Here we provide a detailed procedure for large-scale production of research-grade AAV vectors using adherent HEK 293 cells grown in cell stacks and affinity chromatography purification. This protocol consistently yields >1 x 1013 vector genomes/mL, providing vector quantities appropriate for large animal studies.
Adeno-associated virus (AAV) vectors are among the most clinically advanced gene therapy vectors, with three AAV gene therapies approved for humans. Clinical advancement of novel applications for AAV involves transitioning from small animal models, such as mice, to larger animal models, including dogs, sheep, and nonhuman primates. One of the limitations of administering AAV to larger animals is the requirement for large quantities of high-titer virus. While suspension cell culture is a scalable method for AAV vector production, few research labs have the equipment (e.g., bioreactors) or know how to produce AAV in this manner. Moreover, AAV titers are often significantly lower when produced in suspension HEK 293 cells as compared to adherent HEK293 cells. Described here is a method for producing large quantities of high-titer AAV using cell stacks. A detailed protocol for titering AAV as well as methods for validating vector purity are also described. Finally, representative results of AAV-mediated transgene expression in a sheep model are presented. This optimized protocol for large-scale production of AAV vectors in adherent cells will enable molecular biology laboratories to advance the testing of their novel AAV therapies in larger animal models.
Gene therapy utilizing adeno-associated virus (AAV) vectors has made huge strides over the past three decades1,2. Demonstrated improvements in a diverse range of genetic diseases, including congenital blindness, hemophilia, and diseases of the musculoskeletal and central nervous system, have brought AAV gene therapy to the forefront of clinical research3,4. In 2012, the European Medicines Agency (EMA) approved Glybera, an AAV1 vector expressing lipoprotein lipase (LPL) for the treatment of LPL deficiency, making it the first marketing authorization for a gene therapy treatment in either Europe or the United States5. Since then, two additional AAV gene therapies, Luxturna6 and Zolgensma7, have received FDA approval, and the market is expected to expand quickly over the next 5 years with as many as 10-20 gene therapies expected by 20258. Available clinical data indicate that AAV gene therapy is a safe, well-tolerated, and efficacious modality making it one of the most promising viral vectors, with above 244 clinical trials involving AAV registered with ClinicalTrials.gov. The increasing interest in clinical applications involving AAV vectors requires robust and scalable production methods to facilitate the evaluation of AAV therapies in large animal models, as this is a critical step in the translational pipeline9.
For AAV vector production, the two main requirements are the AAV genome and the capsid. The genome of wild-type (wt)-AAV is single-stranded DNA that is approximately 4.7 kb in length10. The wt-AAV genome comprises inverted terminal repeats (ITRs) found at both ends of the genome, which are important for packaging, and the rep and cap genes11. The rep and cap genes, necessary for genome replication, assembly of the viral capsid, and encapsulation of the genome into the viral capsid, are removed from the viral genome and provided in trans for AAV vector production12. The removal of these genes from the viral genome provides room for therapeutic transgenes and all the necessary regulatory elements, including the promoter and polyA signal. The ITRs remain in the vector genome to ensure proper genome replication and viral encapsulation13,14. To improve the kinetics of transgene expression, AAV vector genomes can be engineered to be self-complementary, which mitigates the need for conversion from single-stranded to double-stranded DNA conversion during AAV genome replication, but reduces the coding capacity to ~2.4 kb15.
Beyond AAV genome design, capsid serotype selection determines the tissue and cell tropism of the AAV vector in vivo2. In addition to tissue tropism, different AAV serotypes have been shown to display different gene expression kinetics16. For example, Zincarelli et al.17 classified different AAV serotypes into low expression serotypes (AAV2, 3, 4, 5), moderate expression serotypes (AAV1, 6, 8), and high expression serotypes (AAV7 and 9). They also categorized AAV serotypes into slow-onset expression (AAV2, 3, 4, 5) or rapid-onset expression (AAV1, 6, 7, 8, and 9). These divergent tropisms and gene expression kinetics are due to amino acid variations in the capsid proteins, capsid protein formations, and interactions with host cell receptors/co-receptors18. Some AAV capsids have additional beneficial characteristics such as the ability to cross the blood-brain barrier following intravascular administration (AAV9) or reside in long-living muscle cells for durable transgene expression (AAV6, 6.2FF, 8, and 9)19,20.
This paper aims to detail a cost-effective method for producing high-purity, high-titer, research-grade AAV vectors for use in preclinical large animal models. Production of AAV using this protocol is achieved using dual-plasmid transfection into adherent human embryonic kidney (HEK)293 cells grown in cell stacks. Furthermore, the study describes a protocol for heparin sulfate affinity chromatography purification, which can be used for AAV serotypes that contain heparin-binding domains, including AAV2, 3, 6, 6.2FF, 13, and DJ21,22.
A number of packaging systems are available for the production of AAV vectors. Among these, the use of a two-plasmid co-transfection systems, in which the Rep and Cap genes and Ad helper genes (E1A, E1B55K, E2A, E4orf6, and VA RNA) are contained within one plasmid (pHelper), has some practical advantages over the common three-plasmid (triple) transfection method, including reduced cost for plasmid production23,24. The AAV genome plasmid containing the transgene expression cassette (pTransgene), must be flanked by ITRs, and must not exceed ~4.7 kb in length. Vector titer and purity can be affected by the transgene due to potential cytotoxic effects during transfection. Assessment of vector purity is described herein. Vectors produced using this method, which yield a 1 x 1013 vg/mL for each, were evaluated in mice, hamsters, and ovine animal models.
Table 1: Composition of required solutions. Necessary information, including percentages and volumes, of components needed for various solutions throughout the protocol. Please click here to download this Table.
1. Double plasmid transfection of HEK293 cells in cell stacks
Figure 1: Maneuvering of cell stack for cell seeding and transfection. For seeding cell stack, start by removing one of the vent caps and pouring in 1 L of pre-warmed complete DMEM with needed quantity of HEK293 cells (A). Evenly distribute cells and media by tightening both vent caps and bring all media to the corner of the cell stack with one of the vent caps and place it in that corner (B), place the cell stack on its side (C), and then turn the cell stack 90° (D) so that the vent ports are up (E). Gently lower the cell stack to its normal horizontal position and ensure all the chambers of the cell stack are completely covered in media (F). When transfecting, unscrew both vent caps and slowly pour out old media into a waste sterile waste container for even flow not to disturb the monolayer of the cells (G). Please click here to view a larger version of this figure.
2 Harvesting AAV and chemical lysis of the transfected HEK293 cells
3 AAV Vector purification using heparin affinity chromatography
Figure 2: Set up for peristaltic pump for AAV purification. Run the tubing from the crude lysate, through the peristaltic pump, and into the heparin matrix column. Please click here to view a larger version of this figure.
NOTE: Ensure not to introduce bubbles or allow the column to run dry, as this will compromise the column and prevent the elution of AAV. Discard the column if it runs dry and use a new column for the remainder of crude lysate.
4 AAV genomic DNA extraction
Component | Volume |
Purified AAV vector | 5 μL |
10x DNase Buffer | 2 μL |
DNase | 1 μL |
ddH2O | 12 μL |
Final Volume | 20 μL |
Table 3: DNase treatment master mix formula. Recommended components and volumes required for DNase treatment of AAV viral vectors during DNA extraction.
5 Titration of AAV vector genomes using quantitative polymerase chain reaction and a Simian Virus 40 (SV40) probe
NOTE: Perform all qPCR work in a PCR hood using filtered pipette tips to avoid external DNA contamination. If the AAV genome does not encode an SV40 polyA sequence, use a probe against the ITR described elsewhere25. Ensure the plasmid DNA selected as standard contains SV40 polyA sequence.
Component | Volume |
Universal qPCR master mix (2X) | 10 μL |
Molecular grade water | 4.5 μL |
40x SV40 polyA primer/probe | 0.5 μL |
Final Volume | 15 μL |
Table 4: qPCR master mix for AAV titration. Recommended components and volumes required for qPCR of DNA extracted from AAV viral vectors.
Component | Sequence |
Forward primer | 5’-AGCAATAGCATCACAAATTTCACAA-3’ |
Reverse primer | 5’-CCAGACATGATAAGATACATTGATGAGTT-3’ |
Probe | /56-FAM/AGCATTTTT/Zen/TTCACTGCATTCTAGTTGTGGTTTGTC/3IABkFQ |
Table 5: Primer sequences against the SV40 polyA DNA sequence. Sequences of the primers and probe used for qPCR titration, which bind to specific areas of AAV viral vectors that contain the SV40 polyA sequence.
Figure 3: Plate layout for qPCR AAV titration. Blue indicates the placement of the serial dilution of the standard; green indicates the placement of the negative control; purple indicates the placement of the dilution of the samples. Each standard, negative, or sample is added in replicate. An example for the concentration of the standard has been added to show the dilution series of the standard, and placement of sample dilutions have been added to their respective wells. Please click here to view a larger version of this figure.
Section | Cycles | Time | Temperature | Description |
Pre-incubation | 1x | 5 min | 95 °C | DNA denaturation. |
Amplification | 38x | 15 s | 95 °C | Amplification of DNA. Settings can be modified if using alternative primers with different annealing temperatures. |
60 s | 60 °C | |||
Cooling | 1x | 60 s | 40 °C | Plate Cooling. End of run. |
Table 6: Thermocycler protocol for hydrolysis probe-based qPCR titration. Recommended thermocycler protocol for use of probe-based qPCR titration of DNA extracted purified AAV vectors.
NOTE: For qPCR AAV titration worksheet see Table 7.
6 Assessment of vector quality and purity
Figure 4: Western blot showing AAV capsid proteins. Lane A; MW ladder, Lane B; AAV6.2FF-hIgG01, Lane C; AAV6.2FF-hIgG02, Lane D; AAV6.2FF-hIgG03, and Lane E; AAV6.2FF-hIgG04. 6 x 1010 vg of each AAV6.2FF-hIgG was loaded into their respective lanes. Please click here to view a larger version of this figure.
Figure 5: Coomassie-stained gel. Lane A; MW ladder, Lane B; AAV6.2FF-hIgG01, Lane C; AAV6.2FF-hIgG02, Lane D; AAV6.2FF-hIgG03, Lane E; AAV6.2FF-hIgG04, Lane F; AAV6.2FF-hIgG05, and Lane G; AAV6.2FF-hIgG06. 6 x 1010 vg of each AAV6.2FF-hIgG was loaded into their respective lanes. Please click here to view a larger version of this figure.
Translation from small rodent models to larger animal models and eventual clinical application presents a significant challenge due to the large amount of AAV required to transduce larger animals and achieve therapeutic effects. To compare transduction efficiency of the rationally designed AAV6.2FF capsid, previously demonstrated a 101-fold increase in transduction efficiency in murine muscle cells compared to AAV63, mice, hamsters, and lambs were all administered AAV6.2FF expressing a human monoclonal antibody (hIgG). The AAV6.2FF-hIgG-expressing vector contained a CASI promoter4, a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a SV40 polyA sequence. Six-week-old female BALB/c (n = 4) mice were intramuscularly (IM) administered 1 x 1011 vg of AAV6.2FF-hIgG in 40 µL, and blood was collected on days 0, 7, 14, 21, and 28 post AAV administration for hIgG monitoring. Four-week-old Syrian hamsters (two females and two males) were IM administered 1 x 1012 vg of AAV6.2FF-hIgG in 40 µL, and blood was collected weekly to monitor for hIgG expression. Lastly, 10-day-old male Dorset lambs (n = 3) were IM administered 1 x1013 vg /kg of AAV6.2FF-hIgG in two to three 1 mL injections in the rump. Weekly blood collection was completed by jugular bleeds and hIgG was monitored weekly for 28 days. All animal experiments were approved by the Institutional Animal Care Committee of the University of Guelph.
Mice IM administered 5 x 1012 vg/kg of AAV6.2FF-hIgG expressed between 171-237 µg/mL of hIgG in the serum by day 28 post-administration (Figure 6). Hamsters IM administered 2 x 1013 vg/kg of AAV6.2FF-hIgG expressed much higher levels than the mice, with serum hIgG levels of 495-650 µg/mL by day 28 post-administration. Lastly, sheep IM administered 1 x 1013 vg/kg expressed serum hIgG levels of 21-46 µg/mL by day 28 post-administration. Across all animal models, the vector did not appear to impede any health indices, such as weight, proving the safety and quality of the produced vectors. It is well known that AAV-mediated monoclonal antibody (mAb) expression varies considerably depending on the species and the mAb. To the best of the authors' knowledge, there are no reports of AAV-mAb expression in ovine species thus, there is no benchmark for expected expression levels. It is noteworthy that the sheep in this study doubled in weight over the 28-day post-injection period and that the animals in Figure 6 were all transduced with different AAV-mAbs and thus cannot be compared.
Figure 6: Intramuscular delivery of AAV6.2FF-hIgG leads to sustained serum hIgG expression in mice, hamsters, and sheep. Female BALB/c mice (n = 4) were IM administered 5 x 1012 vg/kg of AAV6.2FF-hIgG, Syrian hamsters (2 male, 2 female), were IM administered 1 x 1013 vg/kg of AAV6.2FF-hIgG, and 10-day-old male Dorset lambs (n = 3) were IM administered 1 x 1013 vg/kg. Serum was monitored for hIgG expression over a period of 28 days. Data are represented as the mean ± standard deviation. Please click here to view a larger version of this figure.
Transfection of Cell Culture Chamber | |||||
Protocol | |||||
1. Allow DNA, OptiMEM and PEI to warm to room temperature prior to transfection | |||||
2. Input the number of cell stacks to be transfected (no overage required) | |||||
3. Ensure the DNA concentrations are correct as this will change the volumes required | |||||
Number of Cell Culture Chambers | 1 | ||||
Concentration of transgene plasmid | 1 | mg/mL | |||
Concentration of pDGM6.2FF plasmid | 1 | mg/mL | |||
Amount Per cell culture chambers | Transfection Mastermix | ||||
OptiMEM | 48.1 | mL | 48.1 | mL | |
1:3 ratio pTransgene:pHelper | pTransgene | 475 | µg | 475 | µL |
pHelper | 1425 | µg | 1425 | µL | |
4. Add required volumes of OptiMEM and plasmid DNA to a 50 mL conical tube | |||||
5. Invert 10 times to mix | |||||
3:1 PEI:DNA ratio | PEI Max | 5.7 | mL | 5.7 | mL |
6. Add the required amount of PEI to the 50 mL tube containing the OptiMEM and plasmid DNA | |||||
7. Immeditely close the 50 mL tube and vortex and invert 3–5 times to mix. | |||||
8. Set a timer for 10 min for the PEI complexes to incubate | |||||
9. Move cell stacks to be transfected into the BSC | |||||
10. After 10 min, pour the trasnfection mix into one orange port. | |||||
11. Gently mix liquid throughout the cell stack | |||||
12. Return the transfected cells stack to the incubator ensuring equal volume on each layer | |||||
13. Harvest cell culture chamber 72 h later |
Table 2: Transfection calculator for cell culture chamber. Interactive worksheet to determine correct concentration of pTransgene, pHelper, and PEI for transfection of cell culture chamber.
Table 7: AAV Titration Calculator. Interactive worksheet to determine final concentration of AAV samples from qPCR, expressed as vg/mL. Please click here to download this Table.
The production of recombinant AAV (rAAV) vectors described in this paper uses common materials, reagents, and equipment found in majority of the molecular biology research labs and facilities. This paper allows for high-quality in vitro and in vivo grade rAAV to be produced by the reader. Above all, this protocol for rAAV production, compared to more tedious protocols involving cesium chloride purification, is efficient and avoids the use of ultracentrifugation. Once HEK293 cells have been transfected, purified AAV is ready to use within 5 working days.
During the rAAV production and purification process, many steps can influence the purity and final titer of the vector. The first and most critical step is the health of the HEK293 cells, which has a direct effect on the vector titer. The use of HEK293 cells as opposed to other cell types or systems, such as HEK293T cells, is advantageous in that HEK293 cells have a greater ability to adhere to the plastic coating of plates and cells creating robust cell networks for continual cell growth. It is recommended to monitor cells for mycoplasma contamination and to avoid passaging HEK293 cells past ~40 passages or once their doubling time appears to be slowing down as this will lead to lower AAV yields. Furthermore, confirming cells are approximately 80% confluent prior to transfection ensures that cells are not too sparse or overgrown. With regard to the transfection components, the use of high-quality plasmid DNA (e.g., commercially prepared plasmid DNA) is highly recommended to ensure DNA remains in solution and interacts properly with chemical transfection delivery components, such as PEI. Lastly, the transfection reagent has a significant impact on rAAV yields. Here, PEI is used as the transfection agent. PEI is a stable cationic polymer that delivers exogenous DNA to the nucleus of cells through production of plasmid-polymer complexes, which are then taken up by cells and trafficked to the nucleus through host-cell processes29. PEI-based transfections are quick and easy in comparison to other methods of DNA delivery, including calcium phosphate or lipofectamine. However, the ratio of PEI:DNA must be optimized.
The use of cell stacks opposed to traditional adherent plates provides a more convenient and consistent method for the production of rAAV. Cell stacks involve less technical manipulation during transfection, mitigating the possible dislodgement of cells from the adherent monolayer, allowing for stronger cell networks, and better production of rAAV. Post transfection, the collection of supernatant and lysis of cells is efficient and consistent. To harvest rAAV from cells, physical cell lysis methods such as freeze-thawing are inefficient and inconsistent as there is no way to ensure that all the cells are lysed. Here, a chemical lysis procedure is described. The use of chemical lysis buffer ensures that all the cells are exposed to the lysis agent at a specific concentration, as well as additives such as protease inhibitor to prevent capsid degradation. This method more consistently lyses cells allowing for a more efficient harvest of rAAV. Furthermore, the pelleting and removal of cellular debris eliminates possible contaminants from the crude lysate, which can increase the time and cost of filtering lysate prior to purification.
Though rAAV might be present in high amounts in the crude lysate, the act of capturing and cleaning the rAAV can differ among various purification methods, such as cesium chloride gradients. Here, the use of heparin sepharose affinity chromatography purification provides a rapid and easy method for vector purification that results in ultrapure virus and does not require gradient ultracentrifugation. However, not all AAV serotypes contain heparin-binding domains, so for those serotypes this purification method would not be appropriate. For example, while AAV2 and AAV6 bind heparin sulfate, AAV4 and AAV5 do not30. Though this method is not universal, it is efficient and easy for those capsids that do bind heparin sulfate. Unlike ultra-centrifugation gradients such as iodixanol, all rAAV particles are bound to the membrane of the column until they are eluted using high salt concentration washes, avoiding issues such as mixing of gradients and skill needed to recover fractions from the gradient. Elution through high salt concentration washes allows for controlled and precise elution of the virus into fractions that can be further concentrated. Only concentrating certain fractions of the eluted virus further removes potential contaminants while recovering >97% of the eluted virus. One limitation of heparin sepharose affinity chromatography purification is that it does not distinguish between empty and full particles. Additional analytical ultracentrifugation steps would need to be incorporated in order to remove empty capsids31.
qPCR is a cost-effective and rapid method for determining the number of vector genomes in an AAV vector prep. Though qPCR is a sensitive quantification method, it does not provide any information about the number of infectious particles in the prep. Moreover, the sensitivity of this assay can result in variability as technical errors during pipetting can lead to inter-assay variability. Thus, for any experiment that involves comparing different AAV vectors, it is critical that the vectors be titered on the same qPCR plate. Despite these limitations, qPCR is the most accurate method developed for titering AAV and is currently the most widely used and accepted method for quantification of AAV vectors32. The use of a primer/probe that binds to the SV40 polyA of a rAAV genome is based on the fact that this sequence is conserved across many AAV genome plasmids engineered in the laboratory. Beyond choosing a conserved sequence among the reader’s rAAV genome plasmids, qPCR probes can be designed for all parts of the rAAV genome, including, but not limited to, promoters, transgenes, or post-transcriptional factors.
Assessing the purity and quality of the AAV product is an important step in the production process. Western blotting can be used to detect the VP1, VP2, and VP3 structural proteins that make up the capsid of AAV, as well as any altered forms of VP. VP1, VP2, and VP3 are typically present at a ratio of 1:1:10, but this can vary from 1:1:5 to 1:1:20 depending on the serotype. Therefore, it is critical to determine the ratio for each system empirically, particularly because the N-terminal region of VP1 has been shown to be important for infectivity and transduction33. SDS-PAGE coupled with Coomassie staining is a rather straightforward method for detecting VP1, VP2, and VP3, as well as host cell protein contaminants. The use of fluorescent protein stains such as SYPRO Ruby offer higher sensitivity detection of host cell protein contaminants than Coomassie; however, not all laboratories have access to the imaging equipment required to visualize the gel. Finally, commercial ELISAs can be used to quantify host cell protein contaminants in AAV vectors produced in HEK293 cells.
This protocol provides an in-depth overview of producing high titer, high purity rAAV for serotypes that bind to heparin. In the representative results section, the use of novel AAV6.2FF expressing hIgG in a variety of animal models shows the safety and efficiency of this rAAV in vivo. Though the AAV6.2FF vector was administered IM, these high-quality, high purity viruses can be administered via a variety of different routes in vivo, as possible inflammatory contaminants, such as host-cell protein, have been eliminated through our purification processes.
The authors have nothing to disclose.
Amira D. Rghei, Brenna A. Y. Stevens, Sylvia P. Thomas, and Jacob G. E. Yates were recipients of Ontario Veterinary College Student Stipends as well as Ontario Graduate Scholarships. Amira D. Rghei was the recipient of a Mitacs Accelerate Studentship. This work was funded by the Canadian Institutes for Health Research (CIHR) Project Grant (#66009) and a Collaborative Health Research Projects (NSERC partnered) grant (#433339) to SKW.
0.22 μm filter | Millipore Sigma | S2GPU05RE | |
0.25% Trypsin | Fisher Scientific | SM2001C | |
1-Butanol | Thermo Fisher Scientific | A399-4 | CAUTION. Use under a laminar flow hood. Wear gloves |
10 chamber cellstack | Corning | 3271 | |
1L PETG bottle | Thermo Fisher Scientific | 2019-1000 | |
30% Acrylamide/Bis Solution | Bio-Rad | 1610158 | |
96-well skirted plate | FroggaBio | FS-96 | |
Adhesive plate seals | Thermo Fisher Scientific | 08-408-240 | |
Ammonium persulfate (APS) | Bio-Rad | 161-0700 | CAUTION. Use under a laminar flow hood. Wear gloves |
Blood and Tissue Clean up Kit | Qiagen | 69506 | Use for DNA clean up in section 4.6 of protocol |
Bromophenol blue | Fisher Scientific | B392-5 | CAUTION. Use under a laminar flow hood. Wear gloves |
Cell Culture Dishes | Greiner bio-one | 7000232 | 15 cm plates |
Culture Conical Tube | Thermo Fisher Scientific | 339650 | 15 mL conical tube |
Culture Conical Tube | Fisher Scientific | 14955240 | 50 mL conical tube |
Dulbecco's Modified Eagle Medium (DMEM) with 1000 mg/L D-glucose, L-glutamine | Cytiva Life Sciences | SH30022.01 | |
ECL Western Blotting Substrate | Thermo Fisher Scientific | 32209 | |
Ethanol | Greenfield | P016EA95 | Dilute ethyl alcohol(95% vol) to 20% for section 3.7.4 and 70% for section 6.1.1.1 |
Fetal Bovine Serum (FBS) | Thermo Fisher Scientific | SH30396.03 | |
Glacial acetic acid | Fisher Scientific | A38-500 | CAUTION. Use under a laminar flow hood. Wear gloves |
Glycerol | Fisher Scientific | BP229-1 | |
Glycine | Fisher Scientific | BP381-500 | |
HBSS with Mg2+ and Ca2+ | Thermo Fisher Scientific | SH302268.02 | |
HBSS without Mg2+ and Ca2+ | Thermo Fisher Scientific | SH30588.02 | |
HEK293 cells | American Tissue Culture Collection | CRL-1573 | Upon receipt, thaw the cells and culture as described in manufacturer’s protocol. Once cells have been minimally passaged and are growing well, freeze a subfraction for future in aliquots and store in liquid nitrogen. Always use cells below passage number 30. Once cultured cells have been passaged more than 30 times, it is recommended to restart a culture from the stored aliquots |
HEK293 host cell protein ELISA kit | Cygnus Technologies | F650S | Follow manufacturer’s instructions |
Heparin sulfate column | Cytiva Life Sciences | 17040703 | |
Kimwipe | Thermo Fisher Scientific | KC34120 | |
L-glutamine (200 mM) | Thermo Fisher Scientific | SH30034.02 | |
Large Volume Centrifuge Tube Support Cushion | Corning | CLS431124 | Support cushion must be used with large volume centrifuge tubes uless the centrifuge rotor has the approriate V-bottom cushions |
Large Volume Centrifuge Tubes | Corning | CLS431123-6EA | 500 mL centrifuge tubes |
MgCl2 | Thermo Fisher Scientific | 7791-18-6 | |
Microcentrifuge tube | Fisher Scientific | 05-408-129 | 1.5 mL microcentrifuge tube, sterilize prior to use |
Molecular Grade Water | Cytiva Life Sciences | SH30538.03 | |
N-Lauroylsarcosine sodium salt | Sigma Aldrich | L5125 | CAUTION. Wear gloves |
NaCl | Thermo Fisher Scientific | BP35810 | |
Optimem, reduced serum medium | Thermo Fisher Scientific | 31985070 | |
Pasteur pipets | Fisher Scientific | 13-678-20D | Sterilize prior to use |
PBS (10x) | Thermo Fisher Scientific | 70011044 | Dilute to 1x for use on cells |
Penicillin-Streptomycin Solution | Cytiva Life Sciences | SV30010 | |
pHelper plasmid | De novo design or obtained from plasmid repository | NA | |
Pipet basin | Thermo Fisher Scientific | 13-681-502 | Purchase sterile pipet basins |
Polyethylene glycol tert-octylphenyl ether (Triton X-100) | Thermo Fisher Scientific | 9002-93-1 | CAUTION. Wear gloves |
Polyethylenimine (PEI) | Polyscience | 24765-1 | Follow manufacturer’s instructions to produce a 1L solution. 0.22μm filter and store at 4°C |
Polypropylene semi-skirted PCR Plate | FroggaBio | WS-96 | |
Polysorbate 20 (Tween 20) | Thermo Fisher Scientific | BP337-100 | CAUTION. Wear gloves |
polyvinylidene difluoride (PVDF) membrane | Cytiva Life Sciences | 10600023 | Use forceps to manipulate. Wear gloves. |
Primary antibody | Progen | 65158 | |
Protein Ladder | FroggaBio | PM008-0500 | |
Proteinase K | Thermo Fisher Scientific | AM2546 | |
pTrangene plasmid | De novo design or obtained from plasmid repository | NA | Must contain SV40 polyA in genome to be compatible with AAV titration in section 5.0 |
Pump tubing | Cole-Parmer | RK-96440-14 | Optimize length of tubing and containment of virus in fractions E1-E5 |
RQ1 Dnase 10 Reaction Buffer | Promega | M6101 | Use at 10x concentration in protocol from section 4.0 |
RQ1 Rnase-free Dnase | Promega | M6101 | |
Sample dilutent | Cygnus Technologies | I700 | Must be purchased separately for use with HEK293 host cell protein ELISA kit |
Secondary antibody, HRP | Thermo Fisher Scientific | G-21040 | |
Skim milk powder | Oxoid | LP0033B | |
Sodium dodecyl sulfate (SDS) | Thermo Fisher Scientific | 28312 | CAUTION. Use under a laminar flow hood. Wear gloves |
Sodium hydroxide (NaOH) | Thermo Fisher Scientific | SS266-4 | |
SV40 polyA primer probe | IDT | Use sequence in Table X for quote from IDT for synthesis | |
Tetramethylethylenediamine (TEMED) | Thermo Fisher Scientific | 15524010 | CAUTION. Use under a laminar flow hood. Wear gloves |
Tris Base | Fisher Scientific | BP152-5 | |
Trypan blue | Bio-Rad | 1450021 | |
Ultra-Filter | Millipore Sigma | UFC810024 | Ultra-4 Centrifugal 10K device must be used, as it has a 10000 molecular weight cutoff |
Universal Nuclease for cell lysis | Thermo Fisher Scientific | 88702 | |
Universal qPCR master mix | NEB | M3003L | |
Whatman Paper | Millipore Sigma | WHA1001325 | |
β-mercaptoethanol | Fisher Scientific | 21985023 | CAUTION. Use under a laminar flow hood. Wear gloves |
CAUTION: Refer to the Materials Table for guidelines on the use of dangerous chemicals. |