Stratagene’s Complete
Control inducible mammalian expression system.1 While successful with many cell lines,
this type of vector has its shortcomings, principally the fact that many
cell lines are refractory to transfection. Our new retroviral-based system
expands the versatility of our inducible mammalian expression system by
providing a method of gene delivery for difficult-to-transfect cell lines
including primary human cell lines, lymphocytes, neurons, and other
nondividing cells.
Additionally, with the retroviral
method, copy number can be easily controlled by varying the multiplicity
of infection, which is important in cases where copy number affects both
basal expression and induction ratios.
High-Efficiency Expression in Difficult-to-Transfect
Cells
Fig.1
The retroviral system works by
activating a target gene that is stimulated by the interaction of the
ecdysone insect molting hormone and two receptor proteins derived from
Drosophila melanogaster.2 The
system’s two vectors are the pFB-ERV vector (Figure 1), an MMLV-based replication-defective retroviral
vector that supplies the ecdysone receptor proteins RXR and VgEcR, and the
pCFB-EGSH retroviral vector (Figure 1), which carries an inducible cassette with a multiple cloning
site to insert the target gene.
Mammalian cells are first transduced with pFB-ERV and
pCFB-EGSH retroviral vector supernatants. Expression is then induced with
the ponasterone A (ponA) ecdysone analog. The receptor is then competent
and activates transcription of the target gene. Induction ratios can
exceed 1,000-fold. Because the ecdysone components are insect-derived and
genetically modified, the Complete Control system offers transcriptional
activation that is highly specific, with minimal cross talk between the
exogenous inducer and endogenous cellular pathways. Furthermore, the ponA
inducer penetrates cells and tissues efficiently, does not affect
mammalian physiology, and rapidly induces gene expression.
Retroviral Delivery of the Inducible Expression
Cassette
The ecdysone-inducible expression
cassette of the pCFB-EGSH vector (Figure 1) is positioned between the viral LTRs. The cassette
is in antisense orientation relative to the viral promoter within the 5'
LTR, with the CMV promoter###
replacing the U3 portion of the LTR. This replacement increases production
of viral RNA in packaging cells and thereby increases the titer of the
viral supernatants. The inducible expression cassette includes a multiple
cloning site that contains three contiguous copies of the hemagglutinin
epitope (3×HA) that are positioned for fusion at the C-terminus of the
protein of interest. Thus, expression can be conveniently monitored by
Western blot analysis. A second expression cassette contains the
hygromycin-resistance gene for selection and maintenance of stable cells.
Inducible, High-Level Expression
We assessed the levels of inducible
expression using two cell lines. In the first experiment (Figure 2), we prepared tissue culture supernatants from the control
pCFB-EGSH-Luc control vector. The supernatants were concentrated and used
to infect the ER-CHO cell line, which carries the VgEcR and RXR ecdysone
receptor proteins. Two days following infection, the cells were induced
for 20 hours with either the ecdysone-analog ponA (10 µM) or were
mock-induced for 20 hours with an equivalent volume of ethanol. When we
assayed cells for luciferase activity, we determined that the ER-CHO
cells, infected with the 100X-concentrated supernatant and induced with
ponA, achieved a 1,000-fold induction.
Fig.2
To further demostrate the low background
and sensitive induction of this system, we repeated the procedure in the
pFB-ERV transduced cell line A610-20. Two days following infection, the
cells were induced for 40 hours with either ponA or a mock vehicle, then
and assayed the cells for luciferase activity. Figure 2 shows that luciferase activity from the ponA-induced,
pCFB-EGSH-Luc supernatant-infected cells was also 1,000-fold above
background. Thus, the two retroviral vectors function extremely well in
concert with each other.
Conclusions
Accomplish high-level gene expression in a wide range of
cell lines with Stratagene’s Complete Control retroviral inducible
mammalian expression system. Use this novel system as an alternative to
plasmid-based systems when dealing with difficult-to-transfect cells. With
Stratagene’s retroviral system, the high transduction efficiencies of
retroviral vectors are combined with the tightly regulated induction
achieved by the ecdysone-inducible system. Furthermore, by varying the
multiplicity of infection, copy number can be controlled.
REFERENCES
1. Wyborski, D. and Vaillancourt, P. (1999)
Strategies 12: 1-4.
2. No, D., Yao, T.P., and Evans, R. (1996)
Proc. Natl. Acad. Sci. USA
93:
3346-3351.
3. Miller, A.D. (1997) In Retroviruses (Eds.
Coffin, J.M., Hughes, S.H., and
Varmus, H.E.)
Cold Spring Harbor Laboratory Press, Cold Spring
Harbor.
pp. 437-473.
4. Felts, K., et al.
(1999) Strategies 12: 74-77.
5. Vaillancourt, P. and Felts,
K.A. (2000) Strategies 13: 34-36.
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