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viral-icon-01.png Viral Production


Overview of Viral Production

Viruses are generated using standard methods that have been optimized for each specific virus in order to generate high quality preparations. After production, all virus preps are titered and subjected to quality control by Addgene before being distributed to customers. Details about our production protocols, titering methods, and quality control are described below.

Lentivirus

Production

Lentiviral preparations (-LV catalog items) are produced in the Lenti-X 293T cell line using the 2nd generation psPAX2 and pMD2.G packaging system developed by the Trono lab. Cell culture medium (DMEM + 10% FBS) containing lentivirus is first cleared by low speed centrifugation and then by filtration through a 0.45 µm polyethersulfone membrane. The preparations are then aliquoted, snap frozen in liquid nitrogen, and stored at -80°C. Concentrated lentiviral preparations (-LVC catalog items) are generated from lentiviral preparations (described above) that are subject to a concentration step prior to being frozen. Specifically, lentiviral particles are collected from the lentiviral preparation by precipitation in 10% polyethylene glycol (PEG) followed by centrifugation. Precipitated pellets containing viral particles are then resuspended in PBS, aliquoted, snap frozen in liquid nitrogen, and stored at -80°C.

Titer

All titering is performed on lentiviral preparations that have been stored at -80°C and thawed. This ensures that any loss of titer associated with the recipient's initial thaw will be accounted for in our reported titers.

Lentiviral vectors carrying selectable markers are titered using the standard colony formation assay. Briefly, A549 cells are transduced with serial dilutions of a lentiviral vector, exposed to antibiotic, and resistant colonies are allowed to expand for 2 weeks. Culture dishes are then stained and macroscopic colonies are counted to determine infectious titer.

In addition to the colony formation assay, lentiviral vectors carrying puromycin and blasticidin resistance undergo a relative titering assay based on cell proliferation after antibiotic addition. Briefly, Lenti-X 293T cells are transduced with serial dilutions of a control lentiviral stock of known titer (typically pRosetta) in addition to the lentiviral stock to be titrated. After 48 hours the cells are treated with the appropriate dose of antibiotic and incubated for an additional 2-3 days. Cell proliferation is then measured using resazurin dye. Relative titers are extrapolated from a standard curve generated by the control lentiviral stock.

Lentiviral vectors carrying fluorescent markers undergo additional titering based on expression of the fluorescent protein. Briefly, Lenti-X 293T cells are transduced with serial dilutions of a lentiviral vector and fluorescent cells are quantified by microscopy 96 hours post-transduction.

Quality Control

Mycoplasma

The Lenti-X 293T and A549 lines were obtained directly from Clontech and Sigma Aldrich, respectively, and are routinely tested for mycoplasma contamination using the protocols described by Uphoff and Drexler (PubMed: PMID 23179822). Lines are maintained for 15 passages before being discarded and replaced with a new vial of early passage cells. Approximately 2-3 weeks post-thaw, cell culture supernatant is tested for mycoplasma contamination. To date, Addgene has never had a case of mycoplasma contamination. In the event of contamination, all of the virus produced in the line will be taken off-line and discarded.

Confirmation of Transfer Plasmid

Addgene uses a rigorous barcode matching system that follows the sample from the DNA tube all the way to the cleared viral preparation pool.

In addition, the final viral preparation undergoes PCR with primers targeting the transfer plasmid used in the transfection. PCR products are analyzed by gel electrophoresis to ensure the correct banding pattern and sizes.

Diagram of viral production

Schematic of the barcode matching system and PCR-based confirmation of transfer plasmids used in Addgene's viral production.

Barcoding allows our DNA and virus to be monitored throughout production, ensuring the identity of each sample from the DNA tube all the way to the viral preparation. After production, we confirm the identity of the transfer plasmid in the viral preparation by performing PCR against unique regions of the transfer plasmid.

Sterility

The majority of our lentiviral vectors are titered using the colony formation assay. This assay involves transducing cells with serial dilutions of a lentiviral vector, and allowing antibiotic-resistant colonies to expand for 2 weeks. During this long-term cultivation, cells are routinely checked for signs of microbial contamination.

Purity

When possible, all plasmids used for viral production are propagated in the endA-mutated NEB Stable strain of E. coli. In addition, plasmids are typically prepared using endotoxin-free plasmid purification kits.

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AAV

Production

AAV distributed by Addgene has been produced either in-house by Addgene scientists or through collaboration with viral vector manufacturing facilities. Transfections are performed using the transfer plasmid, a plasmid encoding rep and serotype-specific cap, and a plasmid encoding adenoviral helper sequences.

AAV preparations are purified by iodixanol gradient ultracentrifugation or cesium chloride gradient ultracentrifugation and concentrated to purity and titers adequate for in vivo studies. Preparations are then aliquoted and stored at -80°C.

Titer

AAV particles are titered by real-time quantitative PCR using primers targeting the ITR. The amplicons are detected using SYBR green technology. Titer values are then determined by comparison to a standard curve of a plasmid sample of known concentration. The qPCR assay and corresponding titer determinations are also validated using AAV Reference Material (ATCC).

Quality Control

Confirmation of Transfer Plasmid

The final AAV preparation undergoes PCR with primers targeting the transfer plasmid used in the initial transfection. Most of our primers are targeting the promoter region and/or the transgene. For DIO or FLEX plasmids, we also validate the orientation of the cassette using 2 or more primer pairs. PCR products are analyzed by gel electrophoresis to ensure the correct banding pattern and sizes.

When possible, we perform next-generation sequencing on viral genomes isolated from the final AAV preparation. Sequencing results are analyzed to confirm the identity and integrity of the viral genome and the absence of unexpected DNA contaminants.

Purity

Purity of AAV preparations is assayed by comparing the relative stoichiometric ratios of the viral capsid proteins VP1, VP2 and VP3. Samples of viral preparations are subjected to polyacrylamide gel electrophoresis (PAGE) followed by silver staining and the molecular weight and relative intensity of the viral capsid proteins are analyzed. The abundance of viral capsid proteins as a fraction of total protein present in the sample is also determined and used to determine purity of the AAV preparation.

Silver stain of AAV

Silver staining of purified and non-purified AAV subjected to gel electrophoresis. Viral capsid proteins VP1, VP2,
and VP3 are shown relative to the total protein present in the sample.

Transducibility

Whenever possible, viruses are tested in vitro and in vivo for reporter gene expression to validate transduction. Fluorescence microscope images showing reporter gene expression are posted on the material page for each viral catalog item for which this validation was performed.

Electron Microscopy

The ratio of empty to full (i.e., genome containing) AAV particles within representative vector preparations was quantified with electron microscopy after negative staining. Empty vector particles can be identified after negative staining and appear darker than full vector particles.

Electron microscope image of AAV

Electron micrograph of AAV vector preparation shows that the vast majority of the vectors consist of
full particles (white arrowhead) relative to empty particles (black arrowhead). Scale bar = 100 nm.

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