Neuroscience Gene Vector and Virus Core
Viral vector request form for lentiviruses. Fill out form and deliver or e-mail to mlochrie@stanford.edu
Date:
.
Investigator:
Envelope desired (e.g., VSV-G):
.
Lab head:
.
.
Production scale desired (Number and size of flasks of cells to be transfected to produce the virus? Often
one to three T-75s are used to produce lentiviruses.):
. (Alternatively you can
request a specific volume and concentration.)
Purity level desired (crude or pelleted?):
.
(Crude lentivirus can be
used for many cell culture experiments. It will be ~300x less concentrated than pelleted but is less
expensive.)
Name of transgene vector (e.g., pLenti CMV-GFP):
.
Description of transgene vector (e.g., lentivirus vector with GFP gene driven by CMV promoter):
_____________
.
Does your vector encodes oncogenes, select agents, toxins, apoptotic genes, etc. that may be more
hazardous than usual? (yes/no)
.
If your vector encodes a fluorescent reporter, will the promoter driving reporter gene expression be
active in 293 cells? (yes/no)?
. (such that transfection and infection efficiencies can be assessed)
Are you preparing the vector plasmid (yes/no)?
.
If yes, at least 12 g is needed per T-75 transfected for lentivirus preps. The DNA should be high
quality (e.g., purified using ion exchange, not silica-based columns). The exact plasmid prep to be used
for transfection to produce virus should be well characterized (e.g., sequenced, digested with several
restriction enzymes, functional tests if practical) especially if it is a lentivirus vector.
Do you have a complete predicted sequence for your vector (yes/no)?
.
If yes, please e-mail a text or Vector NTI (preferred) file with as much information about
landmarks as is available. The sequence is used to determine a) if the viral genome encoded by the
plasmid is oversized and b) if the sequence can be detected by Q-PCR probes (if necessary) that are instock (pCMV, hGH poly A, WPRE) for genomic and/or infectious titering.
What “generation” is your vector? (1st, 2nd, or 3rd?):
. (This is useful to know because different
“helper plasmids” are used along with the vector plasmid to produce virus depending on which
generation it is. There are also other implications. For example second generation vectors often produce
less virus than third generation vectors, third generation vectors are more like to have a “SIN”
mechanism to inactivate the upstream viral promoter, etc.)
What characterization of the final virus preparation do you desire:
infectious titer by infection of 293 cells (yes/no)?
.
(applicable only if genome encodes a fluorescent reporter that is expressed in 293 cells.
The cost of this assay is included in the cost of virus production.)
infectious titer by Q-PCR detection of viral genome integrated in infected cells (yes/no)?
.
(If your genome does not encode a fluorescent reporter that is adequately expressed in 293 cells then this
would be the only method that an infectious titer could be determined. The cost of this assay is not
included in the cost of virus production.)
Probe that can be used for Q-PCR (pCMV, hGH poly A, or WPRE):
Would you like the final virus preparation aliquoted and if so what size?
.
________
.
(Lentiviruses are very unstable. It is best to aliquot to minimize repeated freezing and thawing since each
freeze-thaw may reduce infectious titer by 3-fold or more. For pelleted lentiviruses final volumes may be
from 20-50+ microliters depending on the scale of production. Usually 5 microliter aliquots are made,
but other sizes may be requested. For crude lentiviruses final volumes will typically be 10-20 ml per T-75
transfected. Usually 1 ml aliquots of crude lentiviruses are made, but other sizes may be requested. )
What date do you need the virus by?
Notes or comments:
.
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