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Addgene

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.

AAV

Production

AAV distributed by Addgene has been produced either in-house by Addgene scientists or through collaboration with viral vector manufacturing facilities, such as the University of Pennsylvania Vector Core (Link opens in a new window). Transfections are performed using the transfer plasmid, a plasmid encoding Rep and serotype-specific Cap genes, and a plasmid encoding adenoviral helper sequences.

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

Titer

Titering is either performed by Addgene or by the University of Pennsylvania Vector Core. In general, titering is performed by the facility that produced the viral vector lot. Contact us to determine which facility produced your viral vector lot.

At Addgene, AAV particles are titered by droplet digital PCR (ddPCR) using primers and probes targeting the ITR elements and an internal control of known titer (protocol modified from Lock et al. (2014) (Link opens in a new window)). Previous experience suggests that titers obtained using ddPCR are generally 2–3 fold higher than those achieved using the standard qPCR method.

Titering by the University of Pennsylvania Vector Core is (as of April 2016) also performed by droplet digital PCR (ddPCR).

Quality Control

Addgene ensures high quality viral vectors by optimizing and standardizing production protocols and performing rigorous quality control (QC). The specific QC experiments performed varies for each viral lot. To learn which specific QC experiments were performed on your lot, please contact us.

  • Full sequencing of the Viral Genome

    Next-generation sequencing is performed 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. The data is also used to determine recombination rates in FLEX/DIO/fDIO constructs.

  • Endotoxin

    Endotoxin contamination in vector preparations can alter the immunogenic properties of the final product, particularly in large animal studies. Endotoxin contamination is minimized by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic properties of the final vector preparation, the quantity of gram-negative bacterial endotoxin is ensured to be less than 5 endotoxin units per mL. The endotoxin levels are determined using a chromogenic endotoxin detection assay based on the amebocyte lysate method.

  • 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 or SYPRO Ruby 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

Figure 1: 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.

  • Sterility

    Viral vector preparations are added to cells in culture. Three to five days later, cell cultures are inspected for sterility.

  • Transducibility

    Some viral vectors are tested in vitro and in vivo for gene expression and/or function. These data are sometimes reported or posted on the material page for the corresponding catalog item (see maps section for images).

Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre (55634-AAVrg) alone at 1.7E6 viral genomes (vg)/cell, pAAV-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] (Addgene 84445-AAVrg) alone at 1.1E6 vg/mL, or both. Two days later, Cre-dependent GFP expression was detected with direct fluorescence. GFP was not detected in the absence of Cre. mCherry expression alone was detected. pAAV-Ef1a-mCherry-IRES-Cre was a gift from Karl Deisseroth (Addgene viral prep # 55632-AAVrg). pAAV-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] was a gift from Edward Boyden (Addgene viral prep # 84445-AAVrg).

  • 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

Figure 3: 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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Lentivirus

Production

Lentiviral preparations are produced in 293T cells using the 2nd generation psPAX2 and pMD2.G packaging system developed by the Trono lab. Cell culture medium (OptiPro SFM) containing lentivirus is first cleared by low-speed centrifugation and then by filtration through a 0.45 µm polyethersulfone (PES) membrane. The preparations are then aliquoted, frozen, and stored at -80 °C.

Titer

All titering is performed on lentiviral preparations that have been stored at -80 °C and thawed. Lentivirus titration is performed using a 293T cell line in the presence of 10.0 μg/mL polybrene. This ensures that any loss of titer associated with the recipient's initial thaw will be accounted for in our reported titers.

Lentiviral vectors are titered using a lentiviral droplet digital PCR (ddPCR) titration protocol (modified from Wang et al. (2018) (Link opens in a new window)). Briefly, 293T cells are transduced with serial dilutions of a lentiviral vector, incubated for 72 h, and genomic DNA is extracted. Primers and probes targeting integrated copies of the lentiviral Rev responsive element (RRE) gene and the cellular ribonuclease P/MRP 30 kDa subunit (RPP30) as a control for normalization purposes are being used.

Quality Control

  • Mycoplasma

    The 293T cell line was obtained from Takara, and is routinely tested for mycoplasma contamination using mycoplasma detection kits. The cell line is maintained for ~20 passages before being discarded and replaced with a new vial of early passage cells. Approximately 2 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.

  • Proviral Identity Confirmation and Sterility

    All of our lentiviral vectors are confirmed to have the correct integrated provirus using PCR. This assay involves transducing cells with serial dilutions of the lentiviral vector, harvesting cells several days later, and isolating genomic DNA. This DNA is then used as a template to amplify a portion of the intended lentiviral insert. During this 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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