- protocol
- AAV Production in HEK293T Cells
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introduce
This protocol can be used from aFive-compartment battery pack (CF5)(3,180 cm2- Same surface area as 21 x T-150 flasks). The battery pack provides an efficient means of scaling up without having to deal with a large number of T-150 flasks.
Last Updated: May 24, 2019
Workflow Timeline
Day 0:Seed cells in CF2
Day 2:Seed cells in CF5
Day 3 (morning):transfected cells
Day 7 (morning):harvest cells
equipment
- biological safety cabinet
- pipette
- pipette
- pipette
- incubator
- sterile stir bar
Reagent
- Adherent HEK293T cells (preferably AAV-293T clones)
*expert tip*Although adherent, these cells are very loosely attached to the dish surface and should be handled with care. Avoid touching cells when changing media.
- T-150 Flask (Corning, 150 cm2)
- Five-compartment battery stack (CF5, Corning, 3180 cm2)
- Heat-inactivated fetal bovine serum (HI-FBS)
*expert tip*Different brands and amounts of FBS can promote or inhibit transfection. Test various brands and tons of FBS to find one that fits your plan. FBS can be purchased heat-inactivated, or can be inactivated by heating to 56°C for 30 minutes in the laboratory.
- 0.45 μm polyethersulfone (PES) filter system
*expert tip*Do not use filters made of materials other than PES. AAV particles will adhere to many other surfaces, but not PES. Using the PES filter will maximize thepotency.
- DMEM high glucose with L-glutamine and 1 mM pyruvate
- optimize memory
- 0.05% Trypsin/EDTA
- 1x PBS pH 7.4
- 1 mg/mL polyethyleneimine (PEI) 25 KDa MW.
*expert tip*Other transfection reagents may be used in this protocol, but their conditions must be optimized.
- Transfection plasmid: pHelper; pRC (Rep-Cap),Plasmids expressing your gene of interest
- Triton X-100
- Benzonase / DNAse I
- 40% Polyethylene Glycol 8000 (PEG)
- RNase free
- pipette
Reagent preparation
DMEM complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine. In a 500 mL DMEM high glucose bottle, add 55 mL of heat-inactivated FBS and 11 mL of 200 mM L-alanyl-L-glutamine. Store at 4°C.
1 mg/mL polyethyleneimine (PEI) solution:
- Dissolve 100 mg of PEI powder into 100 mL of deionized water.
- While stirring, hydrochloric acid was slowly added until the solution was clear.
- Check the pH of the solution and adjust the pH to 7.0 with hydrochloric acid or sodium hydroxide.
*expert tip*The pH of this solution can drift fairly quickly after the addition of an acid or base. Add only a few drops at a time. Let them mix and recheck the pH to prevent going above or below the desired pH.
- Let the solution mix for 10 minutes, then recheck the pH to make sure it has not drifted.
- Filter the solution through a 0.22 µm membrane.
- Aliquot 500-1000 µl into sterile tubes.
- Store the tubes at -80 °C.
- Once thawed, the solution can be stored at 4°C for up to 2 months. After 2 months, discard the tube and thaw the new working stock.
40% polyethylene glycol (PEG) 8000 solution:
(Video) AAV Transfer Plasmids - Viral Vectors 101- Dissolve 400 g of PEG 8000 and 24 g of NaCl into ddWater and adjust to a final volume of 1,000 mL.
- Stir at room temperature until completely dissolved.
*expert tip*This step is challenging due to the high viscosity of PEG. Stirring over medium heat will promote faster dissolution.
- Adjust the pH to ~7.4.
- Autoclave or sterile filter.
*expert tip*Stirring during cooling is recommended, otherwise the solution may separate into phases.
- Aliquot and store at 4°C.
Considerations before starting
The health of HEK293T cells is critical for optimal AAV production.
- Don't overgrow your cells. Passage the cells twice a week during the maintenance phase and do not allow the cells to reach 100% confluency (80-90% is ideal).
- Passage and plate cells the day before transfection.
- Thaw a new vial of cells after 30 passages.
program
- Trypsinize and resuspend HEK293T packaging cells from 2 x T-150 flasks. Cells should be at ~80% confluency. For each T-150 flask:
- Aspirate the medium and rinse once with 10 mL of PBS.
- Aspirate the PBS and add 4 mL of 0.05% trypsin/EDTA. Wait ~2 minutes.
- Neutralize trypsin by adding 4 mL of HI-FBS.
- Pipette vigorously back and forth several times to obtain a single cell suspension (no cell clumps).
- Collect cells from 2 x T-150 flasks. Adjust the volume to 200 mL with DMEM complete medium and mix.
- Seed all cells in 1 CF2. Return to the incubator for 48 h.
- Trypsinize and resuspend cells in CF2. Cells should be at ~80% confluency.
- Aspirate the medium and rinse once with 60 mL of PBS.
- Aspirate the PBS and add 35 mL of 0.05% trypsin/EDTA.
- While waiting for the cells to lift, aliquot 5 mL of HI-FBS into sterile 100 mL plastic bottles.
- Gently tap the side of the CF2 to help detach the cells, then transfer the cells to a bottle containing 5 mL of HI-FBS to neutralize the trypsin.
- Rinse CF2 with 30 mL of DMEM complete medium and pool and use the cells harvested in the previous step.
- Pipette vigorously back and forth to obtain a single cell suspension (no clumps).
- Add 435 mL of complete medium (final volume 500 mL).
- Seed 250 million cells from CF2 into 1 CF5. Return to the incubator for 24 h - 36 h.
- To transfect:
Calculate the amount of each plasmid required to have a 1:1:1 molar ratio and 2 mg total DNA per CF5
plasmid Plasmid size (bp) DNA concentration (µg/µl) DNA volume (μl) representative cap 7,265 1.00 727.6 helper 11,854 1.00 1,185 transfer plasmid 5,842 people 1.00 584.2 Total BP 24,961 In total we need 2.5 mg of DNA or 2,500 micrograms.
Using the total number of base pairs for all three plasmids, we can determine the total μg/bp required to achieve a 1:1:1 molar ratio of each plasmid:
2,500 micrograms/24,961 bp = 0.10 micrograms/bp
Therefore, for each plasmid we need:
Sample calculation
Representative cap:0.10 micrograms/bp × 7,265 bp = 727.6 micrograms
Required volume: 727.6 μg/ 1.0 μg/μl = 727.6 μl
helper:0.10 micrograms/bp × 11,854 bp = 1185.4 micrograms
Required volume: 1185.4 μg/ 1.0 μg/μl = 1185.4 μl
Transfer plasmid:0.10 micrograms/bp × 5,842 bp = 584.2 micrograms
(Video) AAV Titration by qPCRRequired volume: 584.2 μg/ 1.0 μg/μl = 584.2 μl
(Video) 2) Adeno Associated Virus (AAV) - Production and Modification of AAV- Aliquot 176 mL of OptiMEM into 250 mL sterile bottles.
- Aliquot 350 mL of DMEM + 2% HI-FBS into sterile 500 mL bottles.
- Add the plasmid DNA to the bottle containing Opti-MEM. Mix well.
- Add 7.5 mL of PEI (1:3 μg DNA to μg PEI ratio). Shake the bottle vigorously up and down for 30 seconds (bubbles are okay).
- Incubate for 15 min at room temperature. Note that longer incubation times will reduce transfection efficiency.
- Add the OptiMEM + DNA + PEI solution to the bottle containing 350 mL DMEM + 2% HI-FBS. Mix well.
- Remove the CF5 from the incubator and pour the medium into a waste container.
- Carefully add OptiMEM + DNA + PEI solution to CF5. Make sure that all five layers are covered with medium. 293T cells are fragile and detach easily - added media should always be kept away from the cells (not poured over them) and can be adjusted by carefully tilting the CF5 back and forth.
*expert tip*To help distribute media between the five layers, tilt the CF5 so the media faces the lid. If the media touches the lid, replace with a new one before placing the CF5 in the incubator.
- Return the CF5 to the incubator for 96 h. This incubation time can be adjusted according to the serotype. Typically AAV2 is harvested at 48-72 hours, while other serotypes are harvested at 96-120 hours.
- Filter through a 0.45 µm PES membrane.
- Add 25 mL of PEG solution per 100 mL of supernatant. Divide into 2 x 500 mL sterile bottles as needed.
- Add a stir bar and stir slowly at 4°C for 1h, then stand at 4°C for 3h without stirring to fully precipitate. The pellet of virus can be incubated overnight at 4 °C if desired.
- Transfer the entire sample to a 50 mL conical tube and centrifuge at 2,818 g for 15 min at 4 °C.
- Discard the supernatant and resuspend the virus (small pellet) in 10 mL of PBS + 0.001% pluronic F68 + 200mM NaCl. Pipette back and forth to completely resuspend the virus.
- Keep the resuspended pellet on ice.
- Resuspend and lyse the cells by adding 10 mL of PBS + 0.001% pluronic F68 + 200mM NaCl and sonicating 4 x 1 s pulses with at least 15 min on ice between each pulse at 50% amplitude. Return to ice between each sonication to avoid overheating of the sample.
- Pellet cell debris by centrifugation at 3,220 g for 15 min at 4 °C.
- Transfer the cleared lysate to the tube containing the resuspended virus from step 12 above.
Note: A portion of the solution can be reserved for qPCR to determine vector loss due to purification. Note that reagents present in the medium may affect PCR efficiency.
sample
FAQs
How long does AAV production take? ›
The entire AAV production process takes ~4-5 weeks starting from AAV cloning.
How do you increase AAV yield? ›AAV production yields vary depending on the serotype and the gene of interest. The best way to increase production of a given AAV is to go over critical parameters that directly have an impact on the yield: plasmid DNA, transfection reagent, cells and medium.
Why is AAV replication deficient? ›During the AAV packaging process, many particles that lack the genomic DNA are formed. These particles lack the ability to transduce the cells they come into contact with and are therefore non-functional.
Is AAV replication incompetent? ›AAV is, however, naturally replication-incompetent and requires additional genes from a helper virus infection, which in nature is generally complemented by Ad or HSV coinfection.
What are the steps in AAV production? ›There are multiple strategies to develop AAV vectors, but typical steps include plasmid development and production, cell expansion, plasmid transfection, viral vector production, purification, and fill and finish.
How long is AAV stable at 4c? ›This study examined the stability of AAV serotype 1 (AAV1) vectors under different conditions. First, transducibility after storage at 4°C decreased 20% over 7 weeks. Over 10 freeze–thaw cycles, the resulting transduction efficiency became variable at 60–120% of a single thaw.
Which cell line is best for AAV production? ›HEK293 cells are most frequently used for rAAV production due to the expression of adenoviral E1a/b genes (Graham et al., 1977) which are essential for AAV production (Richardson and Westphal, 1984).
How much AAV do you inject? ›Inject the AAV vector at a steady rate of ~1 mL/min. Once the AAV vector is injected, carefully remove the needle and apply pressure to the injection site to avoid bleeding (see Note 11).
What is the limit of AAV gene? ›Adeno-associated virus (AAV) has many features which make it a great viral vector, but its packaging capacity is limited to ~4.7kb, or roughly half the packaging limits of lentiviral and adenoviral vectors.
What is a major disadvantage of AAV vectors? ›A major limitation of using AAV vector is a relatively small transgene size (4 kb), a necessity of helper Ad or herpesviruses for successful transduction, and integration into the genome of target tumor cells as well as host cells that could lead to mutagenesis.
What are the disadvantages of AAV? ›
As with LV vectors, AAV vectors come with several drawbacks that affect their applications and efficiency. Firstly, AAV vectors are limited by their restricted capacity for insertion of transgene DNA; because of their relatively small transgene size, they are unable to deliver genes larger than 4.8 kilobytes.
Can AAV replicate on its own? ›As the genus name suggests, AAV can only replicate in the presence of helper factors, which are provided by coinfections by helper viruses from the herpesvirus family (e.g., HSV-1 and human cytomegalovirus, HCMV), adenoviruses (e.g., AdV5), and papillomaviruses (e.g., human papillomavirus type 16, HPV-16), as well as ...
What are potential disadvantages of using AAV vectors for gene therapy? ›Disadvantages to using the Adenovirus as the vector in gene therapy include non- integration, immunogenicity, replication competence, no targeting, and small insert size.
How long is AAV stable at room temperature? ›Environmental Stability: AAV is stable at temperatures ranging from 4-56˚C and at pH 5.5-8.5. AAV has been shown to remain infectious for at least one month at room temperature after desiccation.
What is a major disadvantage of adenovirus compared to AAV vectors? ›Although adenovirus benefits a great deal of disease therapies, it does present some drawbacks. 1) The major drawback is its limited cloning capacity (less than 4.7kb) of the vector, which restricts its use in gene delivery of large genes.
What plasmids are needed for AAV production? ›- the packaging plasmid which contains the AAV structural and packaging genes,
- the adenoviral helper plasmid which contains the proteins needed for the virus to replicate,
- and the transfer plasmid which contains the viral genome.
The VP composition of the AAV capsid is estimated to be a molar ratio of 1:1:10,4,5 indicating that one AAV capsid likely has 5 VP1, 5 VP2, and 50 VP3.
Which engineering method improve the efficiency of AAV? ›Vector engineering can increase AAV transduction efficiency (by optimizing the transgene cassette), vector tropism (using capsid engineering) and the ability of the capsid and transgene to avoid the host immune response (by genetically modifying these components), as well as optimize the large-scale production of AAV.
What pH is AAV stability? ›AAV1, AAV2, and AAV8 were shown to be most stable at pH 5.5 while AAV5 was most stable at pH 7.5.
What temperature should AAV be stored at? ›AAV Virus Handling & Storage
All viral vectors are shipped frozen on dry ice and should be stored at -80° C upon receipt and for long term storage. Vectors can be stored for short periods of time at -20 or +4°C.
Is AAV bsl1 or bsl2? ›
Historically, the IBC has assigned all work with AAV/rAAV to Biosafety Level 2 (BSL-2) or Animal Biosafety Level 2 (ABSL-2).
What buffers for AAV? ›Recombinant AAV stocks are provided in formulation buffer containing PBS 1X, 35mM sodium chloride, 0.001% pluronic F-68 and 5% glycerol (Final NaCl is 172mM). Never dilute the viral sample in low salt buffer, water or ethanol. We strongly recommend that you test the virus prior to diluting it.
What is the most common AAV? ›AAV Serotypes
Eleven serotypes of AAV have thus far been identified, with the best characterized and most commonly used being AAV2.
AAV2 is probably the most widely used AAV serotype for in vitro and in vivo gene delivery. However, many other serotypes of AAV have been isolated, and every year there are new serotypes being discovered.
Does AAV need to be higher than drain? ›An AAV should be located within the maximum developed length permitted for the vent. It must be located a minimum of 4” above the horizontal branch drain, 6” above any insulation material and within 15 degrees of vertical.
How high above trap should AAV be? ›To work properly, the bottom edge of the AAV needs to be at least 4 inches above the top of the drain trap.
How many fixtures can an AAV vent? ›AAVs can be used to service an individual fixture or several - what's permitted depends on local code, and the model being used - units like the Redi-Vent can accommodate a mere 20 DFUs (Drainage Fixture Units) while large units like the Maxi-Vent can handle up to 500 DFUs.
What is the 260 280 ratio for AAV? ›260/280 Ratio
The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA.
What can AAV titers tell you? Physical titer: the concentration of viral particles containing viral genomes. Physical titers are measured by quantifying the concentration of viral genomes (by qPCR or other DNA quantification methods - see below), since each viral particle typically contains one viral genome.
What is the AAV titer protocol? ›The AAV Titration protocol can be used to determine the number of genome-containing particles of an AAV prep using SYBR green technology. This protocol was validated using an internal reference AAV of known titer, 100837-AAV1, and by measuring the titer of samples obtained from academic viral vector cores.
How long do AAV vectors last? ›
Due to the high stability of the capsid, AAV can remain infectious for at least a month at room temperature following simple dessication or lyophilization.
Is AAV better than lentivirus? ›AAVs are smaller particles than LVs, which gives them advantage on their spreading efficiency within particular tissues. But this benefit also brings a major drawback limiting the size of the expression cassette to 4,5 kb max whereas a lentiviral vector can carry a 10kb insert.
What is AAV failure? ›American Express Advanced Verification (AAV) Results
Either: No results have been returned to us; The service is temporarily unavailable; The transaction is high risk and has been stopped by the Risk Management service before proceeding to authorisation.
| Virus | Expression | Immune Response |
|---|---|---|
| Lentivirus | Stable | Low |
| AAV | Transient or Stable* | Very Low |
| Adenovirus | Transient | High |
| γ-Retrovirus | Stable | Moderate |
The isoelectric point (pI) of empty and full AAV capsids is around 6.3 and 5.9, respectively. Therefore, AEX separation of AAV6. 2 was evaluated in the pH range from 7.5 to 9.0. At higher pH, AAV was more negatively charged, which resulted in longer retention time and better separation (Figure S1).
How much DNA is in AAV? ›Simply put, AAV is a protein shell surrounding and protecting a small, single-stranded DNA genome of approximately 4.8 kilobases (kb).
Is AAV RNA or DNA? ›AAV is a small (25-nm), nonenveloped virus that packages a linear single-stranded DNA genome. It belongs to the family Parvoviridae and is placed in the genus Dependovirus, because productive infection by AAV occurs only in the presence of a helper virus, either adenovirus or herpesvirus.
Does AAV need helper virus? ›Adeno-associated virus (AAV) is a member of the parvovirus family of single-stranded small DNA viruses that require a helper virus such as adenovirus or herpes simplex virus for replication (Siegl et al., 1985).
What are the limitations of viral vector gene therapy? ›The main drawbacks of using virus vectors are its immunogenicity and cytotoxicity. The first related fatality of gene therapy clinical trial was related to the inflammatory reaction to the viral vector (Adenovirus).
What is one of the dangers in gene therapy using Adenovirus vectors? ›Gene therapy with viral vectors has been successful, but it does carry some risk. Sometimes the virus triggers a dangerous immune response. In addition, vectors that integrate the genetic material into a chromosome can cause errors that lead to cancer.
What are the cons of viral vectors? ›
General Cons of Viral Vectors:
Immune response- Despite not carrying the ability to infect the host with the virus's original infectious material, the foreign nature of viral vectors can elicit an immune response – both innate and adaptive.
Q: How long will it take to detect expression? A: GFP expression is typically detectable 48 hours after infection with AAV. Expression of most genes is expected within 2-7 days after in vitro infection; however protein expression levels may vary based on the protein being expressed, the promoter, and the cell type.
Does AAV go to the nucleus? ›Studies of AAV biology reveal that AAV accumulates in the perinuclear region of cells, presumably unable traffic into the nucleus. The mechanism by which AAV enters the nucleus is largely unknown.
Why is AAV better than adenovirus? ›...
Adenovirus vs. AAV.
| Adenovirus | AAV | |
|---|---|---|
| Protein Expression | High | Low |
| Gene Expression | Transient | Potentially Long Lasting |
| Target Cell's Immune Response | High | Very Low |
| Onset of Expression | 16-24 Hours | 2-7 Days (in vitro) 3-21 Days (in vivo) |
The major disadvantage is that adenoviral proteins are highly immunogenic and many people have circulating antibodies and cellular immune responses toward the virus. Other drawbacks are that transgene expression is relatively unstable because the virus remains episomal, and that the virus may be toxic at high doses.
What is the maximum length of DNA that can be inserted with adenovirus? ›| Viral system | Adenovirus (Ad5) | Retrovirus |
|---|---|---|
| Biosafety level | BSL-2 | BSL-1/2 |
| Insert size | 8–36 kb | 8 kb |
| Max titer (particles/mL) | 1 × 1013 | 1 × 109 |
| Tropism | Broad, low for blood cells | Broad (pan or psuedo-typed) |
Time after infection. An adequate amount of time needs to pass between injection and tissue processing to detect AAV-mediated gene expression. Timing will highly depend on the capsid type and on the tissue you're infecting. Waiting ~2 weeks is a good starting point for many tissues.
What is the life cycle of AAV? ›Adeno Associated Virus (AAV) AAV transduces cells through several stages: (1) viral binding to cell surface receptor/coreceptor, (2) endocytosis of the virus, (3) intracellular trafficking of the virus through the endosomal compartment, (4) endosomal escape of the virus, (5) intracellular trafficking of the virus to ...
How are AAVs produced? ›AAV viral vectors are produced from packaging cell lines following transfection of the AAV construct and the co-infection with a helper virus, such as adenovirus (Ad) or Herpes Simplex Virus (HSV) or via a single infection with a recombinant helper viral vector containing the rAAV genome.
How is AAV vector produced? ›In the absence of helper virus coinfection, AAV genome is either integrated in the host genome or maintained as double stranded circular episomes (3–5). Recombinant AAV vector is generated by replacing the wild type AAV open reading frames with a target (therapeutic or marker) gene expression cassette.
How long does it take to complete the entire process of gene expression? ›
Consistent with the PCA analyses, most expression changes occur from 0 to 120 min. For most genes, a new steady-state expression level is established by 120 min, as shown by minor expression changes between 120 and 240 min.
How long does AAV gene expression last? ›and Xiao et al. showed that an AAV vector would continue to express its transgene for 6–12 months in vivo. Subsequently, expression from an AAV vector in a canine eye persisted unabated for up to 12 years (William Hauswirth, unpublished), and similar results have been reported for muscle and brain transductions.
How long does AAV transduction last? ›On average, AAV needed 1.2 s from the first contact to the cell membrane to the final entry. After 2 minutes, AAV was observed in the cytoplasm. 15 min later, AAV was detected in the nucleus of half of the infected cells. 102 trajectories of viral particles in the nucleus were analyzed.
What is the recovery rate of AAV? ›Ours kits result in a higher purity compared to ultracentrifugation, which is critical when using AAV in vivo. 3. Our AAV purification kits have a 60% or greater yield; ultracentrifugation has about 40% recovery.
What are the drawbacks of AAV? ›As with LV vectors, AAV vectors come with several drawbacks that affect their applications and efficiency. Firstly, AAV vectors are limited by their restricted capacity for insertion of transgene DNA; because of their relatively small transgene size, they are unable to deliver genes larger than 4.8 kilobytes.
What cells are used for AAV production? ›The health of the HEK293T cells is critical for optimal AAV yield.
Does AAV have DNA or RNA? ›AAV is a helper-dependent parvovirus with an approximately 4.7 kb, single-stranded, linear DNA genome that contains two open reading frames encoding the replication and capsid proteins (rep and cap, respectively) [1,2].