Transfection Transduction FAQs
1.My DharmaFECT Duo transfection reagent arrived and the ice was melted. Is the reagent
still ok to use?
All DharmaFECT formulations, including DharmaFECT Duo reagent, have undergone substantial
stability and functional testing, showing excellent performance even following storage at
less-than-ideal conditions. The results are detailed in a product bulletin available for download.
2. How do I determine MOI?
MOI stands for Multiplicity of Infection which refers to the number of viral particles per cell. To
calculate, take the number of viral particles used per well then divide by the number of cells
originally seeded in the well. This equals the MOI. An MOI of 5 indicates that there are five
transducing units for every cell in the well. It is important to note that different cell types may
require different MOIs for successful transduction and knockdown of the target gene. For instance,
HEK293T cells are highly susceptible to lentiviral transduction (MOI of 5-20) while neuronal
cells such as SHSY5Y often require higher MOIs of 10-50.
3. Can/should I linearize your viral vectors for stable integration?
Linearizing the DNA reduces the uptake into the cell, as circular DNA has a 4X greater chance of
cellular uptake. However, once in the cell, linearized DNA does show better integration than
circular DNA. pLKO.1 - NcoI - based on our current map, this should cut just downstream of the
polyA signal. pGIPZ - FspI at base 10,755 (cuts inside ampicillin marker) NOTE: Was changed
from the previously recommended PmeI since it cuts in the poly A signal which might not be good.
pTRIPZ - PmeI at position 8404 should work. This lies just downstream of the 3' LTR so it will
cut the entire viral transcript (LTR to LTR). pSMP** - NdeI at position 6149, which is outside the
LTRs pSM2** - ApaI at base 5055. It lies between the 3LTR and the RK6 promoter. ** Please
note the pSM2 and pSMP libraries, constructs, gene sets and families and RNAintro kits have
been discontinued. Note: We do not linearize our vectors in house, but offer the above suggestions
as to which sites to use. All these sites should linearize the respective plasmid in an area that is
inconsequential to those components that function post integration into the mammalian genome.
The maps used to determine these sites are of EMPTY vectors. Since each shRNA clone contains
a unique target sequence as well as a unique barcode there is a small chance that any one clone
could contain a second restriction site for the enzyme given. Therefore, when the digest is done a
small aliquot should be run on a gel to ensure a single cut occurred, linearizing the vector.
4. Can I use the supernant of the transfected packaging cells to re-infect the packaging cells
and get higher titer virus?
No. The reason why re-infecting the transfected cells does not lead to higher titer is because our
lentiviral vectors are SIN vectors. Following infection and reverse transcription, the deletion in the
U3 region gets copied into the 5'LTR resulting in little if any transcription from the LTR.
Transcription from the 5'LTR is what generates the viral RNA that gets packaged into the
lentiviral particles.
5. What is the purpose of the Central Polypurine tract (cPPT) on your viral vectors?
The Central Polypurine tract helps with transduction into non-dividing cells. Although no one
really understands the mechanism by which the Central Polypurine tract helps translocation into
the nucleus of nondividing cells at this time, it appears to allow the virus to penetrate the nuclear
membrane. Some links to literature: http://www.bloodjournal.org/cgi/content/full/100/3/813
http://www.liebertonline.com/doi/abs/10.1089/104303401750214311?journalCode=hum
6. Can HEK293FT cells be used as producer cells for lentiviral packaging?
Yes, they are similar to HEK293T cells and can be used.
7. What cell line do I need to use for lentiviral packaging?
You can use any 293T cell line to package lentivirus. You do not have to use the HEK293T cell
line included with the Trans-Lentiviral Packaging System to package lentivirus. The key element
that your cell line needs to contain is the Large T antigen. 293T cells have been altered to express
the Large T antigen from SV40 virus. The main function of the Large T antigien is to help the
plasmids with an SV40 origin of replication to replicate and be retained in the cell after a
tranfection. This will in turn allow the cell to make more of the virus during packaging. We are
unable to help trouble shoot if you choose to package with any other cell line but the HEK293T,
but that does not mean it will not work.
8. Instead of purchasing the empty pGIPZ vector for subcloning, can I use one of the
controls or just a random hairpin in pGIPZ?
Please note that we do NOT recommend this! This is because the restriction digests may not work
completely and with an insert so small (~345bp), you may not be able to tell the difference
between which vector band still has a hairpin (if the digest didn't work to completion) and which
band is actually an empty vector. MANY colonies would have to be sequenced to confirm the
presence of the new hairpin. Even if the empty pGIPZ vector is used to subclone into, we
recommend sequencing one or two colonies. Also, there is a unique BamHI site in the stuffer of
the empty vector that can be used as a diagnostic cut to determine if the clone still has the original
stuffer, or if a hairpin has been cloned in. If the hairpin has been cloned in, no cut will be made.
9. What are the advantages/disadvantages of using fibronectin vs. polybrene?
A while back fibronectin was used to increase transduction/infection of hematopoietic stem cells
and other hard to transduce primary cells. They would first coat the plate with fibronectin, add
virus to the plate which will bind to the fibronectin, then plate the cells on top. Polybrene works
by countering the electrostatic charges between the virus and cell membrane, which both have a
negative charge on its surface. Polybrene is a cation. Polybrene is toxic to some cell lines and
primary cells, whereas fibronectin probably is not. For primary cells, there is no standard
transduction protocol because primary cells differ greatly in the way the culture conditions can be
manipulated. Fibronectin has been used to increase transduction/infection of hematopoietic stem
cells and other hard to transduce primary cells. First coat the plate with fibronectin, add virus to
the plate which will bind to the fibronectin, then plate the cells on top. Fibronectin is non toxic for
most cells. Another option to consider is adding polybrene or DEAE-dextran to the virus stock
prior to infection. This is used to increase transduction efficiency by counterbalancing the
charge-charge interactions between the virus particle and the surface of the cell. However, cationic
reagents such as these are very toxic to primary cells in culture and should not be used. A
reduction in serum concentration in the culture can also facilitate transduction, but some primary
cells do not handle a reduction in serum over the time frame of infection. To avoid these problems,
generally one can reduce the volume of the culture media to where it just covers the cells and add
straight virus directly onto the culture media. Let the infection proceed for 6-8 hours with gentle
rocking every hour or so, and then add the normal culture media to the cells. This way there is
little perturbation to the cells. The amount of virus added needs to be determined empirically for
each cell type. We recommend setting up three infections with multiplicity of infections equal to 5,
10, and 50. Not all cell types transduce equally well.
10. Why can you not package lentivirus using a retroviral packaging system and vice versa?
There are many reasons why not, most of them at the molecular level. For instance the packaging
signal (psi, located on the lentiviral vector RNA) which facilitates incorporation into the viral
particle through an interaction with the gag polyprotein, is not recognized by the gag polyprotein
of the retroviral packaging system. However, it's more complicated than that, because just simply
switching the packaging signals doesn't work either. Many researchers have tried unsuccessfully
to get this to work.
11. Can I infect a mammalian cell multiple times with different viruses?
It is possible to infect a cell with multiple viruses. You will need different selectable markers on
whatever it is you are trying to introduce so that you can be sure that both constructs have gone
into the cell.
12. Is a packaging cell line immortal? How much should I order for infection?
Yes, all our 293T cell lines are immortal. You should not need to reorder these cells as they will
grow continually while selection is being maintained with hygromycin (100ug/ml).
13. Can you see a viral pellet?
The virus pellet cannot actually be seen. The pellet that one does see in the bottom of the tube is
derived from serum proteins present in the media of the transfected culture. To separate the virus
from the protein pellet, simply leave the DMEM (200 碌 l) on the pellet for 5-10 minutes at room
temperature, then gently pipette this suspension up and down about 30 times being careful not to
generate air bubbles. Then transfer the virus-protein suspension into a sterile microfuge tube or
cryovial and microfuge for 4 minutes at high speed. This centrifugation step will pellet the protein
in the bottom of the tube (it sticks rather tight to the wall of the tube). Then transfer the
supernatant, which contains the virus particles, into another vial. From there, aliquot the virus in
smaller volumes. It doesn't really matter how much you resuspend the virus in after centrifugation.
It depends on how concentrated you need the virus. We do not recommend resuspending in less
than 100。
14. Is the Tat protein (which is required for packaging of pGIPZ because of the wt 5' LTR)
secreted by the packaging cells into the media?
Yes, Tat is secreted by the packaging cells and it can affect the expression of eukaryotic genes. If
you think this may be a problem when transducing your target cells, you can concentrate the virus,
which will separate the viral particles from any cellular proteins (including Tat).
15. Is VSV-G (which is used to pseudotype viral particles) toxic to cells?
Yes VSV-G is toxic to cells and can induce cell fusion. When we use HEK293T cells to generate
virus, cells eventually die and VSV-G toxicity is one of the contributors. However, we harvest the
virus and discard the cells before this happens. There is also some toxic effect on the target cells
as well, though it is generally transient and the cells will recover.
16. Can shRNAmir/TRC constructs be packaged using a 293G cell line?
We are familiar with the 293G cell line but have not worked with it directly. It is a cell line that
expresses the VSV-G envelope which is used to pseudotype the lentiviral particle. We don't
believe that the 293G cell expresses any of the packaging components other than the VSV-G
envelope. The VSV-G envelope expressed on the surface of the viral particle is what binds the
virus to the surface of the target cell. When you co-transfect the 293G cells with the other
lentiviral packaging components (Gag-Pol, Rev, and the gene transfer vector), it will then produce
the lentiviral vector particles that have the VSV-G envelope. We use a similar cell line, 293T, to
generate our virus stocks. The difference is that our cell line does not express the VSV-G envelope.
Instead, we transfect an additional plasmid that encodes the VSV-G envelope cDNA along with
the other packaging components. The short answer is that both the shRNAmir and TRC constructs
can be packaged in the 293G cell line.
17. How long after transfection/transduction do you wait before adding puromycin?
We recommend that you wait 24-48 hours post tranfection/transduction before selecting with
puromycin.
18. What are the differences in HEK293 vs 293T cell lines for packaging?
HEK293 cells are the parental line for 293T. The "T" means that it expresses the Large T antigen
which is important for replicating plasmids containing an SV40 origin of replication to high copy
number in the transfected cell. The SV40 origin of replication is present on our lentiviral
packaging and vector constructs and therefore should be transfected into 293T cells for virus
production. Parental 293 cells do not contain the T antigen and would not produce sufficient titers.
19. Can you target more than one gene in the same cell using the shRNA system if interested
in co-silencing two target genes in the same cell?
The answer is yes, by two methods: 1. You could transduce with one type, select with puro and
then transduce with the second type. Remember that after the second transduction puro selection
will no longer be effective as the cells already have the marker from the first transduction. 2. You
can simply mix the two virus types together and transfect at a high MOI and statistically get some
cells with both types integrated. As with method 1, cells with either one or two types of virus will
grow with puromycin selection so you will have to rely on statistics to ensure you have a large
population with both virus types.
20. Is the ligation ratio correct in the pGIPZ empty vector product insert where it says
250ng of pGIPZ to 7.4ng of insert?
Yes, it is correct. You must have more of the pGIPZ vector because ng is a unit of weight. Since
the pGIPZ vector is ~11 kb and the insert is ~0.35 kb, you will have about 31 times less vector
than insert per ng. You must increase the amount of vector used to equal the number of pieces of
insert used.
21. Do you know the maximum gene size that can be inserted into a lentiviral vector?
10-11Kb is about the maximum size that cen be inserted between the LTRs before you start seeing
a reduction in packaging efficiency and therefore reduction in viral titers. The reference below
shows a logarithmic inverse relationship between packaging size and titer. Depending on where
the sequence is inserted, you could see a drop of 1 log in titer for every 2Kb of insert. Systematic
Determination of the Packaging Limit of Lentiviral Vectors Mukesh Kumar, Brian Keller, Ndeye
Makalou, Richard E. Sutton Human Gene Therapy. 2001, 12(15): 1893-1905.
22. Do you recommend transfection or transduction to more precisely regulate the
induceable promoter on the TRIPZ vector?
When transfecting an inducible lentiviral vector like our pTRIPZ vector, it is possible to end up
with so many copies of the vector in a cell that the leakiness associated with all inducible vectors
is magnified, which may mask the effect of doxycyline on the cells. It is possible to use the TRIPZ
vector in a transfection based assay but you may have to optimize the amount of plasmid DNA
used to minimize this effect. With transduction, you can control the number of viral particles (the
MOI) going into the cells to achieve a more accurately inducible system. For the reasons
mentioned above, although more precise integration can be achieved by transduction, pTRIPZ can
also be transfected if preferred.
23. What techniques are critical to successful packaging with Trans-Lentiviral packaging
system?
A few of the more critical techniques are listed below: 1. Make sure the HEK293T cells are not
split too thin. In other words, do not split the cells more than 1:5. And in the days leading up to
transfection, split or expand the cells 1:2 so that the cells are in a "rapid" replication state. Do not
split the cells back so far that it takes 5 days to become confluent. It just slows down the cellular
machinery to a crawl. You want the cell's transcriptional and translational machinery functioning
at their highest level when generating virus. 2. Transfect the cells within 24 hours after seeding
and at a confluency of no less than 90%. This is why you need the cells rapidly replicating. 3. If
using Arrest-In as the transfection reagent, do not use media containing serum or antibiotics. 4.
The transfer vector DNA (i.e. pGIPZ, pTRIPZ) should be of transfection quality. If using Qiagen,
we prefer maxiprep DNA over midiprep or miniprep.
24. What concentration should I use for miRIDIAN Mimics and Hairpin Inhibitors?
Concentration curves should always be conducted to empirically determine the best concentration
for each miRNA and each cell system. Suggested starting concentrations are 50 nM for the Mimic
and 25 nM for the Hairpin Inhibitor when using a lipid-based delivery system such as the
DharmaFECT transfection reagents. The recommended range for optimizations is 0.4 to 100 nM.
25. What is the best method to transfect miRIDIAN microRNA Mimics and Inhibitors?
The miRIDIAN microRNA Mimics and Inhibitors can be delivered using standard lipid-mediated
or electroporation delivery methods. The optimal methods and conditions for delivery will depend
on the specific cell line or cell type that you are using. They should be similar to those for the
delivery of other small nucleic acids (e.g. single-stranded oligos or siRNAs) into the same cell line.
DharmaFECT Transfection Reagents can be used to transfect mimics and inhibitors. Cell
line-specific transfection conditions are available in the DharmaFECT General Transfection
protocol.