Construction of Adenoviral Vectors using DNA Assembly Technology

NOTE: Adenoviruses are classified as Biosafety Level 2 (BSL-2); all the steps to use the viruses were carried out in a biosafety level 2 laboratory. The wild-type HAdV-7 was isolated in 2017 from the nasopharyngeal aspirate specimen of a 10-month-old infant who was hospitalized with acute respiratory tract infection in Beijing Children's Hospital²⁰. The virus stocks were stored at -80 °C.

1. Extraction of HAdV-7 genomic DNA

1. Seed 2.0 x 10⁶ of the HEK293 cells in one T-25 flask containing 5 mL of Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS) and culture at 37 °C in a humidified atmosphere supplemented with 5% CO₂. Cells should be 80%-90% confluent and ready for infection in 18-24 h.
2. Inoculate the cells with wild-type HAdV-7 for 2 h. Remove the virus-containing medium by aspiration and add 5 mL of fresh DMEM containing 2% FBS to the cells.
3. Extract the viral genomic DNA with the modified Hirt's method²¹
   1. In 2 or 3 days post-infection, when cytopathic effect (CPE) is observed on 50%-90% of cells, discard the culture supernatant and rinse cells once with phosphate buffered saline (PBS).
      NOTE: Cytopathic effect (CPE) is defined as a phenomenon where the virus-infected cells round up, swell, and loosely attach or detach from the culture flask into grape-like clusters²².
   2. Lyse cells in the flask in 1.0 mL of lysis buffer containing 25 mM Tris-HCl, 0.5 mM EDTA, 50 µg/mL protease K, and 0.8% SDS (pH7.6) for 5 min on ice. Transfer the lysate to a 1.5 mL microcentrifuge tube and incubate the tube in a water bath at 50 °C for 30 min.
   3. Add 120 µL of precipitation buffer to the tube. Place the tube on ice for another 30 min after gently mixing.
      NOTE: The precipitation buffer is composed of 3 M CsCl, 1 M potassium acetate, and 0.67 M acetic acid. It will help precipitate the cellular debris and cellular chromosome DNA after centrifugation while keeping the virus genome in the supernatant.
   4. Centrifuge the tube for 25 min at 15,000 x g at 4 °C, and then transfer the supernatant (about 1.1 mL) to a 15 mL polypropylene tube.
   5. Add 2.2 mL of ice-cold absolute ethanol to the tube. Mix gently and let the tube stand on ice for 15 min. Centrifuge for 5 min at 2000 x g at room temperature.
   6. Aspirate the supernatant carefully and wash the precipitate (virus genomic DNA) once with 800 µL of 70% ethanol. Centrifuge the tube for 6 min at 15,000 x g at room temperature. Discard the supernatant and air-dry the DNA pellet at the bottom. Do not overdry the DNA pellet since it may cause difficulty in redissolving.
   7. Dissolve the pellet in 200 µL of TE buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA), add 1 µL of RNase A (10 mg/mL), and incubate the tube at 37 °C for 15 min. Purify the dissolved DNA by using a commercial genomic DNA clean and concentrator kit (see Table of Materials).
      ​NOTE: Quantitate the viral genomic DNA with the commonly used method in the laboratory. Generally, 1-5 µg of genomic DNA can be obtained in a T-25 flask of HEK293 cells infected with adenovirus.

2. Construction of infectious clone of HAdV-7 by DNA assembly

1. Design PCR primers to amplify the plasmid backbone (Figure 1). Add the overlap sequences that are needed for DNA assembly to the 5' ends of PCR primers. In addition, include non-cutter restriction sites (e.g., Pme I site) on both ends of HAdV-7 genome to help release the viral genome from the infectious clone.
   NOTE: The plasmid backbone containing antibiotic resistant gene and replication origin is amplified by PCR and fused to HAdV-7 genome. Adenoviruses can be rescued from packaging cells with higher efficiency when linearized virus genome is used instead of a circular adenoviral plasmid. After considering above mentioned factors, primers are designed as shown in Figure 1.
2. Perform PCR to amplify a fragment (Kan-Ori) containing kanamycin resistance gene (Kan) and pBR322 origin (Ori) using pShuttle-CMV as the template with primers of Ad7KanF1 and Ad7KanR1 (Table S1). Set the PCR reaction as follows: one cycle at 98 °C for 30 s, then five cycles of denaturing at 98 °C for 8 s, annealing at 68 °C for 30 s, extension at 72 °C for 90 s, and finally 25 cycles of denaturing at 98 °C for 8 s, annealing and extension at 72 °C for 2 min.
3. Run the PCR product in a 1% agarose gel by electrophoresis, and purify the fragment using a commercial DNA gel recovery kit.
4. Add the recovered Kan-Ori and viral genomic DNA of HAdV-7 to the commercial DNA Assembly Master Mix. Set up the assembly reaction in a total volume of 20 µL and a molar ratio of vector to viral genomic DNA of 1:2. Incubate at 50 °C for 1 h.
   NOTE: Set the reaction in a total volume of 20 µL with 1-3 µL of vector, 7-9 µL of viral genomic DNA, and 10 µL of DNA Assembly Master Mix.
5. Transform the chemically competent E. coli strain TOP10 cells with 10 µL of the assembly product using heat shock. Spread the transformation mix onto kanamycin-containing (50 µg/mL) Luria-Bertani (LB-Kan) plates. Incubate plates at 37 °C for at least 12 h.
6. Inoculate at least three clones each in 5 mL of LB-Kan medium at 37 °C for 12 h with gentle shaking (200-250 rpm). Extract plasmid DNA using a commercial Plasmid Miniprep Kit.
7. Digest the plasmid using restriction endonuclease, and then check the size of DNA fragments with agarose gel electrophoresis (Figure 2A). Moreover, confirm the fusion sites by sequencing. The correct assembly plasmid is named pKan-Ad7.
8. Grow a 100 mL culture of bacteria from the correct clone. Extract a maxi-plasmid preparation using a commercial Plasmid Maxprep Kit.

3. In silico construction and modification of an intermediate plasmid

NOTE: Generally, an intermediate plasmid should be constructed, and how to construct an ideal intermediate plasmid is the key step. DNA analysis software with a restriction enzyme module, such as pDRAW32 (a free software, available at www.acaclone.com), is needed for the design of an intermediate plasmid.

1. Import the DNA sequence of pKan-Ad7 to pDRAW32 to draw a plasmid map. Annotate the regions of interest on the map. For pKan-Ad7, E1, E3 regions, and plasmid backbone (Kan-Ori) were annotated.
2. Operate the pDRAW32 program. Click Settings > Enzyme Selection firstly, and then click Min/Max Cuts. Input 1 in the box Min and input 2 in the box Max, and tick the box before in the entire sequence. Lastly, click OK to show the unique and dual cutter restriction enzyme sites on the map.
   NOTE: Generally, only the restriction enzymes that recognize a sequence of 6 bp or longer are selected. As shown in Figure 3A, 17 enzymes match the enzyme selection criteria.
3. Consider the first restriction site that flanks the E3 region since the E3 is planned to be modified. Hpa I, Sal I, Avr II-Mlu I, and Spe I satisfied the requirement.
   NOTE: For this experiment, Hpa I is selected because digestion of pKan-Ad7 with it would generate the shortest fragment carrying the E3 region, and a smaller intermediate plasmid would make future modification easier. The selection of Sal I sites also has advantages-such an intermediate plasmid is suitable for the modification of both E1 and E3 regions (Figure S1).
4. Create a final sequence file displaying both DNA strands of the intermediate plasmid pKan-Ad7HpaI. This virtual sequence can be used as a template to design overlapping primers.
   1. Copy the sequence between the two Hpa I restriction sites including the E3 region. For subsequent assembly, it is necessary to copy each ~20 bp sequence (Tm equal to or greater than 48°C) outside the Hpa I restriction site (Figure 3C, overlap-Final 1 and overlap-Final2).
   2. Add the recognition sequence of Pme I (Figure 3C, gtttaaac) at both ends of the above sequence.
   3. Add the sequence of backbone plasmid containing antibiotic-resistant gene and replication origin (Figure 3C, Kan-Ori) outside of the Pme I restriction site.
   4. Import the final sequence to pDRAW32 to draw a plasmid map. Annotate the E3 region on the map.
   5. Operate the pDRAW32 program. Click Settings > Enzyme Selection firstly, and then click Min/Max Cuts. Input 1 in both the box Min and Max, and tick the box before in entire sequence. Click OK lastly to show the unique restriction enzyme sites on the map. It can be seen that a lot of unique cutters are available for E3 modification.
      NOTE: For this experiment, BstZ17 I and Mlu I are selected for the replacement of partial E3. A dual cutter can also be useful. For example, dual cutter Ssp I together with unique cutter BssH II can be selected for the replacement of the whole E3 region by using overlap extension PCR.
5. Design primers using the virtual sequence as a template for the construction of intermediate plasmid (Figure 3C).
   NOTE: To shorten the length of each primer, four primers were designed. The PCR product, which is amplified with primers of Ad7HpaIF1 and Ad7HpaIR1, would be used as the template to obtain the final PCR fragment with primers of Ad7HpaIF2 and Ad7HpaIR2 (Figure 3C).

4. Rescue of infectious recombinant adenovirus in HEK293 cells

NOTE: The three adenoviruses generated in this article are all rescued according to the following protocol.

1. Digest 10 µg of recombinant adenoviral plasmid with 2 µL of Pme I for 5 h at 37 °C in a total volume of 100 µL to linearize plasmid. Check 1 µL of the digested DNA on a 0.8% agarose gel. Digestion should yield a ~34 kb fragment of the adenoviral genome and a ~2.5 kb plasmid backbone of Kan-Ori.
2. Purify the remnant linearized adenoviral plasmid using the genomic DNA Clean and Concentrator kit.
3. Culture HEK293 cells in DMEM plus 10% FBS. Seed HEK293 cells (2 x 10⁶ cells) in a T-25 flask 1 day before transfection so that they reach 70%-80% confluency the next day. Use 5 µg of Pme I digested adenoviral plasmid to transfect HEK293 cells by using commercially available transfection reagents.
   NOTE: The medium of the transfected cells should be replaced every 3 days.
4. Check the cells every day, collect the cells, and culture medium in a 15 mL polypropylene tube when the comet-foci form (Figure 2B, indicating virus replication).
5. Lyse the cells via three freeze-thaw cycles. After centrifugation for 12 min at 1200 x g at room temperature, collect the supernatant and store it at -80 °C for subsequent virus propagation.
   ​NOTE: When the recombinant adenovirus expresses GFP or other fluorescent proteins, it is easier to identify whether the virus is rescued successfully, as the foci formed by fluorescent protein-positive cells could be observed under a fluorescence microscope intuitively.

5. Construction of E3-deleted HAdV-7 genome plasmid

NOTE: The genome of HAdV-7 is 35,239 bp in length, and the E3 region is located between 27,354 bp and 31,057 bp of the genome. To construct an intermediate plasmid, find an endonuclease with two cutting sites on the infectious clone plasmid, or two endonucleases with a single cutting site on the plasmid. There are Hpa I, Sal I, or Avr II and Mlu I available. The sequence between BstZ17 I (28,312 bp) and Mlu I (30,757 bp) is to be deleted and replaced with the CMV-GFP-PA cassette or CMV-mCherry-PA cassette.

1. Digest 600 ng of pKan-Ad7 with Hpa I. Purify the large fragment pKAd7-HpaI-FL (~30 kb) and small fragment pKAd7-HpaI-FS (~7 kb) respectively, using a DNA gel recovery kit after gel electrophoresis. Store the pKAd7-HpaI-FL at -20 °C.
2. Amplify the fragment containing Kan and Ori using pShuttle-CMV as the template with primers of Ad7HpaIF1 and Ad7HpaIR1 (Table S1) by PCR. Recover and dilute the PCR product (2541 bp) to be used as a template for the second round of PCR reaction to amplify Kan-Ori2 with primers of Ad7HpaIF2 and Ad7HpaIR2.
   1. Purify the PCR product (2584 bp) after resolving in a 1% agarose gel by electrophoresis. Set the PCR reaction as follows: one cycle at 98 °C for 30 s, then 30 cycles of denaturing at 98 °C for 8 s, annealing and extension at 72 °C for 2 min.
      NOTE: The central part of Kan-Ori2 is the fragment containing Kan and Ori, followed by the sequences of a unique restriction endonuclease which does not exist in the small fragment pKAd7-HpaI-FS (e.g., Pme I), and the overlapping region with the large fragment pKAd7-HpaI-FL; the outermost sequences is the overlapping region with small fragment pKAd7-HpaI-FS.
3. Add the PCR product Kan-Ori2 and the recovered pKAd7-HpaI-FS in step 5.1 to DNA Assembly Master Mix. Incubate at 50 °C for 1 h. Follow steps 2.4- 2.6. The correct plasmid is named pKan-Ad7HpaI (~10 kb).
4. Amplify the CMV-GFP-PA fragment (~1.7 kb) containing CMV promoter, GFP CDS, and SV40 polyA signal (PA) using pShuttle-GFP²³ as the template with primers of Ad7E3GFPF1 and Ad7E3GFPR1 (Table S1). Purify the PCR product (1650 bp) after resolving in a 1% agarose gel by electrophoresis.
   NOTE: Overlapping regions that can be assembled with BstZ17 I and Mlu I digested pKan-Ad7HpaI should be included in the primers, and a unique restriction site (e.g., Sbf I) which does not exist in the plasmid pKan-Ad7 is added into the primers for further restriction-assembly.
5. Digest 300 ng of pKan-Ad7HpaI with BstZ17 I and Mlu I. Purify the fragment of 7.3 kb (most of the E3 regions are deleted) after resolving in a 1% agarose gel by electrophoresis. Add the recovered 7.3 kb fragment and the recovered CMV-GFP-PA fragment (~1.6 kb) to DNA Assembly Master Mix. Follow steps 2.4-2.6. The correct plasmid is named pKAd7-E3GFP (~9 kb).
   NOTE: All the sequences between BstZ17 I and Mlu I belong to the E3 region.
6. Digest 300 ng of pKAd7-HE3GFP with Pme I. Purify the large fragment (~6.4 kb) using a DNA gel recovery kit after gel electrophoresis. Add the purified fragment and pKAd7-HpaI-FL recovered in step 5.1 to DNA Assembly Master Mix. Incubate at 50 °C for 1 h. Follow steps 2.4- 2.6. The correct plasmid is named pKAd7-E3GFP.
7. Transfect HEK293 cells with Pme I-linearized pKAd7-E3GFP as described in section 3. The rescued adenovirus is named Ad7-E3GFP.
8. Amplify the CMV-mCherry-PA fragment (~1.6 kb) containing CMV promoter, mCherry CDS, and SV40 polyA signal using pKFAV4-CX19A as the template with the primers of Ad7E3CHEF1 and Ad7E3CHER1. Purify the PCR product after resolving in a 1% agarose gel by electrophoresis.
9. Digest 600 ng of pKAd7-E3GFP with Sbf I, and purify the fragment of ~35 kb. Add the 35 kb fragment and the recovered CMV-mCherry-PA fragment to DNA Assembly Master Mix. Incubate at 50 °C for 1 h. Follow steps 2.4-2.6. The correct plasmid is named pKAd7-E3CHE.
10. Transfect HEK293 cells with Pme I-linearized pKAd7-E3CHE as described in section 3. The rescued adenovirus is named Ad7-E3CHE.

6. Amplification and purification of the recombinant adenovirus in HEK293 cells

1. Inoculate an 80%-90% confluent monolayer of HEK293 cells grown in eight 150 mm diameter TC-treated culture dishes with adenovirus. Incubate at 37 °C for 2 h under mild agitation. Replace the supernatant with 25 mL of DMEM containing 2% FBS.
2. Incubate the cells at 37 °C for 2-3 days.
   1. Discard most of the supernatant of the eight 150 mm diameter dishes when a CPE affects the majority of cells. Harvest infected cells using cell lifters with a final elution volume of ~6.0 mL into 15 mL polypropylene tubes.
   2. Disrupt the cells by three freeze-thaw cycles to release the virus. Clear the lysate by centrifugation for 12 min at 1200 x g at room temperature. Collect the virus-containing supernatant.
3. Add MgCl₂ (0.1 mM) and benzonase nuclease (50 U/mL) to the supernatant. Vortex gently for 40 min at 37 °C. Add NaCl (600 mM) to the supernatant and vortex gently for another 40 min at 37 °C. Centrifuge for 12 min at 1200 x g at room temperature. Collect the virus-containing supernatant for purification.
4. Prepare cesium chloride (CsCl) with different concentrations of 1.35 g/mL, 1.30 g/mL, and 1.25 g/mL with 10 mM Tris-HCl.
   1. Add 0.8 mL of 1.35 g/mL CsCl solution into the bottom of a 5.1 mL Ultra-Clear tube using a pipette, and then slowly add 0.8 mL of 1.30 g/mL CsCl solution and 0.8 mL of 1.25 g/mL CsCl solution on top of the first solution sequentially.
   2. Lastly, carefully overlay ~2.5 mL of the supernatant collected in step 6.3 on the top of 1.25 g/mL CsCl solution, and fill the tube to 2-3 mm from the top using 10 mM Tris-HCl.
5. Centrifuge for 2 h at 200,000 x g and 8 °C in a swinging-bucket rotor with slow acceleration and deceleration to separate the intact viral particles from the defective viral particles.
   NOTE: After centrifugation, several white bands can be clearly seen in the CsCl solution.
6. Carefully remove the bands of the empty particles and the layer of cell debris using the pipette, and collect the lowest band containing mature virus using a 2 mL syringe.
7. Transfer the collected virus suspension to a 20 K MWCO dialysis cassette, and dialyze in dialysis buffer containing 10 mM Tris-HCl, 150 mM NaCl, 1 mM MgCl₂, and 2% glycerol (pH 8.0) overnight at 4 °C.
   1. Exchange the dialysis buffer containing 10 mM Tris-Cl, 150 mM NaCl, 1 mM MgCl₂, and 5% glycerol (pH 8.0) the next day and dialyze 5 h at 4 °C.
   2. Collect the dialyzed virus particles using a 2 mL syringe. Prepare multiple aliquots of desired small volumes (20-200 µL) and store the purified virus at -80 °C.
      ​NOTE: Particle titer of the purified virus can be determined by measuring the content of genomic DNA, and infectivity titer can be determined on HEK293 cells by counting GFP-positive cells with the limiting dilution assay.

7. Identification of recombinant adenovirus genome by restriction enzyme digestion

1. Infect an 80%-90% confluent monolayer of HEK293 cells grown in one T-25 flask with recombinant adenovirus. Incubate for 2 h at 37 °C. Replace the culture medium with fresh DMEM containing 2% FBS.
2. Remove the culture supernatant and extract the viral genomic DNA with the modified Hirt's method described in step 1.3 when CPE occurrs within 2-3 days.
3. Digest the genomic DNA of the recombinant adenovirus with indicated enzymes, and analyze on agarose gel electrophoresis. The recombinant adenoviral plasmid served as a control.