MVC Services

Adenovirus Production
- Custom medium to large-scale productions (purification by sorbitol gradient)
- Custom medium to large-scale productions (purification by cesium chloride gradients)
- Standard Adenovirus production sizes range from 2000 cm² to 6000 cm².
Adenovirus Titration
- TCID₅₀
- Particle titer
- Focus Forming Assay (FFA)
- Replication-competent virus (RCV) assay (required for in vivo use)
For custom adenoviral vector production, user clones genetic sequences of choice into a provided shuttle plasmid
Production size can be anywhere from a small pilot stock to a large prep, with purity levels varying from crude lysate to highly purified stock.
Transgene cassette choices available:
- Inducible promoter for expression of toxic proteins
- CMV promoter and V5-tag for high-level expression and easy protein detection
- RNA interference (RNAi) cassettes for expression of short hairpin RNA (shRNA)
- Empty expression cassettes for user-supplied regulatory elements and transgenes
- Pre-made common stock vectors (such as Adeno-CMV-eGFP) available for pilot testing

Custom rAAV Production

- All custom rAAV productions include purification by Iodixanol Gradient Ultracentrifugation, Titer determination Free ITR qPCR, and purity assessment by silver stained SDS-PAGE
- Choice of serotypes include 1, 2, 2 retro, 3, 5, 6 7, 8, 9 and DJ (serotype plasmid for production provided by MVSC). If your serotype of interest is not on our list, please contact us, and we will do our best to accommodate you. For example, we have made AAV vectors with serotypes like LK03 and PHP.B for users.
- Two production sizes are available:
  - Standard Medium Scale [2,000 cm² culture scale, yields depend on serotype, typically ranging between 10¹¹ - 10¹³ viral genomes (vg)]
  - Large-Scale [6,000 cm² culture scale, yields depend on serotype, typically ranging between 10¹² - 10¹⁴ vg)]

For more detailed information on requirements, how to prepare your plasmids for production, and to request a custom production, please refer to our AAV Production Request Form or contact the project lead.

Custom rAAV "Minipreps"
- rAAV "Minipreps" are a cost-effective method developed in our laboratory to test multiple AAV constructs for expression without the need for cumberson iodixanol purification^1,2. This method is recommended for pilot testing/screening of our constructs. Examples of vectors produced by this method include:

- - AAV1 CBA GFP injected in the developing mouse inner ear expressed GFP and showed no apparent toxicity (done in collaboration with Dr. John Brigande's laboratory)
  - AAV8 CMV EGFP injected intraperitoneally in mice showed EGFP expression in the liver without any local inflammation (done in collaboration with Dr. Caroline Enns laboratory)
- rAAV "Miniprep" includes production in one T-150 flask (150 cm²) with partial purification by ultrafiltration, and quantitation by Free ITR qPCR. Yields typically range between 10¹⁰-10¹¹ vg.

For further details please refer to the AAV Miniprep Request Form.

AAV Stock vectors

- Pre-made AAV stock vectors for pilot testing are available for purchase and include:
  - AAV-CMV-EGFP (serotypes 1, 2, 2 retro,3, 5, 6, 7, 8, 9, DJ)
  - scAAV9-CMV-EGFP (self-complementary vector)
  - AAV1 CBA GFP
  - AAV- CMV- Luciferase (serotypes 1, 2, 6, 8 and 9)

To purchase pre-made stocks, please fill out the AAV Stock Request Form and email the project lead.

AAV Neutralizing Antibody (AAV NAb) Assays

- Separate AAV NAb assay for AAV serotypes 1, 2, 5, 6, 8 and 9
- Two types of NAb assay are available: Screening and ID50
  - The screening assay is used to screen animals for NAb presence/absence; 3-dilution range
  - The ID50 assay includes 11 three-fold dilutions globally fitted to determine ID50

For further details please refer to the AAV NAb Assay Request Form or contact the project lead.

References

1. Gomes MM, Wang L, Jiang H, Kahl CA, and Brigande JV. (2015). A Rapid, Cost-Effective Method of Recombinant Adeno-Associated Virus Preparation Enables Efficient Gene Transfer to the Developing Mouse Inner Ear. Auditory and Vestibular Research (Second Edition): Methods in Molecular Biology.

2. Kleven MD*, Gomes MM*, Wortham AM, Enns CA and Kahl CA. (2018). Ultrafiltered recombinant AAV8 vector can be safely administered in vivo and efficiently transduces liver. PLoS One 5; 13(4):e0194728. *Equal contribution

Project Lead: CoreyAyne Singleton

The core provides production of 2nd and 3rd generation lentiviral vector production providing either conditioned medium or concentrated virus preparations. The core will assist with vector design as needed and provides some standard vectors. The core can assist with infection of cell lines and cell sorting if desired.

What steps have to be taken before a lentiviral prep can be started?
The core has a very specific checklist of requirements that must be met before actual production of lentivirus can begin:

The lentiviral core must receive a copy of your current RDRQ with proposed constructs and an electronic (.pdf) copy of IBC approval letter before any work can be started.
Generation of plasmid for lentiviral preps should be transformed in XL-010 gold, STBL 3 or comparable competent cells. Plasmid must be endotoxin free. (We get good results with Qaigen Maxi kits.) This step can also be done by the Core facility if you wish.
Quality control digestion of plasmid should be done to confirm correct band cuts/pattern expected. For any labs doing steps 2 and 3 themselves, they will need to send a copy of gel image to the core for records.
We strongly suggest doing a test plate of all plasmids to confirm through FACS or qPCR of successful packaging of viral vectors before doing a full scale prep. This will help prevent doing a large scale prep that may not package well. Once the Core starts a full scale prep all charges will be incurred.
Actual production of lentivirus can begin at selected scale (see below, 10 µg of DNA is needed for each 10cm dish reaction.)
Fill out the lentivirus production request form.
The Core will gladly work with you to set up any custom preparations that you have in mind.

Project Lead: Jeff Torgerson

RhCMV Production
- Medium to large scale productions (purified by sorbitol cushion)
- Crude infected cell lysate for viral protein
RhCMV Titration
- TCID₅₀
- Plaque Assay
RhCMV Diagnostics
- Virus co-culture from animal tissues
- Viral load quantification (qpcr)
Virus co-culture from animal tissues
Virus stock and vector production
Viral load quantification (qPCR)

Project Lead: Don Siess

Custom virus stock productions for HIV, SIV and SHIV.
SIV and SHIV Titration by TZM-bl (FFA)
SIV and SHIV Diagnostics
- SIV plasma viral load (pVL) quantification (Hybrid-digital qRT-PCR)
- Ultrasensitive SIV detection and quantification in tissues (nested digital qPCR, qRT-PCR)
For lentiviral vectors, please contact CoreyAyne Singleton

Project Lead: Don Siess , Ashley White, and Travis Giobbi

DNA/RNA extraction from small and large tissue samples
Manual and robotic nucleic acid isolation and purification

Virus isolation from tissues and fluids
RhCMV, RRV and SFV viral antigen preparations for ESPF screening
Cell stocks: primary and cell lines
Sample processing and banking

The MVSC also offers data analysis and troubleshooting, custom services, consultation and training. Please inquire if you need additional assays not listed here and for assay development. We are committed to serving you and your research needs.

Rates for internal users

For Federal grants, our internal prices are based on the F& A rate negotiated between the NIH and OHSU/ONPRC. Internal users paying with non-NIH funds (Foundations, charitable organizations, etc.) can calculate their rate by entering the percentage of F&A covered by the funding organization into row 5 of the rate sheet. External users need to pay for services with a purchase order. Please contact Greg Dissen for a quote.