Download Tips for Successful Lentiviral Transduction

Tips for Successful Lentiviral Transduction
The most critical factor for successful lentiviral transduction is viral titer. This page will help you
understand:
How to measure and evaluate viral titer >>
Upstream factors that influence titer >>
Downstream tools that can help improve transduction with lower titer sample >>
Viral Titer
It is important to have an estimation of the concentration of infection forming units (IFU) in the supernatant, as
this measurement will determine how much supernatant needs to be used to achieve a desired multiplicity of
infection (MOI), the number of virions per cell in a transduction.
Functional Titer Versus Non­functional Titer
Titration protocols can be roughly divided into functional and non­functional methods.
Non­functional methods include p24 ELISA and qRT­PCR measurement of viral genomic RNA and Northern
blotting. ELISA and qRT­PCR methods are quick and easy, and are highly consistent between viral preps. Non­
functional methods, however, tend to overestimate infectious titer because of free p24 protein, viral RNA, and
immature or empty particles present in all packaging supernatants. Therefore, non­functional titers should be
used based on previous knowledge of how p24 or genome measurement relates to infectivity.
Functional titer (infectious titer) only measures infectious mature virus; titer measurement is expressed as
infectious units per ml (IFU/ml) or transducing units per ml (TU/ml). Functional methods include assessment of
the number of colony forming units following antibiotic selection, or, if the vector contains a fluorescent protein,
flow cytometry analysis of transduced cells. If the vector does not express a fluorescent protein, determining the
number of integrated proviral DNA copies per cell by qPCR is the fastest, easiest, and best method for
assessing functional titer.
Titration kits
qRT­PCR
The Lenti­X qRT­PCR Titration Kit evaluates viral RNA genome copies/ml using a rapid 4 hour qRT­PCR
assay. The procedure involves isolation of viral RNA, and absolute quantification by real­time PCR. It is
important to note that not all genome copies are infectious, and the actual IFU/ml is orders of magnitude lower.
The exact correlation will vary for each construct, packaging system, and packaging conditions used. However
once this correlation is established, as long as these factors remain identical, IFU/ml can be calculated from
genome copies/ml for the same viral construct.
p24 ELISA
The Lenti­X p24 Rapid Titer Kit is a p24­based ELISA that can be used to determine viral particles/ml in
approximately 4 hours. Similar to the qRT­PCR assay, since each viral particle is not necessarily infectious, the
actual IFU/ml will be orders of magnitude lower than the viral particle number. Again, the exact correlation will
vary for each construct, packaging system, and packaging conditions used. However once this correlation is
established, as long as these factors remain identical, IFU/ml can be calculated from genome copies/ml for the
same viral construct.
Lenti­X GoStix also measures p24 protein in the packaging cell supernatants. This rapid test (> 10 minutes) can
be used to quickly determine if virus production is within a usable range or for selecting the best time to harvest
your virus.
Flow cytometry
For Lenti­X vectors containing a fluorescent marker, cells can be transduced and the number of fluorescent
cells can be determined 24–48 hours after transduction using flow cytometry.
Antibiotic selection
For Lenti­X vectors that contain a selectable marker, cells can be infected with serial dilutions of the virus stock,
and stable transductants can be selected using the appropriate antibiotic. Titer is calculated from the number of
drug­resistant colonies that develop after selection. In general, titers determined using this method are lower
than those determined by flow cytometry due to the extra stress placed on cells during drug selection and to
loss of transductants during colony expansion.
Titration of proviruses
Measuring provirus, the viral genome after integration into the DNA of target cells, provides the most accurate
functional titer. The transduction efficiency of different cell types can vary widely, so measuring virus titer from
packaging supernatant often will not provide an accurate estimate of how much virus is needed to transduce
one cell type versus another. For example, HT1080 cells can be transduced with lentivirus more easily than
Jurkat cells; thus, using the same amount of virus supernatant will result in far fewer integrated proviruses and
lower multiplicity of infection (M.O.I.) for Jurkat cells. To measure the number of integrated virus copies we
recommend the Lenti­X Provirus Quantitation Kit.
Evaluating Titer
Under proper conditions, a titer of >108 IFU/ml can be achieved for lentiviral constructs. The cPPT/CTS and
WPRE sequences and the wild­type HIV­1 LTRs on the pLVX vectors all contribute to higher titers. In 13
independent packaging reactions performed with 10 different fluorescent protein constructs, the average titer
obtained for pLVX vectors packaged using the Lenti­X system was 1.85 x 108 IFU/ml, as determined by flow
cytometric analysis of transduced HeLa cells.
If the IFU/ml titer is low, Lenti­X Concentrator can be used to concentrate the viral supernatant up to 100­fold.
This method is scalable, easier and faster than ultracentrifugation, and requires no specialized equipment.
If the titer is low, additional troubleshooting should be performed to increase titer and increase the likelihood of
successful transduction. To start, evaluate upstream factors that may have led to lower packaging efficiency.
Upstream Factors that Influence Viral Titer
Vector
Features of the transfer vector
The presence or absence of different vector sequence elements (e.g., LTRs, WPRE, cPPT/CTS, etc.)
can affect packaging efficiency and thereby viral titer. Clontech’s pLVX vectors are optimized for both
titer and expression, and will produce higher titers than other vectors. The Lenti­X HTX Packaging
System is likely to produce the highest titer for any HIV­1­based lentiviral vector. When using a vector
system other than Clontech’s, be sure to evaluate whether permission from any third party is required
for your intended use.
Vector integrity
Due to homologous sequences in the LTRs, all lentiviral vectors have a propensity for recombination in
certain strains of E. coli. Use strains with a low recombination frequency (e.g., Stellar Competent
Cells). Use diagnostic restriction enzyme digests to ensure vector integrity after propagation in E. coli.
Target gene
Your gene may affect the titer if it alters the normal growth of 293T cells (e.g., a toxic gene). Also, if
the gene is very large, the increased distance between the LTRs may reduce packaging efficiency.
Wild­type lentiviruses have a 9.7 kb genome from the start of the 5' LTR to the end of the 3' LTR;
creating constructs containing an insert larger than this can result in unstable viral particles and a
reduction in viral titer. Also, poly A signals should be avoided in order to eliminate the chance of
premature truncation of the viral genomic RNA during packaging.
Collection time
Collect virus 24–48 hours after the media change that follows packaging transfection. Virus titers will
typically be highest 48 hours after the start of transfection. You can useLenti­X GoStix to determine if
the supernatant has sufficient lentivirus, or if you should wait longer before harvesting.
Packaging cells
Cultures of healthy cells at an early passage provide the best titers. We recommend theLenti­X 293T
Cell Line.
shRNA­mediated cleavage
shRNAs have been reported to reduce titers by as much as 30­fold, due to shRNA­mediated self­
cleavage of the RNA genome. However, we have not observed this phenomenon with the Lenti­X
System, and we routinely generate titers of 108 IFU/ml with our pLVX­shRNA vectors.
Transfection
Quality and amount of plasmid used for transfection
We strongly recommend using NucleoBond Xtra Maxi gravity columns to purify “transfection­grade”
transfer vector DNA. Our protocol requires 7 µg of transfer vector DNA and 36 µl of the Lenti­X HTX
Packaging Mix.
Tetracycline­contaminated serum
The Lenti­X HTX Packaging System uses Tet­Off transactivation; packaging must be performed under
tetracycline­free conditions, otherwise the activity of the Tet­Off transactivator will be reduced, resulting
in lower titers.
Transfection reagent
Xfect Transfection Reagent is recommended for transfection of Lenti­X 293T cells. We routinely
achieve >99% transfection efficiency in Lenti­X 293T cells when using our optimized reagents.
Navigating Transduction Problems: Downstream Tools
When you have high viral titer (>108 IFU/ml), small volumes of viral supernatant can be used,
minimizing introduction of transduction inhibitors that may be present in the conditioned media. If low
transduction efficiency results despite high titer, one of the following downstream tools can help
improve transduction.
Removing inhibitors
The Lenti­X Maxi Purification Kit will remove transduction inhibitors, and result in up to a 10­fold
concentration of virus, depending on the starting supernatant volume.
Improving cell­virus contact
Depending on the cell type, consider using RetroNectin reagent. The RetroNectin bound virus (RBV)
protocol increases colocalization of viral particles and cells while removing transduction inhibitors from
conditioned media. RetroNectin reagent is the transduction enhancer of choice for suspension cells and
VLA­4­ and VLA­5­expressing cells including T cells, B cells, monocytes, NK cells, eosinophils, bone
marrow monocytic cells, lymphoid progenitors, thymocytes, activated T­cells, and mast cells.
Another option, Lenti­X Accelerator can be used to speed up transduction, thereby limiting the
exposure of sensitive cells to virus­containing conditioned media and Polybrene. Traditional lentiviral
transductions require overnight incubation of cells and virus with Polybrene, a transduction enhancer.
But with Lenti­X Accelerator, transduction is complete in only 30 minutes without the use of Polybrene.
Ecotropic Receptor Boosters can be used to temporarily elevate the density of the ecotropic receptor
protein mCAT­1 on the surface of any target cell that you wish to infect with ecotropic virus. This
allows, for example, efficient transduction of human cells with ecotropic retrovirus or lentivirus that
could otherwise only infect rodent cells. The technology can be used to increase infection efficiency of
any cell type that is resistant to viral infection.
Finally, to maximize cell­virus contact, we recommend a brief centrifugation step during infection (i.e.,
spinfection). Spinfection can improve tranduction efficiency by 2–10 fold. Refer to the Lenti­X
Expression System User Manual for additional details.
http://www.clontech.com/US/Products/Viral_Transduction/Selection_Guides/Lentiviral_Transduction_Tips