Transgene Expression in Cultured Cells Using Unpurified Recombinant Adeno-Associated Viral Vectors

1. Plasmid acquisition

NOTE: This protocol will clone a gene of interest (GOI) into an ITR-containing plasmid with a cytomegalovirus (CMV) promoter and a SV40 poly-adenylation sequence (pAAV.CMV.Luc.IRES.EGFP.SV40). However, this plasmid can be used without further cloning to produce rAAVs, packaging a convenient dual EGFP and Luciferase reporter. Plasmids with different regulatory elements or for different applications, such as for CRISPR-based experiments, are available online and will follow similar cloning steps as those below (see discussion for additional cloning steps). Additionally, rep/cap, or trans, plasmids for different serotypes can be used—this protocol will use rep/cap for AAV serotype 2.

1. Purchase bacterial stabs containing an ITR-containing cis plasmid, an adenovirus helper plasmid, a rep/cap plasmid, and a plasmid containing a GOI. Streak the bacteria on individual agar plates containing antibiotics specific to the resistance of each plasmid. Incubate the bacteria at 30 °C overnight to grow.
   NOTE: If a plasmid with a GOI is not readily available for purchase, a synthetic DNA fragment (see Materials Table) can be purchased online. Additionally, a synthetic DNA fragment can be used to skip the entire PCR process if desired.
2. Pick a single colony from each plate and grow in 3 mL of Luria Bertani (LB) buffer supplemented with the corresponding antibiotic at 30 °C overnight, shaking at 180 rpm. Transfer 1 mL of the bacteria into a sterile 250 mL Erlenmeyer flask containing 50 mL of LB buffer supplemented with the corresponding antibiotic. Shake at 30 °C, 180 rpm overnight.
3. Transfer the bacteria to a 50 mL conical tube and centrifuge for 20 min at 3,000 x g, room temperature. Discard supernatant. Isolate the plasmid using an endotoxin-low or -free midiprep kit as per manufacturer's instructions.
   ​NOTE: ITRs are unstable structures. To prevent ITR deletions or mutations, bacterial cells should be grown at 30 °C to slow cellular division and mitigate errors in replication. To increase plasmid yield and purity, a midiprep or maxiprep kit is ideal. It is recommended to use an endotoxin-low or -free kit to reduce downstream endotoxin contamination of cells. It is not necessary to perform plasmid isolation inside a laminar flow hood, as contamination of plasmid preparation is not likely if performed on a standard bench.

2. Cloning gene of interest into AAV ITR-containing plasmid

1. Open the plasmid map containing the GOI in a DNA viewing software.
   NOTE: The full cassette, including the ITRs, should not exceed 4.9 kb due to physical packaging limitations of the capsid. Additionally, PCR amplification cannot be achieved through ITRs due to secondary structures. As such, Gibson assembly is not recommended for rAAV transgene cloning.
   1. Click the Sequence tab to reveal the full sequence of the plasmid. Scroll to the 5' most region of the GOI sequence and click on the First Nucleotide while dragging inward towards the gene body to create a forward primer. Release once a melting temperature (Tm) of ~55 °C has been reached.
   2. Click Forward Primer. Manually include the sequence for an EcoRI restriction site (5' GAATTC 3') at the 5' most end of the primer sequence. Additionally, add six extra random bases at 5' end of this restriction site to allow for the enzyme to efficiently interact with the DNA. Click Add Primer. Refer to Figure 2 for a representative example.
   3. Check the primer to ensure that it cannot form hairpins or primer dimers (except for the restriction site that is palindromic). If hairpins form, change the random sequence of the six extra bases. Additionally, ensure the primer does not bind anywhere else on the plasmid.
   4. Repeat the steps to create a reverse primer at the 3' most region of the GOI inward to the gene body. Click on Reverse Primer and include a NotI restriction site (5' GCGGCCGC 3').
      NOTE: The GOI cannot contain EcoRI or NotI restriction sites – if so, different restriction enzymes will need to be used.
2. Amplify the GOI in a 50 µL PCR reaction. Use the required individual components from Table 1.
   1. Place the reaction in a thermocycler and follow the cycle parameters from Table 2 for programming. If primers have different T_m, use the lower T_m for the program.
   2. Verify amplification of correct PCR fragment by adding 1 µL of gel loading dye (6x) to 5 µL of PCR reaction. Run a DNA ladder and the PCR mixture on a 0.8% agarose gel containing ethidium bromide and visualize with UV light.
      CAUTION: Ethidium bromide is a known mutagen.
   3. If a single, specific band appears on the gel, purify the remaining PCR product in the PCR strip tube using a column-based PCR cleanup kit as per manufacturer's instructions. If multiple bands appear (due to non-specific amplification), excise the desired band, and purify the DNA using a gel extraction kit as per manufacturer's instructions.
3. Digest the purified PCR product and pAAV.CMV.Luc.IRES.EGFP.SV40 backbone plasmid in a 50 µL reaction with the required individual components from Table 3 for 1 h at 37 °C. A larger quantity of pAAV plasmid DNA is required due to expected inefficient recovery following gel extraction.
   NOTE: The restriction enzymes used are high fidelity (HF) versions. Regular NotI cannot be used with CutSmart buffer. If different enzymes are being used, a different incubation temperature and buffer may be required.
   1. Purify the digested PCR product with a column-based PCR cleanup kit as per manufacturer's instructions.
   2. Prepare the digested pAAV.CMV.Luc.IRES.EGFP.SV40 backbone plasmid for gel electrophoresis by adding 10 µL of gel loading dye (6x) to 50 µL of digestion reaction. Load a DNA ladder, digestion reaction, and 250 ng of undigested plasmid (negative control) on a 0.8% agarose gel containing ethidium bromide with wide-teeth wells. Visualize with UV light, excise the desired fragment (~4.5 kb), and purify the DNA using a gel extraction kit as per manufacturer's instructions.
4. Ligate the digested PCR product and digested pAAV backbone plasmid in a 20 µL ligation reaction with the required individual components from Table 4. To calculate the amount of PCR products needed, use Formula 1. Typically, use a 3:1 molar ratio of PCR product (insert) to backbone (pAAV), with a mass of 50 ng backbone.
        Formula 1
5. Perform a negative control by replacing the PCR product with water. Incubate ligation reactions at 16 °C overnight, or at room temperature for 2 h.
6. Thaw a vial of competent, recombination-deficient bacterial cells (such as Stbl3 cells) on ice and place 50 µL into a 1.5 mL tube. Add 3 µL of the ligation reaction directly onto the cells and incubate on ice for 30 min.
   NOTE: ITRs are unstable structures and therefore require recombination-deficient bacterial strains to limit ITR deletion and recombination events. DH5α cells may be used intermittently for propagation (one or two times) but are not recommended for long-term use. ITR integrity should be checked regularly after midiprep purification.
   1. Heat-shock the bacteria in a 42 °C water bath for 30 s then immediately transfer to ice for 2 min. Add 200 µL of LB buffer and shake for 1 h at 30 °C, 180 rpm.
   2. Spread 125 µL of the mixture onto an agar plate with the corresponding antibiotic and incubate overnight (~18 h) at 30 °C. Pick multiple clones and place each into a sterile plastic culture tube with 3 mL of LB containing the corresponding antibiotic. Incubate overnight shaking at 30 °C, 180 rpm.
      ​NOTE: To prevent ITR deletions or mutations, bacterial cells should be grown at 30 °C to slow cellular division and mitigate errors in replication. Additionally, smaller colonies should be picked as those without ITRs may have a growth advantage over others.
   3. Pipette 1.8 mL of each culture into a 2 mL tube and centrifuge at 6,000 x g for 3 min, room temperature. Isolate the DNA using a miniprep kit. Store the remaining 1.2 mL of culture at 4 °C. Verify correct clones by DNA sequencing or diagnostic restriction enzyme digest.
      NOTE: Sanger sequencing of the insert is recommended, but processivity through ITRs is not achievable by standard methods. ITRs should be verified by restriction digest as described below.
   4. Add 50 mL of LB buffer containing the corresponding antibiotic to a sterile 250 mL Erlenmeyer flask. Add 500 µL of the remaining culture to the flask and shake at 30 °C, 180 rpm until an OD₆₀₀ of 0.2-0.5 is reached (~18 h). Transfer the bacteria to a 50 mL conical tube and centrifuge for 20 min at 3,000 x g, 4 °C. Isolate the DNA using an endotoxin-low or -free midiprep kit.
   5. Check ITR integrity with 20 µL of diagnostic restriction enzyme digest using XmaI (or SmaI). Prepare a reaction containing 500 ng plasmid, 0.5 µL of enzyme, and 1x buffer; incubate for 1 h at 37 °C. Run the reaction on a 0.8% agarose-gel. If ITRs are intact, digestion will give a band at ~2.9 kb and another band the size of the cassette. If this distinct band is not observed, the ITR may not be intact, and cloning will need to be redone.
      NOTE: Plasmids containing XmaI (or SmaI) restriction sites (in addition to those in the ITRs) will have additional bands appear on the gel.

Figure 2: Representative example for primer design. Forward and reverse primer design for an mScarlet transgene (representative example). Please click here to view a larger version of this figure.

Table 1: PCR amplification reagents. The required components for a successful PCR reaction.

Table 2: PCR amplification programming. The required cycling parameters for a successful PCR reaction.

Table 3: Digestion reagents. The required components for a successful digestion reaction.

Table 4: Ligation reagents. The required components for a successful ligation reaction.

3. Vector production with triple-plasmid transfection

NOTE: The following values are optimized for a single well of a 6-well plate that yields a final crude preparation volume of 2 mL. All values can be scaled up by 10x for a 15 cm plate that yields a final volume of 20 mL or scaled down by 4x for a 24-well plate with a final volume of 500 µL.

1. Make a 1 µg/µL stock of polyethylenimine hydrochloride (PEI) MAX by dissolving 100 mg of PEI MAX in 100 mL of distilled water. Adjust pH to 7.1 with NaOH. Filter sterilize the mixture with a 0.22 µm filter and freeze 1 mL aliquots at -20 °C for long-term storage. Once thawed, store the reagent for 1 month at 4 °C.
2. Seed 3 x 10⁵ HEK293 or HEK293T cells into a 6-well plate with pre-warmed Dulbecco's modified eagle medium (DMEM, 4.5 g/L glucose, 110 mg/L sodium pyruvate) supplemented with 10% fetal bovine serum (FBS). Grow to ~75%-90% confluency in an incubator set at 37 °C and 5% CO₂ (Figure 3).
   NOTE: Cells cultured with penicillin/streptomycin (P/S) have been observed to decrease the vector production yield. Using proper sterile technique bypasses the need to use P/S, and thus it is recommended not to culture HEK293 cells with antibiotic²⁰. If P/S is required in downstream transductions, it is recommended to add P/S to crude preparation after harvesting.
3. In a 2 mL tube, prepare a mixture containing 1.3 µg pAAV2/2 (Rep/Cap, serotype 2), 1.3 µg pAAV.GOI, 2.6 µg pAdΔF6, and serum-free (SF) DMEM (4.5 g/L glucose, 110 mg/L sodium pyruvate) into a total volume of 100 µL (see Supplementary Table 1 for convenient calculations).
4. Prepare a negative control in a separate tube by replacing pRep/Cap with any unrelated plasmid. The negative control accounts for unpackaged pAAV.GOI plasmid present in the crude preparation that could possibly transfect cells during transduction, although rare.
   NOTE: Endotoxin-low or -free maxiprep or midiprep plasmid is ideal for triple-transfection and generally produces higher titer vector compared to miniprep DNA, although miniprep DNA can be used for quick-and-dirty preliminary testing.
5. Add 5.2 µL of PEI MAX to the plasmid mixture; this is a plasmid:PEI mixture with a ratio of 1:1. Mix well by pulsing 10-15 times on a vortex mixer set to 7. If multiple vector preparations are produced, add PEI to each plasmid mixture at staggered time intervals of 1 min (i.e., preparation 1 at t = 0 min, preparation 2 at t = 1 min, etc.) to allow for sufficient timing to aspirate wells and dilute the reaction in the following steps.
   ​NOTE: A 1:1 ratio of plasmid:PEI has been observed to be optimal. However, individual users can optimize this ratio for maximum efficiency. Refer to the discussion for how to perform PEI optimization (also see Supplementary Table 2).
6. Incubate each tube for exactly 15 min and then dilute the reaction with 1.9 mL of SF DMEM (4.5 g/L glucose, 110 mg/L sodium pyruvate) for a final volume of 2 mL. Gently pipette 2x to mix. Overmixing will disrupt Plasmid/PEI complexes and result in lower transfection efficiency.
7. Aspirate media from well and add plasmid:PEI mixture gently on well sides to prevent cellular detachment. Incubate cells for 72 h at 37 °C and 5% CO₂. Cell lysis and detachment during incubation is a normal process of rAAV production (Figure 4).

Figure 3: HEK293 cells ready for transfection. The ideal confluency (75%-90%) of HEK293 cells needed for triple-plasmid transfection. Please click here to view a larger version of this figure.

Figure 4: HEK293 cells after transfection. The appearance of HEK293 cells before and after 1, 2, or 3 days post transfection with pAAV.CMV.Luc.IRES.EGFP.SV40, pAAV2/2 , pAdΔF6, and 1:1 ratio of plasmid:PEI . Scale bars: 400 µm. Please click here to view a larger version of this figure.

4. Harvesting crude vector preparations

1. Freeze the entire plate of transfected cells for 30 min at -80 °C, followed by a thaw for 30 min at 37 °C. Repeat for a total of three freeze/thaw cycles. Do not remove the lid—the plate must remain sterile inside. Plates can remain at -80 °C until ready to proceed to next steps.
   NOTE: A non-humidified incubator works best for thawing, which minimizes condensation on the outside of plates that can lead to accidental contamination.
2. Perform the following two steps in a laminar flow hood using aseptic techniques. Mix each well by pipetting to ensure maximum disruption of cells. Transfer lysate to a 2 mL tube and centrifuge for 15 min at 15,000 x g, room temperature to remove cell debris.
3. Carefully transfer supernatant to a new 2 mL tube. This crude preparation can be used immediately without titration or purification. Vector can be stored at 4 °C for several months or -20°C for years.
   ​NOTE: If needed, titers may be calculated by quantitative PCR (qPCR) by quantifying the number of vector genomes (VG) inside of DNase-resistant particles. As such, titers are given in units of VG/mL. Vector stored for several months at 4 °C should be re-titrated before use as vector can aggregate and/or bind tube walls, reducing the titer of the preparation.

5. Transduction

1. Plate the desired cell type (e.g., Huh7) in a 96-well plate at a target confluency of 50%-75%, depending on the duration of transduction. Greater confluency may result in reduced AAV transduction efficiency.
2. Add vector (e.g., CMV.mScarlet) to cells without knowing the titer of the crude preparation for applications where dose is not relevant, such as testing a panel of capsids for transduction in a new cell type. Perform a 1:3 dilution series of the crude preparation in serum-free (SF) media to obtain the optimal amount of vector required for the application. Aspirate media from 96-well plate and add 50-100 µL of diluted crude preparation to wells. Incubate cells at 37 °C and 5% CO₂.
   ​NOTE: Serum may contain antibodies that can neutralize the AAV vector and reduce transduction efficiency. However, serum may be used during transduction for sensitive cell types.
3. Remove vector after 48 h post-transduction (hpt) and wash cells once with pre-warmed phosphate buffered saline (PBS). Vectors can also be removed and replaced with serum-containing media as soon as 2 hpt for sensitive cell types. For many applications, perform overnight incubation to transduce cells and replace wells with fresh serum-containing media in the morning. Robust cell types, such as Huh7 and U2-OS, do not require a media change.
4. Terminate transduction by fixing cells in 4% paraformaldehyde (PFA) for 10 min. Various termination methods can also be used based on the application (e.g., lysis). For most cell types, peak expression occurs by 48 hpt and is thus a common experimental endpoint.

6. Variations on transduction method by cell type

NOTE: Different cell types require different culture conditions. Therefore, addition of vector for transduction should be optimized based on the needs of the cell type. Below are very specific transduction protocols for the cell types included in the representative results, illustrating some variations of transduction protocols that a researcher may wish to try for their own needs.

1. Mouse small intestinal organoids
   1. Derive mouse small intestinal organoids (mSIOs) from a small intestinal crypt prep from C57BL/6J mice. Embed organoids in 90% basement membrane matrix and culture in 24-well plates containing either organoid growth medium (without antibiotics) or pre-transduction medium (50% Wnt3a-conditioned medium [produced in-house using L Wnt-3A cells in organoid growth medium], 10 mM nicotinamide, 10 µM ROCK inhibitor, and 2.5 µM CHIR99021) for one passage (5-7 days) before transduction at 37 °C and 5% CO₂.
   2. Prior to transduction, rinse organoids with D-PBS, disrupt basement membrane matrix domes by pipetting, and dissociate organoids into small cell clusters by incubating them in dissociation medium for 10 min at 37 °C and further pipetting.
   3. Stop the cell dissociation process by adding 5% FBS in DMEM/F-12 with 15 mM HEPES, transfer cell clusters into centrifuge tubes and collect cell clusters by centrifugation for 5 min at 1000 x g, room temperature.
   4. Resuspend the cell clusters in transduction medium (pre-transduction medium containing 10 µg/mL polybrene or organoid growth medium containing 10 µg/mL polybrene) and transfer into 48-well plates, followed by the addition of AAV crude preparations packaging CMV.mScarlet for transduction.
   5. Perform transduction of organoids by centrifuging the plate for 1 h at 600 x g and 37 °C and then incubate at 37 °C for an additional 6 h. Collect transduced organoids in 1.5 mL microcentrifuge tubes, centrifuge for 5 min at 1000 x g, room temperature, and put on ice.
   6. After removal of the supernatant, resuspend transduced organoids in 90% basement membrane matrix in pre-transduction medium or organoid growth medium and seed droplets of 20 µL into one well of a pre-warmed chambered cover glass.
   7. After incubation for 10 min in the incubator, embed the droplet in 350 µL of pre-transduction medium or organoid growth medium and incubate at 37 °C in the incubator. Determine the transduction efficiency 2-5 days after transduction by imaging the transduced organoids on a confocal microscope and estimate the percentage of red fluorescent cells per organoid.
2. BeWo cells
   1. At 24 h before transduction, plate human placental choriocarcinoma BeWo cells at 10,000 cells per well of 96-well plate. Dilute crude AAV preparations packaging CMV.mScarlet to 1:1 in serum-free F12-K BeWo medium up to a total volume of 50 µL per well. Aspirate medium from well and replace with 50 µL diluted AAV per well.
   2. Incubate cells at 37 °C overnight (~18 h) and then add 100 µL of F12-K BeWo medium containing 10% FBS to bring up the total volume to 150 µL. Incubate cells for an additional 6 h for a total of 24 h post-transduction (hpt).
   3. For imaging, stain live cells with Hoechst dye for 30 min in F12-K medium, then exchange the medium for phenol-free DMEM containing 10% FBS, 1x glutamax supplement, and 1x sodium pyruvate supplement for imaging. Conduct live-cell imaging on a confocal microscope using DAPI (for Hoechst imaging) and mCherry (for mScarlet imaging) filter sets with 37 °C and 5% CO₂.
3. Hepa1-6 and Huh7 cells
   1. Plate murine Hepa1-6 liver cells and human Huh7 liver cells in DMEM (4.5 g/L glucose, 110 mg/L sodium pyruvate, 10% FBS) at 5,000 cells per well of a 96-well plate 24 h before transduction.
   2. Add 50 µL of undiluted crude AAV preparations packaging CMV.mScarlet directly to cells and incubate at 37 °C for 48 h. Wash cells once in PBS, fix in 4% PFA, and stain with Hoechst dye for 10 min. Conduct imaging on a confocal microscope using the DAPI (for Hoechst imaging) and mCherry (for mScarlet imaging) filter sets.
4. C2C12 and HSkMC cells
   1. Plate undifferentiated murine C2C12 myoblasts and undifferentiated human primary skeletal muscle cell (HSkMC) myoblasts at 5,000 cells per well of a 96-well plate, 72 h before transduction.
   2. Dilute crude AAV preparations packaging CMV.mScarlet to 1:1 in DMEM (4.5 g/L glucose, Penn/Strep, 20% FBS) for C2C12 myoblasts or skeletal muscle growth media for HSkMC myoblasts. Differentiate HSkMC myoblasts to myotubes by changing the media to differentiation media.
   3. Incubate myoblasts and myotubes in diluted AAV preparations for 24 h at 37 °C. Stain live cells with Hoechst dye for 10 min and image on a confocal microscope using the DAPI (for Hoechst imaging) and mCherry (for mScarlet imaging) filter sets.