Penn Medicine
Penn Vector Core
Department of Pathology and Laboratory Medicine
Division of Transfusion Medicine
Perelman School of Medicine
University of Pennsylvania
Custom Vector Request Form
Request #:
Request Date: _____ / _____ / ______
Month
Day
(For Penn use)
Year
Principal Investigator: __________________________________________
Institution:
__________________________________________
Research Area/ Disease Application/Target tissue (e.g heart, liver, lung, blood, muscle, eye, CNS,
other):__________________________________________________________________________________
(This information is useful to the Penn Vector Core to determine whether center grant support might apply)
FOR INVESTIGATORS EXTERNAL TO THE UNIVERSITY OF PENNSYLVANIA:
Requestor
Name: ________________________________
Shipping
Address:___________________________________
Requestor Email
Address:_______________________________
__________________________________________
Requestor Phone
Number:_______________________________
__________________________________________
ShippingAcct#:_____________________________
FOR INVESTIGATORS WITHIN THE UNIVERSITY OF PENNSYLVANIA:
Requestor
Name: ________________________________
26 Digit
Acct #: ___________________________________
Requestor Email
Address:_______________________________
Account
Name:____________________________________
Requestor Phone
Number:_______________________________
Expiration
Date:___________________________________
PI Signature: __________________________________________
TRL Suite 2000, 125 S. 31 s t Street | Philadelphia PA 19104-3403 | Tel: 215-573-0633 | Fax: 215-261-2442
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FOR CUSTOM VECTOR PRODUCTION
(To request vectors available through our inventory, please use our Inventory Request Form)
Please check (9) one vector type per request form:
□AAV Vector Production
□Adenoviral Vector Production
□Lentiviral Vector Production
Please choose AAV Serotype:
□ AAV1 □ AAV2 □ AAV5 □ AAV7 □ AAV8 □ AAV9 □ AAV other
Please choose Lentiviral Vector Pseudotype:
□ VSVG □ Mokola □ LCMV □ MuLV □ EboZ □ NTDL4 □ Other
* For Adenoviral Vector and Lentiviral Vectors, a single scale of production only is offered*
* For AAV Vectors, three scales of production are offered (see our website for release criteria)
AAV VECTOR PRODUCTION SCALE
Please check (9) one scale per request form:
□ Small Scale (2.5 x 10 GC anticipated yield or greater; approx. 1 ml in 100 ul aliquots)
□ Routine Scale (5 x 10 GC anticipated yield or greater; 1.5 - 2 ml in 100 ul aliquots)
□ Mega Scale (5 x 10 GC anticipated yield or greater; 4.5 - 6 ml in 100 - 500 ul aliquots)
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12
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SUPPORTING PLASMID DNA SERVICES REQUESTED (check (9) as many as apply):
□ Cloning (gene of interest provided but must be inserted into an AAV cis plasmid)
□ DNA amplification (AAV cis plasmid provided and further amplification required)
□ DNA structure/sequence analysis (AAV cis plasmid sequence must be verified)
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PLEASE COMPLETE THE FOLLOWING IF SOURCE MATERIAL WILL BE PROVIDED:
Penn Vector provides DNA cloning, production and analysis services or can produce vectors from source material
supplied by the investigator. For investigators wishing to provide sufficient plasmid DNA to go directly into
production, please submit a restriction enzyme analysis of the DNA you are submitting indicating that the following
elements: cis acting sequences (e.g. ITRs or LTRs), promoter, transgene, polyadenylation signal (polyA) where
applicable and any other element are intact. Please note that plasmids containing repeated elements can be unstable
and verifying the integrity of DNA submitted is essential. Please attach or send electronically a map and sequence of
your vector indicating the above elements and restriction enzymes used for analysis. Sequence information is required
to enable Penn Vector to correctly titer vectors using taqman PCR. (Penn Vector uses Vector NTI available online at
www.invitrogen.com).
FOR CUSTOM AAV VECTOR PREPARATIONS PLEASE PROVIDE THE FOLLOWING:
□ At least 1.2 mg AAV cis plasmid (i.e. containing AAV ITRs flanking the transgene cassette) and
□ Maintained in a recombinase (rec minus cell line (e.g. stbl2, SURE) and
□ Maintained in the presence of carbenicillin (for plasmids carrying the ampR gene) and
□ Purified using an endotoxin-free method (e.g. Qiagen endo-free mega kit) with a
□ Gel Image confirming both AAV 5’ and 3’ ITRs and other important elements (see next page) and
□ Confirmation of a genome coding size of 4.9 kb or less (from ITR-ITR, including ITRs)* and
□ Electronic map and sequence information (map indicating restriction sites used for analysis)
-)
Note that our standard release criteria will not apply for vectors with over-size genomes (>4.9 kb),
double-stranded (self-complimentary) genomes or for genomes that express shRNA sequences*
FOR CUSTOM LENTIVECTOR PREPARATIONS PLEASE PROVIDE THE FOLLOWING:
□ At least 1.2 mg Lenti transfer DNA (i.e. containing LTRs flanking the transgene cassette) and
□ Maintained in a recombinase (rec minus cell line (e.g. stbl3, HB101) with carbenicillin and
□ Purified using an endotoxin-free method (e.g. Qiagen endo-free mega kit) with a
□ Gel Image confirming both 5’ and 3’ LTRs and other important elements (see next page) and
□ Confirmation of a genome coding size of 9.7 kb or less (from LTR-LTR, including LTRs)* and
□ Electronic map and sequence information (map indicating restriction sites used for analysis)
-)
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PLEASE COMPLETE THE FOLLOWING IF SOURCE MATERIAL WILL BE PROVIDED:
*Visit http://www.med.upenn.edu/gtp/vector_core.shtml for sample restriction enzyme analysis*
TOTAL GENOME SIZE (from ITR to ITR) ______________ (Note: AAV must be 4.9 kb or less)
Map of the plasmid showing the restriction sites used for analysis (please paste below):
Restriction Enzyme Digests: Each plasmid characterization must confirm the following elements: 3’ITR/LTR , 5’
ITR/LTR, promoter, transgene and polyadenylation signal if applicable. Note for AAV plasmids, a SmaI (or XmaI)
digest either alone or in combination is mandatory for every plasmid. MscI digests are mandatory for plasmids with sc
or ds vector genomes. Additional digests should be selected for each plasmid and uncut as well as linearized plasmid
DNA should be included on the gel.
Copy the fragment lists from vNTI file into the table below. Highlight bands containing the relevant elements as
follows: 3’ITR 5’ ITR 3’ ITR /PolyA PolyA Promoter Transgene
*TS = Too small to visualize
Restriction enzymes
Enzyme(s) A
For AAV
Sma I
Combination
Enzyme(s) B
Element
5’ ITR
3’ ITR
5’LTR
3’LTR
promoter
Enzyme(s) C
Enzyme(s) D
transgene
(intact, correct
orientation )
poly A (if appl)
Enzyme(s) E
other element
Expected bands
Obtained bands
No
Faint
Yes
No
Faint
Yes
Yes
No
Faint
No
Faint
Yes
Yes
No
Faint
No
Faint
Yes
No
Faint
Yes
No
Faint
Yes
No
Faint
Yes
No
Faint
Yes
Yes
No
Faint
No
Faint
Yes
No
Faint
Yes
Yes
No
Faint
No
Faint
Yes
No
Faint
Yes
Extra bands
TS
TS
TS
TS
TS
TS
TS
TS
TS
TS
TS
TS
TS
TS
TS
TS
Interpretation of Restriction Enzyme Digest Results:
□ Intact 5’ ITR (or LTR)
□ Intact 3’ ITR (or LTR)
□ Complete promoter
□ Transgene (intact, correct orientation)
□ Intact polyadenylation signal (if appl)
Comments __________________________________________________________________________
____________________________________________________________________________________
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PLEASE COMPLETE THE FOLLOWING IF SOURCE MATERIAL WILL BE PROVIDED:
Promoter (name and source e.g. human)
________________________________
Transgene (name and source e.g. mouse)
________________________________
Biological Activity of Transgene
________________________________
____________________________________________________________________________________
____________________________________________________________________________________
Poly A sequence, if applicable (e.g. bGH, SV40, other)
________________________________
Any other important element (e.g. intron, IRES, other)
________________________________
* THE FOLLOWING IS FOR CUSTOM ADENOVIRAL VECTOR PREPARATION ONLY *
For adenoviral vector creations from pShuttle plasmids: please provide at least 50 ug of shuttle plasmid (a plasmid
containing I-CeuI and PI-SceI sites flanking the transgene cassette). Penn Vector will supply a pShuttle plasmid free
of charge upon request or investigators may utilize the commercially available pShuttle plasmid from Clontech.
For adenoviral vector rescues from pAd plasmids: please provide at least 50 ug of pAd plasmid (a plasmid containing
a transgene cassette incorporated into an adenoviral genome), maintained in a recombinase minus (rec-) cell line (e.g.
stbl2) and purified using an endotoxin-free method (e.g. Qiagen endofree mega kit) is required. Note that adenoviral
plasmids can be unstable due to size and repeated elements (ITRs); verifying the integrity of the ITRs and genome
structure for each lot of DNA submitted is essential.
For adenoviral vector expansions from purified vector or lysate: please provide at least 1 E+12 particles or 100 ul of
vector or lysate. Information detailing the backbone deletions and presence (if any) of replication competent
adenovirus is also necessary.
Please keep in mind that the success rate of recombinant vector production depends upon the quality of your DNA,
purified vector or lysate. Penn Vector recommends, whenever possible, that the construct’s expression be tested prior
to initiation of vector production.
ADENOVIRAL VECTOR SOURCE MATERIAL SUBMITTED:
□ Purified Adenoviral Vector for expansion (amount _________ )
□ Adenoviral Vector Lysate for expansion (amount _________ )
□ Adenoviral Vector Plasmid DNA for rescue (amount _________ )
□E1 Deletion □E2 Deletion □E3 Deletion □E4 Deletion Coordinates of deletion (bp-bp) ______
For questions regarding payment, please contact: Ree Ree Melko, Business Administrator, Gene Therapy Program
Phone 215.746.8937
Fax 215.573.5655 Email melkoam@mail.med.upenn.edu
For questions regarding production, shipping or material transfer, please contact: The Penn Vector Core
Phone 215.573.0633
Fax 215.261.2442 Email vector@mail.med.upenn.edu
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