Neuroscience Gene Vector and Virus Core Viral vector request form for retroviruses. Fill out form and deliver or e-mail to

Date: .

Investigator: . Lab head: .

Envelopedesired (e.g., VSV-G, ecotropic, etc.): .

Production scale desired (Number and size of flasks of cells to be transfected to produce the virus? Often one to three T-75s are usedto produceretroviruses.): . (Alternatively you can request a specific final volume and concentration.)

Purity level desired (crude or pelleted?): .(Crude retrovirus can be used for many cell culture experiments. It will be ~300x less concentrated than pelleted butis less expensive.)

Name of transgene vector (e.g., pMLV CMV-GFP): . Description of transgene vector (e.g., retrovirus 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 neededper T-75 transfected forretrovirus 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 retrovirusvector. 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 theviral genome encoded by the plasmid is oversized and b) if the sequence can be detected by Q-PCR probes (if necessary) that are in-stock (pCMV, hGH poly A, WPRE) for genomic and/or infectious titering.

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.) infectioustiter by Q-PCRdetection 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? ______.

(Retroviruses 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 retroviruses final volumes may be from 20-50+ microliters depending on the scale of production. Usually 5 microliter aliquots are madethen immediately frozen but other sizes may be requested. For crude retrovirusesfinal volumes will typically be 10-20 ml per T-75 transfected. Usually 1 ml aliquots of crude retroviruses are made, but other sizes may be requested. )

What date do you need the virus by? .

Notes or comments: .