We thank Robert Tjian and Xavier Darzacq for their support and use of laboratory equipment. We thank Mark Kay for his gift of the KP1 and LK03 rep/cap plasmids, and Luk Vandenberghe for AAV4 rep/cap plasmid. Funding was provided by the Howard Hughes Medical Institute (34430, R. T.) and the California Institute for Regenerative Medicine Training Program EDUC4-12790. N.W. acknowledges funding from the Berkeley Stem Cell Center via a Siebel postdoctoral fellowship and from the German Research Foundation (DFG) via a Walter Benjamin fellowship.

Crude Vector Production and Efficient DNA Delivery in Cell Cultures

A.C.M. is a consultant for Janssen Pharmaceuticals and serves on the SAB for NewBiologix. All the other authors have no conflicts of interest to declare.

Cloning 
The cloning protocol is not limited to the pAAV.CMV.Luc.IRES.EGFP.SV40 plasmid used above and can be easily altered based on a researcher's experimental needs. Many ITR-containing plasmids are readily available online for purchase. For example, plasmids containing both Cas9 and an sgRNA cloning site are available but require few additional steps such as oligonucleotide annealing and PNK treatment30. Additionally, plasmids containing a multiple cloning site (MCS) with only ITRs and no inner regulatory elements can be found31. If different plasmids are to be used, the restriction enzymes (RE) used for digestion are typically the only elements that may need to be changed in this protocol. However, a limitation of rAAV is its limited cargo capacity. Due to physical limitations of the capsid, the vector genome should not exceed 4.9 kb, including the ITRs.
When isolating plasmid from bacteria, it is critical to use an endotoxin-low or -free midiprep or maxiprep kit to mitigate harm to cells during triple-plasmid transfection or transduction. Plasmid from miniprep kits often contain higher impurities, reduced concentrations, and fewer supercoiled DNA, all of which can affect the downstream production of rAAV and is thus not recommended.
It is critical to understand the structure and properties of ITRs during cloning. First, it is extremely difficult to use PCR through the ITR. Cloning designs that require PCR amplification through ITRs should be avoided, and additionally limit the use of the Gibson assembly cloning technique. As such, restriction enzyme cloning is the preferred method for cloning into ITR-containing plasmids. Furthermore, certain primers for Sanger sequencing may not be compatible if the sequenced region contains the ITR. Instead, it is recommended to use primers that sequence away from the ITRs and into the vector genome body to get more precise sequencing results. Second, ITRs are prone to deletions, rearrangements, and mutations when transformed into bacteria for plasmid amplification32,33. To mitigate these events, it is recommended to use recombination-deficient competent bacterial strains, such as Stbl3, and to incubate them at 30 °C to slow down cellular divisions. Last, it has been observed that smaller colonies may correspond with clones without rearrangements or deletions, as those without ITRs may confer a growth advantage and be larger. Therefore, it is recommended to pick colonies that are small.
Vector production 
The successful production of rAAV vector can be affected by multiple elements. One critical factor is the health of HEK293 or 293T cells used for transfection. Generally, low passage numbers are ideal, as highly passaged cells may exhibit genotypic and phenotypic variances that can reduce rAAV titers. Additionally, the density of the seeded cells should be 75%-90% confluency for effective production. Sparse cells generate low vector yields because there are less cells available to produce vectors, while overgrown cells will not be efficiently transfected.
Variations between reagent lots, cell stocks, and general lab-to-lab variability contribute to differences in transfection efficiencies and production titer. One optimizable factor that can lead to titer improvements is the plasmid:PEI ratio in transfection reactions. It is critical to use fresh (&lt;1 month old) PEI MAX. It is recommended that a plasmid:PEI ratio of 1:1 be used as a starting point, and if transfection or transduction efficiency appears poor, test several different ratios. Titer optimization is easiest if using a transgene with a visual readout, such as the CMV.Luc.IRES.EGFP reporter trangene used herein as starting material for cloning. To perform the optimization, follow protocol step 3 using a 12-well plate and scaling down the plasmid masses and reagent volumes by two (final plasmid mass is 2.6 µg). Adjust the PEI volume accordingly to correspond to ratios ranging from 1:0.75 to 1:3, with increasing increments of 0.25 (Figure 6). Dilute each reaction with 950 µL of SF media after 15 min. For convenience, a master mix containing the triple plasmids can be made and individually pipetted into 1.5 mL tubes prior to adding PEI-see Supplementary File 2. Harvest the vector, transduce cells of interest, and image. The well with the highest transduction efficiency (proportion of GFP+ cells) corresponds to the highest titer and most optimal ratio of PEI:DNA.
<img alt="Plasmid transfection diagram; HEK293 cell transduction process; PEI MAX; freeze/thaw cycles." class="xfigimg" src="/files/ftp_upload/65572/65572fig06.jpg" data-altupdatedat="1747911167000" data-alt="Plasmid transfection process, vector harvest, cell imaging; includes PEI MAX ratio adjustment, HEK293."> 
Figure 6: PEI optimization workflow. Schematic of the steps required for PEI optimization. Multiple ratios of plasmid: PEI are tested to determine the optimal ratio. <a href="https://www.jove.com/files/ftp_upload/65572/65572fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Harvest and titer considerations 
The freeze/thaw technique used to harvest rAAV vector effectively lyses HEK293 cells in a manner compatible with the direct use of the clarified lysate to transduce cultured cells. Certain rAAV serotypes, such as AAV1, AAV8, and AAV9 are released from cells during vector production and can be harvested from the cultured cell medium without freeze/thaw cycles34. The method described here typically yields titers on the order of 1 x 1010 VG/mL when using AAV2 capsids, and 1 x 1011 VG/mL for AAV8. While higher titers can be achieved by detergent or other chemical-based lysis, these are harmful to cells in downstream use and require rAAVs to be further purified from the lysate. Lower titer is one tradeoff a researcher should consider when determining whether crude preparations are appropriate for their research needs, however, the marginally lower titers produced by the methods described here can transduce many cell types very well (see representative results). In addition to transfection efficiency and cell health, vector titers vary depending on the capsid used during rAAV production and the size and sequence of the transgene within the VG35.
When harvesting crude vector preparations, plasmid DNA that was used during triple-plasmid transfection may be present and, although rare, result in downstream transfection during transduction. Furthermore, unpackaged VGs may bind to the exterior of capsids and invoke an innate immune response to naked and foreign single-stranded DNA36,37. Therefore, sensitive cell types may require vector preparations to be DNase digested and purified to remove unpackaged VGs and plasmid.
If one wishes to calculate the titer of a crude preparation, qPCR can be performed to quantify the number of packaged VG inside DNase-resistant particles (DRP). Briefly, a small amount of crude preparation is DNase-digested to remove plasmid DNA, contaminating nucleic acids, or partially packaged VG. The sample is then subject to qPCR and the protected VG inside of DRPs is quantified, resulting in a titer with units of vector genome per mL of crude preparation38. It is not recommended to perform vector titration using ELISA-based assays that quantify capsid titers. Compared to wild-type AAV virus, rAAV suffers from a proportion of empty and partially packaged capsids39. ELISA will quantify all capsids regardless of their genome contents and will overestimate the transducible units present in a preparation, which requires a packaged VG.
Transduction considerations 
Many factors influence rAAV transductions and proper considerations should be made for any new experiment. Depending on the promoter driving transgene expression, expression onset can occur as early as 4 h post-transduction (hpt), and peak expression is typically achieved by 48 hpt. It is important to keep in mind the duration of time from the initial seeding of cells to the experimental endpoint. This is to estimate the starting confluency of the cells and ensure that they do not overgrow by the end of the experiment. If cells become overconfluent, cellular behavior may be altered due to a stress response and can confound experimental results. Some cell types, like U2-OS, can tolerate overgrowth/contact inhibition quite well. Additionally, they can withstand long periods (48 h+) in serum-free conditioned medium—the product of this production protocol. However, sensitive cell types may require serum addition or dilution of the crude preparation with special growth medium to maintain health during transduction. A slightly reduced transduction efficiency from using serum-containing media is a potential tradeoff for cell health and should be considered by the researcher.
Typically, for rapidly dividing cells, a starting confluency of around 50% is optimal for applications that will be terminated 48 hpt. However, confluency can be adjusted accordingly based on the needs of the experiment. It is not recommended to transduce monolayer-type immortalized cell lines over 75% confluency due to decreased transduction efficiencies. Most cultured cell types are successfully transduced and healthy after overnight incubation with crude rAAV preparations, followed by a change to fresh serum-containing media in the morning.
Capsid serotype is an important factor to consider when producing rAAV to transduce a target cell, as the capsid is the primary determinant of cellular tropism and subsequent transgene expression13. AAV2 is a widely used serotype due to its ability to effectively transduce many types of cultured cells12. This property of AAV2 may be attributed to heparin sulfate proteoglycans (HSPGs) serving as the primary attachment factor for AAV2 and the high levels of HSPGs on cultured cells from the adaptation to growing in a dish40. Other capsids, such as AAV9, are less effective at transducing broad cell types and may be explained by their reliance attachment factors that are not expressed in this setting41. Therefore, we recommend AAV2 as a first-choice capsid in cultured cells if a desired target cell has not been previously tested with rAAV in the literature.
Please note that a major limitation of crude vector preparations is that they are inappropriate for transducing animal models. In vivo studies require preparations to be purified and undergo quality assessment.
Transgene expression and potential integration considerations 
rAAVs do not reliably result in permanent expression of the transgene. Over time, VGs can become silenced and transgenic expression may be shut down following several passages42. Additionally, the majority of VGs remain episomal, and rAAVs do not contain the viral Rep proteins that would mediate frequent integration into the host genome as in a wild-type viral lysogenic infection or promote replication of VGs43. As a result, episomes in transduced cells will eventually be diluted out among daughter cells through divisions.
Basal-level integration is a possibility for all delivered transgenic DNA material. However, ITR-containing VGs are prone to integration at a higher frequency44. Therefore, permanent expression of a transgene may be observed in a small subset of cells. Users should consider this possibility especially when using rAAV to deliver DNA-cutting enzymes, such as Cas9, as double-stranded breaks may result in an even larger frequency of integration and permanent expression45. While this makes rAAV a good candidate for delivering homology directed repair templates for endogenous tagging or gene addition, the possibility of Cas9 insertion should be considered19,46.

Elucidating the molecular bases of cellular functions often requires the expression of transgenic DNA in cell culture. To be expressed, transgenes must penetrate through a cell's selective membrane and reach the nucleus1,2. Therefore, the ability to effectively bypass the cell's physical barriers and manipulate its central processes is a necessity for applying transgenesis to uncover new biological phenomena. One approach capitalizes on the intrinsic ability of viruses to deliver and express foreign DNA3,4.
Adeno-associated virus (AAV) is one of the smallest mammalian viruses: its 4.7 kilobase (kb), single-stranded DNA genome contains two genes, rep (for replicase) and cap (for capsid), packaged inside of a 60-mer icosahedral capsid measuring 25 nm. The rep/cap genes have multiple promoters, reading frames, and splice products which encode at least nine unique proteins required for viral replication, production, and packaging5,6. Additionally, both ends of the genome contain secondary structures called inverted terminal repeats (ITRs) that are necessary for DNA replication, genome packaging, and downstream processing during transduction7,8,9,10. The ITRs are the only DNA elements that are required for the packaging of the genome into the capsid, and therefore AAV can be cloned for transgene delivery purposes by replacing the viral rep/cap genes with a researcher's choice of regulatory elements and/or genes of interest6. The resulting recombinant AAV (rAAV), with an engineered vector genome (VG), is widely used in the clinic for human gene therapy and has amassed successes11. An underappreciated use of the vector is in the laboratory; rAAVs can efficiently achieve transgene expression in cultured cells to fulfill a researcher's experimental needs12.
The most common method for producing rAAV is by triple-plasmid transfection into HEK293 or 293T cells (Figure 1). The first plasmid, commonly called the cis plasmid, contains the desired transgene flanked by ITRs (pAAV). Depending on the application, cis plasmids with common elements, such as strong promoters or CRISPR-based tools, are available for purchase. The second is the pRep/Cap plasmid that contains the wild-type AAV rep and cap genes provided in-trans—i.e., on a separate, non-ITR containing plasmid that expresses regulatory and structural elements that then interact with the cis plasmid—and is thus called the trans plasmid. In addition to physically enclosing the VG, the capsid influences cellular tropism12,13. By providing the serotype-specific cap gene in-trans, researchers are easily able to maximize transduction efficiency by choosing an optimized capsid serotype for their given target cell. Lastly, as a Dependoparvovirus, AAV requires a helper virus to activate rep/cap expression from its viral promoters, achieved by adenoviral helper genes, provided on a third plasmid such as pAdΔF614,15. After 72 h of triple-plasmid transfection, the vector can be released from producer cells into the culture media by repeated freeze/thaw cycles. The entire plate contents are then collected, and large cellular debris is removed by centrifugation; the resulting media supernatant is a crude rAAV preparation ready for downstream transductions.
<img alt="HEK 293(T) cell transfection diagram; vector prep, freeze/thaw cycles, transduction process." class="xfigimg" src="/files/ftp_upload/65572/65572fig01v2.jpg" data-altupdatedat="1747911167000" data-alt="Gene transfection process diagram with HEK 293(T) cells, plasmid vectors, and incubation."> 
Figure 1: Overview of crude rAAV vector production. Crude rAAV production and transduction can be accomplished within 5 days. <a href="https://www.jove.com/files/ftp_upload/65572/65572fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
rAAV may be more favorable for transgene delivery compared to other transfection methods, which are commonly associated with cellular toxicity, low efficiency, and expensive reagents and equipment, such as for electroporation or chemical/lipid-based transfection16,17. rAAV bypasses these obstacles and often provides potent transgene expression with minimal toxicity, and minimal hands-on time. Importantly, producing rAAV and applying it in cell culture is simple and rarely requires purification of the vector from the culture media (Figure 1). Additionally, rAAV does not integrate its VG into the host genome, unlike Lentiviral transgene delivery, and thus lowers the risk of insertional mutagenesis18. Despite the potential benefits of using rAAV for transgene delivery, limitations must be considered. Importantly, the size of the transgene, including the ITRs, should not exceed 4.9 kb due to physical constraints of the capsid, thereby limiting a researcher's ability to effectively deliver large regulatory elements and transgenes. Furthermore, since rAAV is a non-integrating virus, transduction results in transient transgene expression in dividing cells and may not be practical for stable expression. However, methods using dual rAAV-delivered Cas9 and homology-directed repair (HDR) templates may be used to stably insert sequences at specific genomic loci if a researcher wishes19.

<table><tbody><tr><td>Snapgene DNA viewing sofrware</td><td>Snapgene</td><td></td><td></td></tr><tr><td>pAAV.CMV.Luc.IRES.EGFP.SV40</td><td>AddGene</td><td>105533</td><td></td></tr><tr><td>pAAV2/2 (Rep/Cap)</td><td>AddGene</td><td>104963</td><td></td></tr><tr><td>pAd&amp;Delta;F6</td><td>AddGene</td><td>112867</td><td></td></tr><tr><td>LB Agar Carbenicillin</td><td>Sigma-Aldrich</td><td>L0418</td><td></td></tr><tr><td>Boekel Scientific Economy Digital Incubator</td><td>Boekel Scientific</td><td>133000</td><td></td></tr><tr><td>LB medium, powder</td><td>MP Biomedicals</td><td>113002042</td><td></td></tr><tr><td>Carbencillin (Disodium)</td><td>GoldBio</td><td>C-103-5</td><td></td></tr><tr><td>New Brunswick I26 Shaker</td><td>Eppendorf</td><td>M1324-0000</td><td></td></tr><tr><td>50 mL Centrifuge Tubes</td><td>Corning</td><td>430828</td><td></td></tr><tr><td>Centrifuge 5810 R</td><td>Eppendorf</td><td>22627040</td><td></td></tr><tr><td>QIAGEN Plasmid Plus Midi Kit</td><td>Qiagen</td><td>12945</td><td></td></tr><tr><td>PCR PULL-APART 8-TUBE STRIPS</td><td>USA Scientific</td><td>1402-3900</td><td></td></tr><tr><td>Mastercycler nexus</td><td>Eppendorf</td><td>6333000022</td><td></td></tr><tr><td>dNTP</td><td>Thermo Fisher Scientific</td><td>18427013</td><td></td></tr><tr><td>5x Phusion Buffer</td><td>NEB</td><td>B0518S</td><td>Provided with purchase of Phusion Polymerase</td></tr><tr><td>Phusion&amp;nbsp;High-Fidelity DNA Polymerase</td><td>NEB</td><td>M0530S</td><td></td></tr><tr><td>Gel Loading Dye, Orange (6X)</td><td>NEB</td><td>B7022S</td><td></td></tr><tr><td>DNA Clean &amp;amp; Concentrator-100</td><td>Zymo</td><td>D4029</td><td></td></tr><tr><td>Zymoclean Gel DNA Recovery Kit</td><td>Zymo</td><td>D4001</td><td></td></tr><tr><td>NotI-HF</td><td>NEB</td><td>R3189S</td><td></td></tr><tr><td>EcoRI-HF</td><td>NEB</td><td>R3101S</td><td></td></tr><tr><td>CutSmart Buffer (10x)</td><td>NEB</td><td>B6004S</td><td>Provided with purchase of restriction enzyme</td></tr><tr><td>UltraPure Agarose</td><td>Thermo Fisher Scientific</td><td>16500100</td><td></td></tr><tr><td>Ethidium Bromide</td><td>Sigma-Aldrich</td><td>E1510</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>NEB</td><td>M0202S</td><td></td></tr><tr><td>T4 DNA Ligase Reaction Buffer</td><td>NEB</td><td>B0202S</td><td>Provided with purchase of T4 Ligase</td></tr><tr><td>Eppendorf Safe-Lock Tubes (1.5mL)</td><td>Eppendorf</td><td>22363204</td><td></td></tr><tr><td>Precision Microprocessor Water Bath</td><td>Thermo Scientific</td><td>51221046</td><td></td></tr><tr><td>Sterile Plastic Culture Tubes</td><td>Fisher Scientific</td><td>149566B</td><td></td></tr><tr><td>2.0 mL Microcentrifuge Tube</td><td>Thomas Scientific</td><td>1149Y01</td><td></td></tr><tr><td>ZR Plasmid Miniprep - Classic</td><td>Zymo</td><td>D4015</td><td></td></tr><tr><td>Xma1</td><td>NEB</td><td>R0180S</td><td></td></tr><tr><td>Sma1</td><td>NEB</td><td>R0141S</td><td></td></tr><tr><td>HEK 293T cells</td><td>ATCC</td><td>CRL-3216</td><td></td></tr><tr><td>Falcon 6-well</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>Gibco&amp;nbsp;DMEM, high glucose, pyruvate</td><td>Thermo-Fisher</td><td>11995065</td><td></td></tr><tr><td>Sanyo MCO-18AIC(UV) CO2 Incubator</td><td>Marshall Scientific</td><td>MCO-18AIC</td><td></td></tr><tr><td>PEI MAX (Polyethylenimine Hydrochloride)</td><td>Polysciences</td><td>24765-100</td><td></td></tr><tr><td>Mixer Vortex Genie 2</td><td>Electron Microscopy Sciences</td><td>102091-234</td><td></td></tr><tr><td>Sanyo Ultra Low Freezer</td><td>Sanyo</td><td>14656-15267-16219</td><td></td></tr><tr><td>INCU-Line IL 10 with transparent window</td><td>VWR</td><td>390-0384</td><td></td></tr><tr><td>Eppendorf Microcentrifuges</td><td>Eppendorf</td><td>05-400-005</td><td></td></tr><tr><td>Falcon 96-well</td><td>Corning</td><td>353072</td><td></td></tr><tr><td>C57BL/6J mice</td><td>JAX</td><td>strain #000664</td><td></td></tr><tr><td>organoid growth medium</td><td>STEMCELL Technologies</td><td>6005</td><td></td></tr><tr><td>L Wnt-3A cells</td><td>ATCC</td><td>CRL-2647</td><td></td></tr><tr><td>nicotinamide</td><td>Sigma</td><td>N0636-100G</td><td></td></tr><tr><td>ROCK inhibitor</td><td>STEMCELL Technologies</td><td>72302</td><td></td></tr><tr><td>CHIR99021</td><td>STEMCELL Technologies</td><td>72052</td><td></td></tr><tr><td>Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane</td><td>Fisher</td><td>356231</td><td></td></tr><tr><td>24-well plate</td><td>Fisher</td><td>08-772-1</td><td></td></tr><tr><td>D-PBS</td><td>Thermo Fisher Scientific</td><td>14-190-250</td><td></td></tr><tr><td>TrypLE Express</td><td>Fisher</td><td>12604013</td><td></td></tr><tr><td>DMEM/F-12 with 15 mM HEPES</td><td>STEMCELL Technologies</td><td>36254</td><td></td></tr><tr><td>polybrene</td><td>Millipore Sigma</td><td>TR-1003-G</td><td></td></tr><tr><td>48-well plates</td><td>Fisher</td><td>08-772-3D</td><td></td></tr><tr><td>Thermo Scientific&amp;nbsp;Nunc Lab-Tek Chambered Coverglass</td><td>Fisher</td><td>12-565-470</td><td></td></tr><tr><td>BeWo cells</td><td>ATCC</td><td>CCL-98</td><td></td></tr><tr><td>F-12K Medium</td><td>ATCC</td><td>30-2004</td><td></td></tr><tr><td>Hepa1-6</td><td>ATCC</td><td>CRL-1830</td><td></td></tr><tr><td>Huh7</td><td>UC Berkeley BSD Cell Culture Facility</td><td>HUH-7</td><td></td></tr><tr><td>C2C12</td><td>ATCC</td><td>CRL-1772</td><td></td></tr><tr><td>HSkMC</td><td>ATCC</td><td>PCS-950-010</td><td></td></tr><tr><td>Skeletal Muscle Cell Growth Medium</td><td>Sigma</td><td>C-23060</td><td></td></tr><tr><td>Skeletal Muscle Differentiation Medium</td><td>Sigma</td><td>C-23061</td><td></td></tr><tr><td>Invitrogen&amp;nbsp;EVOS Digital Color Fluorescence Microscope</td><td>Fisher Scientific</td><td>12-563-340</td><td></td></tr><tr><td>Perkin Elmer Opera Phenix</td><td>Perkin Elmer</td><td>HH14001000</td><td></td></tr><tr><td>PhenoPlate 96-well</td><td>Perkin Elmer</td><td>6055302</td><td></td></tr><tr><td>DMEM, high glucose, no glutamine, no phenol red</td><td>Thermo-Fisher</td><td>31053028</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Thermo-Fisher</td><td>35050079</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Thermo-Fisher</td><td>11360070</td><td></td></tr><tr><td>pAAV2/5 (Rep/Cap)</td><td>Addgene</td><td>104964</td><td></td></tr><tr><td>pAAV2/8 (Rep/Cap)</td><td>Addgene</td><td>112864</td><td></td></tr><tr><td>pAAV-DJ-N589X (Rep/Cap)</td><td>Addgene</td><td>130878</td><td></td></tr><tr><td>pAAV2/9n (Rep/Cap)</td><td>Addgene</td><td>112865</td><td></td></tr><tr><td>pAnc80L65AAP</td><td>Addgene</td><td>92307</td><td></td></tr><tr><td>KP1 (rep/cap)</td><td></td><td></td><td>gifted by Professor Mark Kay (Stanford University)</td></tr><tr><td>LK03 (rep/cap)</td><td></td><td></td><td>gifted by Professor Mark Kay (Stanford University)</td></tr><tr><td>pAAV4 (rep/cap)</td><td></td><td></td><td>gifted by Professor Luk Vandenberghe (Harvard Medical School)</td></tr><tr><td>pAAV.Cas9.sgRNA</td><td>Addgene</td><td>61591</td><td></td></tr><tr><td>pAAV.MCS</td><td>Addgene</td><td>46954</td><td></td></tr><tr><td>gBlock (synthetic DNA fragement)</td><td>IDT</td><td></td><td></td></tr></tbody></table>

transgene expression in cultured cells using unpurified recombinant adeno-associated viral vectors

Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue types, making them a popular choice for gene delivery in animal models and in clinical settings. In addition to therapeutic applications, rAAVs are a useful laboratory tool for delivering transgenes tailored to the researcher&#39;s experimental needs and scientific goals in cultured cells. Some examples include exogenous reporter genes, overexpression cassettes, RNA interference, and CRISPR-based tools, including those for genome-wide screens. rAAV transductions are less harmful to cells than electroporation or chemical transfection and do not require any special equipment or expensive reagents to produce. Crude lysates or conditioned media containing rAAVs can be added directly to cultured cells without further purification to transduce many cell types&#8212;an underappreciated feature of rAAVs. Here, we provide protocols for basic transgene cassette cloning and demonstrate how to produce and apply crude rAAV preparations to cultured cells. As proof of principle, we demonstrate the transduction of three cell types that have not yet been reported in rAAV applications: placental cells, myoblasts, and small intestinal organoids. We discuss appropriate uses for crude rAAV preparations, the limitations of rAAVs for gene delivery, and considerations for capsid choice. This protocol outlines a simple, low-cost, and effective method for researchers to achieve productive DNA delivery in cell culture using rAAV without the need for laborious titration and purification steps.

Finding optimal capsid for transducing cultured cells of interest 
A variety of cell types were transduced to determine the tropism of various capsid serotypes (Figure 5). The natural serotypes AAV214, AAV421, AAV522, AAV823, AAV924, and the engineered capsid variants Anc8025, DJ26, LK0327, and KP128 were packaged with a vector genome expressing mScarlet under a CMV promoter, cloned using the methods provided in this protocol. Untitrated crude vector preparations were diluted in the appropriate culture media and cells were transduced for 24+ h and imaged (Figure 5B). Transduction efficiency was calculated by the proportion of total cells that were mScarlet+, or the proportion of cells in a single z-plane that were mScarlet+ (for mouse small intestinal organoids; Figure 5C). Transduction efficiencies (mScarlet+ cells) are not provided for differentiated C2C12 and HSkMC myotubes, but rather for undifferentiated C2C12 and HSkMC myoblasts (Figure 5A).
AAV2 and KP1 were the most potent serotypes across cell lines tested (Figure 5A). It was also observed that similar cell types originating from different species are transduced at varying efficiencies. For example, Hepa1-6 (a murine-derived liver cell line) exhibits a marked decrease in transduction compared to Huh7 (a human-derived liver cell line) when using AAV2 (Figure 5B). Moreover, different media conditions play an important role during transduction. Mouse small intestinal organoids (mSIOs) cultured in pre-transduction media are less effectively transduced compared to those cultured in organoid growth media (Figure 5C). Additionally, AAV2 efficiently transduces undifferentiated HSkMC myoblasts and differentiated HSkMC myotubes (Figure 5B), though quantitative analysis of myotubes is difficult and is therefore not provided.
The high transduction efficiencies observed for various cell types in Figure 5 shows that crude vector preparations can be used effectively to transduce a variety of cell types without further steps such as purification and titration. However, careful consideration should be given when choosing a capsid to maximize transgene delivery. Many other cell lines and capsids have been previously tested and are published, though with purified vector preparations12.
A vector particle independent effect of media conditions can affect the efficiency of transduction. However, it is generally accepted that tropism (i.e., cell-type specific uptake and expression of vector) is a property that is capsid specific29. A comparison between dose-matched crude and purified preparations has not yet been observed to affect cellular tropism. Therefore, crude vector preparations can be used in a similar manner as purified vectors in cell culture.
<img alt="Gene editing efficiency chart and fluorescent microscopy images analyzing mScarlet expression in cells." class="xfigimg" src="/files/ftp_upload/65572/65572fig05.jpg" data-altupdatedat="1747911167000" data-alt="AAV transduction efficiency chart and fluorescent microscopy of cell lines with mScarlet-tagged cells."> 
Figure 5: Crude vector transduction of various cell types. (A) Percentage of mScarlet+ following the addition of various AAV vectors containing an mScarlet transgene. (B) Cells expressing mScarlet delivered from AAV serotype 2. Scale bars: 100 µm (Hepa1-6, Huh7, C2C12, and HSkMC), 200 µm (BeWo), 20 µm (mSIO). Abbreviations: hpt = hours post transduction. (C) Mouse small intestinal organoids (mSIOs) were either rAAV treated in pre-transduction media or organoid growth media. <a href="https://www.jove.com/files/ftp_upload/65572/65572fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Table 1: Triple plasmid transfection worksheet. <a href="https://www.jove.com/files/ftp_upload/65572/TriplePlasmidTransfection_Worksheet_RE.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Table 2: PEI optimization worksheet. <a href="https://www.jove.com/files/ftp_upload/65572/PEIoptimization_Worksheet_RE.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Unveiling the Potential of Unpurified Recombinant AAVs in Cell Culture Research at JoVE.com

Author Spotlight: Unveiling the Potential of Unpurified Recombinant AAVs in Cell Culture Research 

Recombinant adeno-associated virus (rAAV) is widely used for clinical and preclinical gene delivery. An underappreciated use for rAAVs is the robust transduction of cultured cells without the need for purification. For researchers new to rAAV, we provide a protocol for transgene cassette cloning, crude vector production, and cell culture transduction.

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

Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue ...

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Research

JoVE Journal

Biology

2.8K Views.  University of California, Berkeley. Recombinant adeno-associated virus (rAAV) is widely used for clinical and preclinical gene delivery. An underappreciated use for rAAVs is the robust transduction of cultured cells without the need for purification. For researchers new to rAAV, we provide a protocol for transgene cassette cloning, crude vector production, and cell culture transduction. 

Video: Author Spotlight: Unveiling the Potential of Unpurified Recombinant AAVs in Cell Culture Research 

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors 

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

Medicine

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Immunology and Infection

Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique combination of beneficial properties1. These include the apathogenicity of the underlying wildtype viruses and the highly advanced methodologies for production of high-titer, high-purity and clinical-grade recombinant vectors2. A further particular advantage of the AAV system over other viruses is the availability of a wealth of naturally occurring serotypes which differ in essential properties yet can all be easily engineered as vectors using a common protocol1,2. Moreover, a number of groups including our own have recently devised strategies to use these natural viruses as templates for the creation of synthetic vectors which either combine the assets of multiple input serotypes, or which enhance the properties of a single isolate. The respective technologies to achieve these goals are either DNA family shuffling3, i.e. fragmentation of various AAV capsid genes followed by their re-assembly based on partial homologies (typically &gt;80% for most AAV serotypes), or peptide display4,5, i.e. insertion of usually seven amino acids into an exposed loop of the viral capsid where the peptide ideally mediates re-targeting to a desired cell type. For maximum success, both methods are applied in a high-throughput fashion whereby the protocols are up-scaled to yield libraries of around one million distinct capsid variants. Each clone is then comprised of a unique combination of numerous parental viruses (DNA shuffling approach) or contains a distinctive peptide within the same viral backbone (peptide display approach). The subsequent final step is iterative selection of such a library on target cells in order to enrich for individual capsids fulfilling most or ideally all requirements of the selection process. The latter preferably combines positive pressure, such as growth on a certain cell type of interest, with negative selection, for instance elimination of all capsids reacting with anti-AAV antibodies. This combination increases chances that synthetic capsids surviving the selection match the needs of the given application in a manner that would probably not have been found in any naturally occurring AAV isolate. Here, we focus on the DNA family shuffling method as the theoretically and experimentally more challenging of the two technologies. We describe and demonstrate all essential steps for the generation and selection of shuffled AAV libraries (Fig. 1), and then discuss the pitfalls and critical aspects of the protocols that one needs to be aware of in order to succeed with molecular AAV evolution.

Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling

We demonstrate the basic technique to molecularly engineer and evolve synthetic Adeno-associated viral (AAV) gene therapy vectors via DNA family shuffling. Moreover, we provide general guidelines and representative examples for selection and analysis of individual chimeric capsids with enhanced properties on target cells in culture or in mice.

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

An Efficient Method for Adenovirus Production

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by &#8220;negative&#8221; and &#8220;positive&#8221; PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

Here, we present a protocol for adenovirus production using the pAdEasy system. The technology includes the recombination of the pAdTrack and pAdEasy-1 plasmids, the adenovirus packaging and amplification, the purification of the adenoviral particles from cell lysate and culture medium, the viral titration, and the functional testing of the adenovirus.

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types ...

Bioengineering

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

Neuroscience

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Purification of AAV Vectors by Single-Step Heparin Affinity Chromatography

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs.
This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

Author Spotlight: Improved Method for Production and Purification of Adeno-Associated Viral Vectors

This manuscript describes a detailed protocol for the generation and purification of adeno-associated viral vectors using an optimized heparin-based affinity chromatography method. It presents a simple, scalable, and cost-effective approach, eliminating the need for ultracentrifugation. The resulting vectors exhibit high purity and biological activity, proving their value in preclinical studies.

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. ...

Simplified and Efficient AAV Production Using HEK293 Suspension Culture

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Author Spotlight: Advancing Gene Therapy with High-Yield AAV Vectors Through HEK293 Suspension Cell Cultivation

Here, a suspension HEK293 cell-based AAV production protocol is presented, resulting in reduced time and labor needed for vector production using components that are available for research purposes from commercial vendors.

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, ...

AAV Isolation by Double Iodixanol Density Gradient Centrifugation

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

Author Spotlight: Efficient Adeno-Associated Virus Isolation for Pre-Clinical Applications

Adeno-associated virus is produced in suspension cell culture and purified by double iodixanol density gradient centrifugation. Steps are included to increase total virus yield, decrease the risk of virus precipitation, and further concentrate the final virus product. Expected final titers reach 1012 viral particles/mL and are suitable for pre-clinical in vivo use.

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

Summary

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