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. Author manuscript; available in PMC: 2022 Oct 20.
Published in final edited form as: Methods Mol Biol. 2022;2455:343–358. doi: 10.1007/978-1-0716-2128-8_26

Generation of Adenovirus for In Vitro and In Vivo Studies of Hepatocytes

Yangyang Liu 1,2, Simiao Xu 2,3, Yun Liu 2,4, Yashaswini Kelagere Mayige Gowda 5, Ji Miao 6
PMCID: PMC9582736  NIHMSID: NIHMS1840723  PMID: 35213006

Abstract

Although non-alcoholic steatohepatitis (NASH) can progress to liver cancer and liver failure, no FDA-approved drugs exist to treat NASH. Deciphering the molecular mechanisms underlying the pathogenesis of NASH will facilitate the development of effective treatments for NASH, and requires loss- or gain-of-function experimental approaches. While genetically modified animals provide important information about the function of a gene, adenovirus is a fast, effective, and versatile tool that allows transient knockdown, knockout, or overexpression of one or more genes of interest (GOIs) in primary hepatocytes in vitro and in mouse liver in vivo. In addition, adenovirus is a promising treatment method in preclinical animal models, including rodents and non-human primates, and is used in many clinical trials. Here, we describe a step-by-step protocol to generate adenovirus for basic medical research. We discuss critical steps during virus propagation and purification and provide notes about how to avoid common pitfalls.

Keywords: Virial construction, Gene transduction, Gain- and loss-of-function, Gradient centrifugation

1. Introduction

Adenovirus is a non-enveloped virus that carries an icosahedral protein capsid and a linear double-stranded DNA genome ranging from 26 to 45 kb [1]. To date, more than a hundred human adenovirus genotypes have been identified and are classified into seven categories (http://hadvwg.gmu.edu) [2, 3]. The infection of a mammalian cell by adenovirus is highly efficient and is mediated by a cell-surface receptor and the virus capsid fiber, followed by endocytosis [4]. The viral DNA then enters the nucleus after disassembling the capsid [5, 6]. In the nucleus, the viral DNA remains epichromosomal and is not incorporated into the host genome [7].

Adenoviral vectors have become a versatile tool in preclinical research and have shown great promise in gene therapy, due to several advantages: (a) high transduction efficiency, both in quiescent and dividing cells, (b) epichromosomal persistence in the host cell and high genomic stability, (c) large cargo capacities, and (d) transient manipulation of one or more GOIs [3, 8]. In fact, more than 500 adenoviral-mediated gene therapies are currently under investigation in clinical trials, accounting for over half of ongoing viral vector clinical trials worldwide (Wiley database on Gene Therapy Trials Worldwide) [3, 9].

The most used adenoviral vectors are derived from human serotype Ad5. Deletion of the adenoviral early region 1 (E1) gene alone, or E1 and E3 genes, leads to the development of replication-deficient first-generation of Ad5 viruses that are safely and widely used in research [3, 9, 10]. The generation of the adenoviral vector requires homologous recombination between a shuttle plasmid containing a cassette for gene overexpression or knockdown/knockout and a plasmid expressing adenoviral components. The generation of adenoviral vectors occurs in human embryonic kidney 293 cells, which contain a viral E1 gene, thus permitting adenoviral packaging after the transfection of recombinant constructs in these cells [11]. Adenovirus can then be propagated further in these E1 protein-containing 293 cells and purified via gradient centrifugation.

In this chapter, we present procedures to obtain recombinant adenoviral constructs by providing three examples of how to perform the homologous recombination reaction in a test tube, in E. coli, or in 293 cells. We also present procedures to generate adenoviral vectors in 293 cells and obtain purified, high-titered adenovirus. How to titrate adenovirus and infect hepatocytes in vitro and in vivo is also discussed. These resources could enable the scientific community to decipher the pathogenesis of NASH and identify therapies to treat NASH [12–14].

2. Materials

Each solution should be prepared using ultrapure water (e.g., the resistivity of 18.2 MΩ cm water by Millipore), autoclaved or filtered (0.22 μM), and stored at room temperature (RT) or at 4 °C, as indicated.

2.1. Adenovirus Generation

2.1.1. Generating Recombinant Adenoviral Constructs Via Homologous Recombination

  1. BlOCK-iT U6 adenoviral RNAi expression system (Invitrogen). Individual components, such as pAd/BLOCK-iT DEST vector, LR Clonase II enzyme, Proteinase K and 293AD cells can also be obtained.

  2. 100 ng/μL of BLOCK-iT U6 vector containing shRNA targeting the GOI or a control shRNA, purified via commercial kits (Qiagen plasmid purification kits or equivalent).

  3. TE buffer: 10 mM Tris-HCl pH 8.0, 1 mM EDTA.

  4. 1.5 mL sterile tubes (DNAse- and RNAse-free).

  5. Chemically competent E. coli (Invitrogen TOP10, Agilent XL10 gold, or equivalent).

  6. 14 mL round-bottom polypropylene Falcon tubes (Thermo Fisher Scientific).

  7. Super Optimal broth with Catabolite repression (SOC) medium for transformation: mix 10 g of tryptone, 5 g of yeast extract, 0.5 g of sodium chloride (NaCl), 5 g of magnesium sulfate (MgSO4)-7H2O in 1 L water. Sterilize by autoclaving for 20 min at 15 psi on a liquid cycle. Add 20 mM glucose and pass through a 0.22 μm filter. Alternatively, mix commercially available SOC powder (MP Biochemicals) with water before autoclaving or filtering.

  8. Luria-Bertani (LB) broth: mix 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl in 1 L of water. Sterilize by autoclaving for 20 min at 15 psi on liquid cycle.

  9. LB-agar plates containing 100 μg/mL ampicillin or 50 μg/mL kanamycin.

  10. pAdTrack-CMV shuttle vector (Addgene or Aligent) with the GOI (pAdTrack-CMV-GOI) or without (which expresses GFP as control).

  11. Pme I restriction enzyme and buffer.

  12. Phenol:chloroform:isoamyl alcohol 25:24:1 v/v/v.

  13. 100% ethanol.

  14. 70% ethanol in water.

  15. 3 M sodium acetate pH 5.0.

  16. Electroporation cuvettes, 0.2 cm gap (Bio-Rad).

  17. BJ5183-AD electroporation-competent E. coli (Agilent).

  18. Pac I restriction enzyme and buffer.

  19. TAE buffer: 40 mM Tris base, 20 mM acetic acid, 1 mM EDTA (pH 8.0).

  20. 0.8% agarose-TAE gel with 1× DNA stain (Biotium Gel Red DNA stain or equivalent).

  21. DNA ladder.

2.1.2. Adenovirus Generation in 293 Cells

  1. RAPAd CMV adenoviral bicistronic expression system (Cell Biolabs): pacAd5-CMV-IRES-GFP and pacAd5 9.2–100.

  2. Pac I restriction enzyme and buffer.

  3. TAE buffer.

  4. 0.8% agarose-TAE gel with 1× Gel Red DNA stain.

  5. DNA ladder.

  6. Phenol:chloroform:isoamyl alcohol 25:24:1 v/v/v.

  7. 100% ethanol.

  8. 70% ethanol in water.

  9. 3 M sodium acetate pH 5.0.

  10. 60 mm cell culture dishes.

  11. 293AD cell culture medium: Dulbecco’s Modified Eagle Medium (DMEM), high glucose, supplemented with 1× Penicillin-Streptomycin (PS, 10,000 U/mL) and 10% (v/v) fetal bovine serum (FBS).

  12. Transfection reagent: Lipofectamine 2000 (Invitrogen), linear polyethylenimine (PEI, Polysciences), or equivalent.

2.2. Adenovirus Propagation

  1. 150 mm cell culture dishes.

  2. 293AD cell culture medium.

  3. 15 and 50 mL sterile conical tubes (DNAse- and RNAse-free).

  4. 1× phosphate-buffered saline (PBS).

  5. 5 cell scraper, sterile.

  6. Dry ice/ethanol bath.

2.3. Adenovirus Purification and Titration

  1. Ultra-clear centrifuge tubes for SW41 rotor (Beckman Coulter).

  2. Low-density cesium chloride (CsCl) solution: dissolve 36.6 g of CsCl in 100 mL of TE. Confirm the density by weighing 1 mL of solution on a balance; it should be 1.25 g/L.

  3. High-density CsCl solution: dissolve 62 g of CsCl in 100 mL of TE. Confirm the density by weighing 1 mL of solution on a balance; it should be 1.40 g/L.

  4. 5 mL syringes, sterile.

  5. 18-gauge needles, sterile.

  6. Slide-A-Lyzer Dialysis cassettes, 3 mL, 10K MWCO (Thermo Fisher Scientific).

  7. Floating buoys for Slide-A-Lyzer Dialysis cassette (Thermo Fisher Scientific).

  8. PBS, store at 4 °C.

  9. 1 L glass beaker, sterile.

  10. Adenovirus 2× storage buffer: 10 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.1% bovine serum albumin (BSA), and 10% glycerol, filter sterilized; store at 4 °C.

  11. Viral lysis buffer: 0.1% SDS in TE buffer.

  12. 96-well cell culture plates.

2.4. Hepatocyte Infection In Vitro and In Vivo with Adenovirus

  1. Freshly isolated primary mouse hepatocytes (at a density of 5 × 105 cells/cm2) in desired well plates (or dishes), cultured in DMEM or equivalent, supplemented with PS and 10% FBS.

  2. Pharmaceutical grade sterile saline (0.9% NaCl).

  3. Pharmaceutical grade isoflurane.

  4. SafetyGlide 31-gauge 5/16 in. 0.3 mL insulin syringe (BD), sterile.

  5. Experimental mice with desired strain, sex, and age.

2.5. Equipment

  1. PCR machine or water bath (capable of heating to 25, 37, 42, and 56 °C).

  2. Electroporator (Bio-Rad Gene Pulser Xcell total system or equivalent).

  3. Bacterial culture orbital shaker.

  4. Bacterial culture incubator.

  5. Spectrophotometer (NanoDrop 1000 or equivalent).

  6. DNA gel electrophoresis system.

  7. DNA gel imaging system (Kodak Gel Logic Imaging System or equivalent).

  8. Mammalian humidified cell culture incubator (37 °C, 5% CO2).

  9. Inverted microscope for cell culture.

  10. Inverted fluorescent microscope (Invitrogen EVOS II or equivalent).

  11. Refrigerated desktop centrifuges for 1.5, 15, and 50 mL tubes.

  12. Beckmann ultracentrifuge.

  13. SW41 swinging-bucket rotor and tubes.

  14. Magnetic stir bar (sterile) and plate.

  15. Precision isoflurane vaporizer and chamber.

3. Methods

3.1. Adenoviral Construct Generation Through Homologous Recombination In Vitro and in E. coli

3.1.1. Using BLOCK-iT U6 Adenoviral RNAi Expression System (In Vitro)

  1. Mix 1 μL of 100 ng/μL U6 entry vector expressing a control shRNA, or an shRNA targeting the GOI, under the control of a mouse U6 promoter (see Note 1) with 1 μL of 150 ng/μL pAd/BLOCK-iT-DEST vector and 6 μL TE buffer in 0.2 μL PCR tubes.

  2. Add 2 μL of LR Clonase II enzyme and mix by pipetting up and down (see Note 2).

  3. Incubate the reaction at 25 °C for 1–18 h in a PCR cycler or a water bath (see Note 3).

  4. Add 1 μL of proteinase K and incubate at 37 °C for 10 min.

  5. Transform 2 μL of the reaction to suitable E. coli chemically-competent cells (Top10, XL10, or equivalent) as in steps 6–8.

  6. Thaw competent cells on ice and transfer 20–50 μL of competent cells in prechilled 14 mL round-bottom Falcon tubes on ice. Add the DNA to the cells and gently tap the tube to mix. Incubate on ice for 5–30 min.

  7. Heat shock the competent cells at 42 °C for 30 s and incubate on ice for 2 min.

  8. Add 500 μL of SOC and incubate at 37 °C on a bacterial orbital shaker for 1 h. Plate transformant cells onto LB-agar plates containing 100 μg/mL ampicillin and incubate at 37 °C overnight (see Note 4).

  9. Pick 2–6 colonies and grow in 2 mL of LB containing 100 μg/mL ampicillin at 37 °C for 12–16 h, and extract plasmids via a commercial plasmid purification kit. A true clone should be ampicillin-resistant and chloramphenicol-sensitive, and express a recombinant vector of >30 kb in size. The insertion of shRNA targeting the GOI and the success of recombination can also be validated by sequencing (see Note 5).

3.1.2. Using AdTrack System in BJ5183-AD (E. coli)

  1. Linearize 1000 ng of each of the pAdTrack-CMV vectors alone (to produce a control adenoviral vector expressing GFP alone) or containing the GOI (pAdTrack-CMV-GOI) with Pme I in a 50 μL reaction at 37 °C for 1–4 h (see Notes 6 and 7).

  2. Purify the digested vector by phenol:chloroform:isoamyl alcohol (25:24:1 v/v/v) extraction followed by ethanol precipitation. In brief, add 250 μL of water to the Pme I digest reaction. Add 300 μL of phenol: chloroform: isoamyl alcohol (25:24:1 v/v/v). Vortex and centrifuge at 13,000 × g for 5 min at RT. Carefully transfer the upper aqueous phase (~250 μL) to a new tube. Add 2.5 volumes of ethanol and a 1/20 volume of 3 M sodium acetate pH 5.0. Mix and centrifuge at 13,000 × g for 30 min at 4 °C. Carefully discard the supernatant and wash with cold 70% ethanol. Centrifuge at 13,000 × g for 5 min at 4°C and discard the supernatant. Air-dry the DNA pellet for 5–10 min.

  3. Dissolve the DNA pellet in 10 μL of H2O and determine the concentration via a spectrophotometer.

  4. Mix 100 ng of the digested DNA with 25 μL of BJ5183-AD cells in a pre-cooled 0.2 cm electroporation cuvette and incubate on ice for 5 min (see Notes 8 and 9). Perform electroporation at 2500 V, 200 Ω, and 25 μF in a Bio-Rad Gene Pulser electroporator.

  5. Resuspend the transformation mix in 500 μL of SOC medium and incubate at 37 °C for 10–20 min. Plate on 3 LB/Kanamycin agar plates and incubate at 37 °C for 16–20 h.

  6. Pick 10–20 of the smallest colonies and grow in 2 mL of LB medium containing 50 μg/mL kanamycin for 16–20 h.

  7. Extract the plasmid using a commercial kit and dissolve in 30–50 μL of H2O. Perform Pac I digestion of at least 1–2 μg of plasmid at 37 °C for 1–2 h. Run samples on an 0.8% agarose-TAE gel containing 1× Gel Red DNA stain along with a DNA ladder. A positive clone should have a large fragment of 30 kb and a smaller fragment of 3 or 4.5 kb.

  8. To propagate the plasmid, transform 1–2 μL of the plasmid extracted from the positive clone into a suitable E. coli strain (Top10, XL10, or equivalent), as described in steps 6–8 in Subheading 3.1.1 using LB/Kanamycin agar plates (see Note 10).

3.2. Adenovirus Generation in 293AD Cells

3.2.1. One-Step Generation of Recombinant Adenovirus from Shuttle Plasmids in 293 Cells Using RAPAd CMV Viral System

  1. Perform Pac I digestion of 6–10 μg each of pacAd5-CMV-IRES-GFP (to produce an adenoviral vector expressing control GFP alone) or pacAd5-CMV-IRES-GFP containing the GOI under a CMV promoter (pacAd5-CMV-GOI-IRES-GFP), as well as 3–5 μg of the pacAd5-9.2-100 vector in a 50 μL reaction at 37 °C for 2–4 h.

  2. Run 5 μL of each reaction on a 0.8% agarose-TAE gel along with a DNA ladder to determine whether the vectors are completely digested. If digested, the pacAd5-9.2-100 vector should yield two fragments of ~30 and 2 kb in size; the pacAd5-CMV-IRES-GFP vector containing the GOI should be linearized to a size of 7.5 kb plus the size of the inserted GOI.

  3. Perform phenol:chloroform:isoamyl alcohol (25:24:1 v/v/v) extraction as described in step 2, Subheading 3.1.2. Dissolve DNA in 15 μL of H2O and measure the concentration via a spectrophotometer.

  4. Co-transfect 1 μg of linearized pacAd5-9.2-100 and 4 μg of either linearized control pacAd5-CMV-IRES-GFP or pacAd5-CMV-GOI-IRES-GFP into 293AD cells (70–80% confluency) in a 60 mm dish (using Lipofectamine, PEI, or equivalent). Include a dish of cells with mock transfection, which should not express GFP and show any cytopathic effect (CPE), as a control (see Notes 11 and 12).

  5. Remove medium the next day and add 3 mL of medium supplemented with 10% FBS and PS. Add more medium every 3–5 days if necessary, as cells typically need to be incubated at 37 °C for 7–14 days post-transfection.

  6. Monitor daily the generation of CPE and the expression of GFP via an inverted microscope and an inverted fluorescent microscope, respectively (see Note 13).

  7. Collect cells and medium when clear CPEs are observed and cells are detached, typically 7–14 days post-transfection (see Note 14). The cells and medium should contain adenovirus expressing GFP alone or the GOI and GFP. Store at −80 °C, or proceed to step 7 in Subheading 3.2.2 to lyse the cells via multiple freeze and thaw cycles (see Note 15).

3.2.2. Adenovirus Generation from Recombinant Adenoviral Plasmids

  1. Perform Pac I digestion of 5–10 μg of the recombinant adenoviral plasmids from Subheading 3.1.1 (using U6 BlOCK-iT system) or Subheading 3.1.2 (using AdTrack-CMV system).

  2. Purify linearized DNA using chloroform:phenol:isoamyl alcohol extraction as step 2 in Subheading 3.1.2. Dissolve in 25 μL of H2O and measure concentration.

  3. Transfect 4 μg of purified DNA into 293 cells (70–80% confluency) in a 60 mm dish using Lipofectamine 2000, PEI, or equivalent. We recommend including a dish of cells with mock transfection, which should not show any CPE, as a control (see Notes 11 and 12).

  4. Remove medium the next day and add 3 mL of medium supplemented with 10% FBS and PS. Add more medium every 3–5 days if necessary, as cells typically need to be incubated at 37 °C for 7–14 days post-transfection.

  5. Monitor daily the generation of CPE and/or the expression of GFP via an inverted microscope or an inverted fluorescent microscope, respectively (see Note 13).

  6. Collect cells and medium by scraping or pipetting into a 15 mL tube when clear CPFs are observed and half of the cells are detached, typically 7–14 days post-transfection (see Note 14).

  7. Perform a freeze and thaw cycle as follows: freeze cells in dry ice/ethanol bath (or −80 °C freezer), thaw in a 37 °C water bath, and vortex vigorously (see Note 16).

  8. Repeat the freeze and thaw cycle three times.

  9. Centrifuge at 7000 × g for 10 min at 4 °C and save the supernatant, which contains adenovirus.

  10. Use 30–50% of the supernatant to infect one 150 mm dish of 293 cells (80–90% confluency).

  11. Collect cells and culture medium when half of the cells are detached, typically 3–7 days post-infection.

  12. Perform three freeze/thaw cycles as described in steps 7 and 8.

  13. Centrifuge at 7000 × g for 10 min at 4 °C and save the supernatant (which contains adenovirus) in the desired volume at 4 °C for short-term or at −80 °C for long-term storage. We recommend validating the virus before further amplification. This can be done by validating the up- or downregulation of the GOI using immunoblotting or qRT-PCR in adenoviral-sensitive hepatocytes (e.g., primary mouse hepatocytes). We also recommend titrating the viruses, including the control virus, before performing experiments using crude lysates containing adenovirus (see Subheading 3.5.2; see Note 17).

3.3. Adenovirus Propagation in 293AD cells

To obtain high-titer adenovirus, further viral propagation is needed.

  1. Use half of the virus from step 13 in Subheading 3.2.2 to infect 10–20,150 mm dishes of 293 cells (80–90% confluency).

  2. Collect cells and culture medium when half of the cells are detached, typically 2–5 days post-infection.

  3. Centrifuge at 1000 × g for 10 min at 4 °C. Discard the supernatant and suspend the cell pellet in 4 mL of sterile PBS. Store at −80 °C or proceed to the next steps and purification.

  4. Perform three freeze/thaw cycles as in steps 7 and 8, Subheading 3.2.2.

  5. Centrifuge at 7000 × g for 10 min at 4 °C and save the supernatant for purification. If a large quantity of virus is needed, infect more 293 cells and perform more rounds of propagation as in steps 2–5, followed by purification and titration (see Notes 18 and 19).

3.4. Adenovirus Purification Via CsCl Discontinuous Gradient Centrifugation

  1. Add 3 mL of the low-density CsCl solution (1.25 g/mL) into clear ultracentrifugation tubes.

  2. Using a 1 mL pipette, slowly add 3 mL of the high-density CsCl solution (1.40 g/mL) to the bottom of the tube. Two layers of solution with a clear interface should be seen (see Note 20).

  3. Overlay 4 mL of the viral solution on the top of the low-density solution. Do not disrupt the CsCl gradient.

  4. Overlay 1 mL of mineral oil on top of the viral solution.

  5. Carefully balance the tubes and centrifuge at 32,000 rpm (175,000 × g) at 4 °C for 16 h or overnight (see Note 21).

  6. Using a syringe connected to an 18-gauge needle, collect the lowest observed white band with a syringe: insert the needle (facing up) into the tube, 5 mm below the viral band; carefully draw the virus out (1–2 mL). Do not take the upper viral band (see Notes 22 and 23).

  7. Remove air from a dialysis bag using an 18-gauge needle connected to a 5 mL syringe. Add viral/CsCl solution to the bag. Mark the opening of the dialysis bag and let it face upwards to prevent the loss of the virus during dialysis.

  8. Attach a buoy to the dialysis bag. In a 1 L beaker, perform dialysis in 1 L of cold PBS in a cold room on a magnetic stirrer for 2 h. Stir gently. Repeat the dialysis 2–3 more times with each fresh cold 1 L of PBS (see Note 24).

  9. Use a 5 mL syringe with an 18-gauge needle to collect the viral solution from the dialysis bag and add 1 volume of cold 2× storage buffer.

  10. Aliquot the virus into prechilled 1.5 mL tubes in the desired volume and store at −80 °C. Avoid repeated freeze and thaw of the virus.

3.5. Adenovirus Titration

This protocol contains instructions for a physical titration method via the quantification of viral DNA and a functional titration method of the end-point dilution assay (see Note 25).

3.5.1. Viral Particle Optical Absorption

  1. Mix 2.5 μL of PBS, 2.5 μL of 2× viral storage buffer, and 95 μL of lysis buffer as a blank solution.

  2. Mix 5 μL of the virus with 95 μL of lysis buffer (20-fold dilution) and incubate at 56 °C for 10 min. Vortex every 2–3 min.

  3. Measure absorption at 260 (A260) via a spectrophotometer using the blank solution made in step 1.

  4. Calculate viral particle number/mL (VP) using the formula below [15]: A260 × 20 (dilution factor) × 1.1 × 1012 (see Note 26).

3.5.2. End-Point Dilution

  1. Seed 293 cells into a 96-well plate and replenish with 100 μL/well of fresh medium containing PS upon reaching 80–90% confluency. Seed each well at the same density.

  2. Make tenfold serial dilutions of the virus from 10−3 to 10−10 into 1 mL of DMEM medium containing PS.

  3. Add 100 μL of virus-containing medium per well to the plate, with 10 replicates per viral dilution. Make sure to include a few control wells with only 200 μL of medium and no adenovirus (see Note 27).

  4. Incubate the 96-well plate in a mammalian cell culture incubator for 10 days. Add more medium to the cells during the incubation if necessary.

  5. Check each well for CPE. For each dilution, count the number of wells with CPE and calculate the fraction of CPE-positive wells.

  6. Calculate the viral titer: titer (pfu/mL) = 10(x+0.8), where x is the sum of CPE fraction at all dilutions, including 10−2 and 10−1 (1; no dilution). If the fraction at dilution of 10−3 is 1, use 1 for the fractions at 10−2 and 10−1 (see Note 28).

3.6. Infection of Primary Mouse Hepatocytes and Mouse Liver

3.6.1. Infection of Primary Mouse Hepatocytes (See Note 17)

  1. Infect freshly isolated primary mouse hepatocytes with adenovirus, 1–10 infectious particles/cell (1–10 multiplicity of infection (MOI)), in serum-free DMEM medium (or equivalent) supplemented with PS (see Notes 29–31).

  2. Change medium 6 h later or the next day.

  3. Perform desired treatments and analyses and collect cells 12–48 h post-infection; determine the expression of the GOI (see Note 32). See Fig. 1a as an example.

Fig. 1.

Fig. 1

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Infection of primary mouse hepatocytes and mouse liver by adenovirus. (a) Primary mouse hepatocytes were isolated from 6-week-old male C57BL/6J mice and incubated in DMEM medium supplemented with FBS and PS. Four hours later, cells were washed with PBS and infected with an adenoviral vector (MOI = 5) expressing GFP in DMEM medium containing PS. GFP expression was monitored using an inverted fluorescent microscope 16 h post-infection (scale-bar: 275 μM). (b) Seven- to nine-week-old male floxed Taz (transcriptional co-activator with PDZ-binding motif) mice (Tazflox/flox) were administered adenovirus (1 × 109 pfu/mouse) expressing control GFP or Cre. Mice were sacrificed 7 days later and the deletion of hepatic TAZ was confirmed by immunoblotting liver lysates

3.6.2. Infection of Mouse Liver (See Note 33)

  1. Anesthetize mice via 2–4% isoflurane and oxygen.

  2. Dilute virus (0.5–2 × 109 pfu/mouse) into saline to 100 μL. In a biosafety cabinet, inject 100 μL/mouse of diluted adenovirus retro-orbitally to fully anesthetized mice using a 31-gauge safety insulin syringe (see Notes 34–36).

  3. Perform desired procedures and collect tissues 3–14 days post-viral treatment for analyses (see Note 37). See Fig. 1b as an example.

Acknowledgments

This work was supported by a National Institutes of Health Award R01DK124328 (Miao, J).

4 Notes

1.

The plasmid containing an shRNA driven by a U6 promoter can be constructed using BLOCK-iT U6 RNAi entry vector kit from Invitrogen.

2.

Keep the LR Clonase at −20 °C and move onto ice just before use. Do not warm the enzyme, since this reduces its activity.

3.

Increasing the incubation duration yields more colonies.

4.

Make sure that the LB-agar-ampicillin plates are freshly made to avoid any false-positive clones.

5.

Use sequencing primers recommended by the manufacturer to detect the success of the homologous recombination and the insertion of shRNA.

6.

pAdTrack-CMV vector contains an additional CMV promoter that drives the expression of GFP.

7.

It is important to completely digest the shuttle vector by Pme I. Incomplete digestion of the vector by Pme I will yield a high number of colonies that do not express the recombinant plasmid.

8.

BJ5183-AD cells can be generated by transforming the pAdEasy-1 plasmid (Addgene) into BJ5183 E. coli (Agilent). Alternatively, perform electroporation of BJ5183 cells with purified Pac I-digested AdTrack vector and 150 ng of pAdEasy-1.

9.

The use of high-quality electroporation-competent cells is essential to successful generation of recombinant adenoviral plasmids. These cells are sensitive to temperature changes and should be stored at −80 °C. Avoid repeated freeze and thaw of the cells. Thaw the cells on ice before performing electroporation.

10.

We recommend using a high-efficiency chemically-competent E. coli strain to perform the transformation, as the recombinant plasmid is >30 kb.

11.

Other commonly used 293 cell lines expressing the E1 protein are suitable for generating adenovirus, including adherent and suspension 293HEK cells.

12.

It is essential to use 293AD cells that have been passaged fewer than 20 times and are free of mycoplasma contamination.

13.

Cells producing adenovirus will initially appear as patches of rounding and dying cells. These cells will lyse and infect neighboring cells. A plaque forms when these adenoviral-producing cells and their neighboring infected cells lyse and die.

14.

Do not keep dishes incubated longer than 14 days.

15.

Adenovirus is a biohazard level 2 (BL2) agent. Work with adenovirus should be in compliance with institutional guidelines. Adenoviral-contaminated medium/cell culture dishes should be disinfected according to institutional guidelines.

16.

Remove tubes from the 37 °C water bath as soon as samples are thawed. Avoid incubating the virus at 37 °C for a longer period, since this reduces viral titer.

17.

Adenovirus can be used to infect established hepatoma cell lines (e.g., HepG2, Hep3B, Hepa1c1c7, AML12, FAO, and others). We recommend increasing MOI when infecting these lines, as they are less sensitive to adenoviruses than primary mouse hepatocytes and are likely to have different responses to adenoviral infection. Therefore, the optimal MOI for these lines needs to be empirically determined.

18.

Each round of amplification should produce 10- to 100-fold more viruses relative to the virus present in the prior round. Typically, three rounds of amplification should produce a viral titer of infectious particles 109 to 1010 (or plaque-forming units (pfu))/mL. However, during the last round of amplification, if cells display CPF before 1 day or after 5 days following infection, the virus amplification is not optimal, and the viral titer is likely to be low. Therefore, the ratio of virus to number of 293AD cells need to be empirically determined to ensure efficient propagation of adenovirus.

19.

We recommend limiting the amplification to fewer than 5–6 rounds to avoid the generation of the replicate-competent virus.

20.

The CsCl gradient is essential to the success of adenoviral purification. It is critical to place the tip of the 1 mL pipette to the bottom of the tube when gently adding the 1.4 g/mL CsCl solution. Avoid air bubbles. Do not mix two layers or disrupt the interface.

21.

Make sure all tubes are carefully balanced. Prepare a blank balance tube if needed (replacing the viral solution with PBS).

22.

Mature adenoviruses can be seen at the interface of the two layers, whereas immature viruses will be seen in the upper, low-density solution.

23.

Adenoviral-contaminated solution should be disinfected with chlorine bleach.

24.

Multiple rounds of dialysis are necessary to remove any trace amount of CsCl, since it is toxic to cells.

25.

Other titration methods include plaque assays, immune-staining infected cells for the expression of viral proteins, and quantification of viral genome copy via real-time PCR. Many commercial kits are also available. While biologic functional titration methods are often more time consuming, they provide more accurate information on the number of infectious viruses and have more biologic relevance when compared with physical titration methods [15].

26.

A260 absorption measures viral DNA and protein. It does not distinguish infectious from non-infectious viral particles and cannot provide information on the expression of transgenes or shRNA; thus, the results are less reliable than functional titration methods. A VP titer is often higher than a PFU titer and the VP/pfu ratio is often between 10:1 and 50:1 for most viral preparations [16].

27.

It is important to include control wells that are not infected with the virus. These wells should not show any CPE.

28.

If cells infected with the highest concentration (10−3) of the virus are not all CPE-positive, lower dilutions of the virus (e.g., 10−2) should be used to repeat the end-point dilution assay. Similarly, if cells infected with the lowest concentration (10−10) of the virus are all CPE-positive, higher dilutions of the virus (e.g., 10−11) should be used to repeat the assay.

29.

It is important to use an established, two-step collagenase perfusion protocol to obtain high-quality primary mouse hepatocytes [17].

30.

Primary mouse hepatocytes are overly sensitive to adenoviral infection. Avoid using excess virus, which leads to cytotoxicity and artificial effects. Determine the optimal MOI empirically.

31.

Simultaneous expression of multiple GOIs or shRNAs can be achieved by infecting cells with several adenoviral vectors.

32.

We recommend performing desired analyses within 48 h of isolation, as typical monolayer hepatocytes lose their morphology, hepatocyte-specific gene expression, and responses to hormones and pharmacologic treatments over time.

33.

The administration of adenovirus to mice should follow institutional guidelines (e.g., perform injection in a biosafety cabinet and use safety syringes).

34.

Administration of adenovirus can also be done via tail vein injection.

35.

Adenovirus efficiently transduces liver, since intravenously-administered adenoviruses are primarily sequestered by the liver (>90%) [18]. Hepatocytes and non-parenchymal liver cells can be efficiently transduced by Ad5 particles [18]. Avoid using excess virus, which leads to excess inflammatory responses and artificial effects. Determine the optimal dose empirically.

36.

When performing mouse studies, the physical titer of adenovirus should also be considered, since adenovirus induces an innate immune response (directed against the capsid protein) and studies show that the response can be lethal in mice [16].

37.

Adenovirus induces transient gene expression, typically less than 7 days [19], due to its ability to induce a strong immune response [20]. However, the duration of the viral-mediated expression of the GOI and its downstream functional impact should be empirically determined.

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