V i r a l
T r a n s d u c T i o n
Retro-X™ Retroviral System FAQs
How to use our premium retroviral systems
What is the cloning/packaging capacity of a retroviral vector from Clontech®?
Wild type moloney murine leukemia virus (MMLV) contains ~8.2 kb of genome,
including both LTRs. Artificially creating a genome larger than 8.2 kb will result
in unstable viral particles and a dramatic drop in viral titer. For recombinant
retroviruses such as those generated using Retro-X systems, much of the viral
genome has been replaced with other useful sequences such as selection markers
or fluorescent proteins, but enough space remains for cloning transgenes.
Since each pRetro-X and pRetro-Q vector contains different useful sequences,
the available space for cloning your transgene varies. To determine the maximum
recommended transgene size, consult your retroviral vector map and determine
the position of the end of the 3' LTR (the end of the 5' LTR is always at position 1),
then subtract that from 8.2 kb. For example:
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•
The 3' LTR of pQCXIP ends at 4.3 kb, so ~3.9 kb of space remains
for you to clone in your gene.
•
The 3' LTR of pRetroX-IRES-ZsGreen1 ends at 3.3 kb, so ~4.9 kb
of space remains for you to clone in your gene.
What fraction of the wild-type MMLV genome is present in Clontech’s
retroviral vectors?
Approximately 12–17%, depending on the vector backbone. pLXRN contains
11.9% of the wild-type MMLV genome, and the MSCV vectors contain 16.9%
of the wild-type MMLV genome.
Do I have to include an internal poly(A) signal in the expression cassette
for my gene of interest (GOI)?
No. The GOI or selection marker expression cassette in the retroviral vector
should not include a poly(A) signal or any other transcription termination
signal, since it is located in the 3' LTR of the retroviral vector. A transcription
termination sequence in the middle of a retroviral construct can lead to the
premature cleavage of viral genomic mRNA, resulting in the loss of downstream sequences that are essential for efficient packaging and transduction
of the target cells.
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V i r a l
T r a n s d u c T i o n
How do bicistronic (IRES) vectors express
two proteins simultaneously?
Bicistronic lentiviral vectors contain an optimized
internal ribosome entry site (IRES) that permits your
gene of interest and a second gene* to be coexpressed
from a single mRNA transcript. Although translation
initiation occurs almost exclusively at the 5' cap
of eukaryotic mRNAs, the IRES attracts ribosomes
to begin translation at a second, internal location.
The result is that two proteins are expressed simultaneously from a single bicistronic mRNA transcript.
*Often the gene for a fluorescent protein or drug
selection marker.
Are there size limitations for cloning into IRES vectors?
Typically, expression of the gene downstream of the IRES
is optimal only if the gene cloned upstream of the
IRES is 0.5–1.5 kb in size. Therefore:
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You can expect reduced expression of the downstream gene* when a gene larger than 2 kb is
cloned upstream of the IRES.
It is better to clone a stuffer into the vector than
to use the empty vector as a control.
How do you guarantee the biosafety of Clontech’s
retroviral packaging cell lines?
Clontech’s retroviral packaging cell lines are routinely
tested to ensure the absence of replication-competent
virus, using control NIH 3T3 pLAPSN stable cells.
Please see each packaging cell line’s Certificate of
Analysis (located under the Documents tab on the
Retroviral Packaging Systems product page) for more
information.
Are Clontech’s retroviral vectors compatible with retroviral
packaging systems from other sources?
Our MMLV-based retroviral vectors are compatible
with MMLV-based retroviral packaging systems.
However, we cannot guarantee the performance or
biosafety of retroviral packaging systems from sources
other than Clontech.
Are Clontech’s retroviral packaging cell lines and
retroviral systems compatible with retroviral vectors
from other sources?
Yes, Clontech’s retroviral packaging cell lines and
retroviral systems are compatible with MMLV-based
retroviral vectors from other sources.
Which retroviral packaging cell line or system should
I use to transduce my target cells?
Viral envelopes are classified by the receptors they
use to enter host cells. Your virus’ range of infectivity
(cell tropism) is determined by its envelope, which is
in turn conferred by the packaging cell line or system.
You should choose your packaging cell line or system
based on your target cells (Table I).
Can primary cells be infected with a retrovirus?
Yes, as long as the cells are mitotically active. For
quiescent or slowly dividing cells, we recommend
lentiviral-mediated gene delivery.
Can I use a retroviral vector to perform regular
plasmid transfections?
Yes. You can use a retroviral vector as a regular plasmid,
via transfection. However, this negates one of the advantages of retroviral transduction: reproducible integration
of individual copies of your transgene. During stable
integration, plasmid vectors break randomly and integrate into the cellular genome nonspecifically with
respect to the original vector backbone. By contrast,
proviral DNA from a retrovirus is integrated into the
transduced cells’ genome precisely via the 5' and 3' LTRs,
ensuring the integrity and linearity of the integrated
expression construct.
Table I: Retroviral Packaging Cell Lines
Integrated Markers
*
Product
Cell Type
Tropism
EcoPack 2-293 Cell Line
HEK 293
Ecotropic
AmphoPack 293 Cell Line
HEK 293
Amphotropic
RetroPack PT67 Cell Line
NIH 3T3
Dualtropic
GP2-293 Cell Line*
HEK 293
**
Envelope
Receptors
gag-pol
env
Host Cell
gp70
mCAT1
4070A
Ram-1 (rPit-2)
Bleo
Hyg
Rat and mouse
Bleo
Puro
10A1
Many mammalian cell types
GALV, RAM
TK
DHFR
Many mammalian cell types
**
**
DHFR
n/a
All cell types
Available as part of the Retro-X Universal Packaging System and the Pantropic Retroviral Expression System.
** The GP2-293 Cell Line requires cotransfection of one of several types of envelope proteins and can produce virus with various tropisms.
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V i r a l
T r a n s d u c t i o n
What are the self-inactivating (SIN) retroviral vectors
(Retro-X Q Vectors)?
SIN vectors have a 5'-LTR that is active in transfected
cells but inactive in transduced cells. During packaging,
the 5'-LTR is active and generates multiple copies of viral
genomic mRNA molecules, which are packaged into retroviral particles. When cells are infected with the packaged
virus, reverse transcription transfers a U3 deletion
from the 3'-LTR to the 5'-LTR, thus inactivating it
in the integrated proviral DNA.
The GOI and drug resistance expression cassettes
of the Retro-X Q Vectors in transduced cells are driven
from internal promoters only.
If a Retro-X Q Vector is introduced into the cells
via transfection, its 5'-LTR will be active.
Can I use the Retro-X Concentrator to concentrate
a lentivirus?
Yes. The Retro-X Concentrator can be used to concentrate
a lentivirus according to the Lenti-X™ Concentrator
Protocol-At-A-Glance. To find the protocol, go to
www.clontech.com/manuals and type “Lenti-X
Concentrator” in the search box.
How does the viral envelope affect the Retro-X
Concentrator’s efficiency?
The Retro-X Concentrator can concentrate retroviral
supernatants created with all commonly used envelopes.
Fold concentration varies for retroviruses pseudotyped with various envelopes (Figure 1).
Can I use the Retro-X qRT-PCR Titration Kit to titer
MSCV-based retroviruses?
No. The Retro-X qRT-PCR Titration Kit is not compatible
with MSCV-based retroviruses. It does work with all
MMLV-based retroviral vectors.
What is MOI?
MOI (multiplicity of infection) is the number of infectious
viral units per cell at the time of infection.
What are IFU?
IFU (infectious units) indicate the ability of the virus
to infect cells and express protein. You can determine
the infectious viral titer (IFU/ml) by measuring the
expression of a reporter or a specific protein by flow
cytometry, immunostaining, etc.
What are CFU?
CFU (colony forming units) determine the virus’ ability
to transduce cells and express protein. Cells harboring
integrated proviral DNA and carrying an antibiotic
resistance gene survive the presence of the drug
and form cell colonies. Lentiviral titer in CFU/ml is
determined by counting drug-resistant cell colonies.
A
B
10
9
IFU/ml
107
106
105
104
96
100
Fold Increase (IFU/ml)
108
116
120
Before
concentrating
After
concentrating
80
60
46
47
10A1
Ampho
40
20
0
10A1
Ampho
Eco
VSV-G
Eco
VSV-G
Figure 1. Retro-X Concentrator can concentrate retroviral supernatants created with all commonly used envelopes. A retrovirus
expressing DsRed2 was packaged with different retroviral envelopes using the Retro-X Universal Packaging System (Cat. No. 631530),
and harvested at 72 hr posttransfection. Retro-X Concentrator was used to concentrate dualtropic (10A1), amphotropic (Ampho),
ecotropic (Eco), and VSV-G (pantropic) pseudotyped retrovirus samples from 10 ml down to 100 µl (Panels A & B). Crude and concentrated
viral stocks were then titrated on HT1080 or NIH 3T3 cells and analyzed via flow cytometry at 72 hr postinfection to determine the percentage
of positive cells.
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