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&#39;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 &#176;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 (&#60;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.&#160;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 &#181;g). 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 &#181;L 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&#160;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="Figure 6" class="xfigimg" src="/files/ftp_upload/65572/65572fig06.jpg" /> 
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&#8212;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&#160;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&#39;s selective membrane and reach the nucleus1,2. Therefore, the ability to effectively bypass the cell&#39;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&#39;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&#39;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&#160;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&#8212;i.e., on a separate, non-ITR containing plasmid that expresses regulatory and structural elements that then interact with the cis plasmid&#8212;and is thus called the trans plasmid. In addition to physically enclosing&#160;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 pAd&#916;F614,15. 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="Figure 1" class="xfigimg" src="/files/ftp_upload/65572/65572fig01v2.jpg" /> 
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&#39;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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#916;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&#160;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 &#38; 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&#160;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&#160;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&#160;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.

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

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

Gene therapy uses a viral capsid to introduce DNA into cells, and then the cells process that DNA through mechanisms that aren't fully understood. By studying how the cell processes AAV delivered DNA, we hope to improve safety, efficacy and longevity of AAV gene therapies. Studying AAV DNA processing at the tissue or organism level was quite difficult.And while we were developing cell culture transduction protocols for AAV visualization via super resolution microscopy, we found that the simplicity of producing and using unpurified AAV preparations in cell culture is not well known. This straightforward method for transient expression can be advantageous for researchers in numerous fields. Creating crude AAV preparations is quite simple, and they have a long shelf life F4C, allowing them to be used in numerous experiments.Applying them is simple, making them a time and cost-effective alternative to standard transfection reagents. Often AAV crude preparations are more efficient and less toxic than transfection. Vectrology has two arms, production and transduction, and both are entirely dependent on host cellular machinery.Our future research aims to identify the cellular machinery involved in these processes and ways to manipulate them so that we can improve production and transduction efficiency. Understanding the basic biology underpinning both arms of vectrology is vital for the success of gene therapies.

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="Figure 5" class="xfigimg" src="/files/ftp_upload/65572/65572fig05.jpg" /> 
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 &#181;m (Hepa1-6, Huh7, C2C12, and HSkMC), 200 &#181;m (BeWo), 20 &#181;m (mSIO). Abbreviations: hpt = hours post transduction. (C) Mouse small intestinal organoids (mSIOs) were either rAAV treated in pre-transduction media or organoid growth media.&#160;<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

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.

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

transgene-expression-cultured-cells-using-unpurified-recombinant

Research

JoVE Journal

Biology

Production of Recombinant Adeno-Associated Viral Vectors and Transduction of Cultured Cells

After cloning the gene of interest in adeno-associated ITR containing plasmid, seed three times 10 to the fifth cells into a six-well plate using pre-warmed DMEM supplemented with 10%FBS. Allow the cells to grow at 37 degrees Celsius and 5%carbon dioxide to attain 75 to 90%confluence. Next, dissolve 100 milligrams of polyethylenimine hydrochloride or PEI max in 100 milliliters of distilled water to make one microgram per microliter stock.Adjust the pH to 7.1 using sodium hydroxide. Then sterilize the mixture using a 0.22 micrometer filter and store the reagent for one month at four degrees Celsius. In a two milliliter tube, add 1.3 microgram rep/cap plasmid, 1.3 microgram ITR containing plasmid, and 2.6 microgram adenovirus helper plasmid to serum free DMEM.The total volume of this mixture should be 100 microliters. Prepare a negative control in a separate tube, replacing rep/cap plasmid with an unrelated plasmid. Now add 5.2 microliters of PEI max to the plasmid mixture.This maintains an equal plasmid to PEI ratio. Gently vortex 10 to 15 times on a vortex mixer set to seven. For multiple vectors, stagger the PEI addition at one minute intervals for sufficient time in subsequent steps.Incubate each tube for exactly 15 minutes at room temperature. Then dilute the reaction by adding 1.9 milliliters of serum free DMEM to achieve a final volume of two milliliters. Gently pipette twice to mix contents.Aspirate media from the well. Carefully add the plasmid PEI mixture to the sides of the well to avoid cell detachment and incubate cells for 72 hours at 37 degrees Celsius and 5%carbon dioxide. Then freeze the plate of transfected cells for 30 minutes at minus 80 degrees Celsius and thaw for 30 minutes at 37 degrees Celsius.Repeat the cycle a total of three times. In a laminar flow hood, aseptically mix each well by pipetting to disrupt cells effectively. Transfer the lysate to a two milliliter tube.Centrifuge at 15, 000 G for 15 minutes at room temperature to remove cell debris and carefully transfer the supernatant to a new two milliliter tube and store the tube at four degrees Celsius. Next, plate the desired cell type in a 96-well plate and incubate for a target confluency of 50 to 75%depending on the transduction duration. The following day, perform a one-to-three dilution series of the crude preparation in serum free media to obtain the optimal amount of vector required for transduction.Then aspirate the media from the 96-well plate and add 50 to 100 microliters of diluted crude preparation to the wells. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide. To determine the transduction efficiency, observe the expression of the fluorescent reporter using a fluorescent microscope at 48 hours post-transduction.For further analysis, remove the vector and wash cells once with pre-warmed PBS. Finally, terminate transduction by fixing cells in 4%paraformaldehyde for 10 minutes. Transduction efficiency data suggested AAV2 and KP1 were the most potent serotypes across all the tested cell lines.HEPA1-6 exhibited a marked decrease in transduction compared to Huh7. AAV2 efficiently transduced undifferentiated HSKMC myoblasts and differentiated HSKMC myotubes. Different media conditions play an important role during transduction.Mouse small intestinal organoids cultured in pre-transduction media were less effectively transduced compared to those cultured in organoid growth media.

1.8K 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. 

Crude Vector Production and Efficient DNA Delivery in Cell Cultures

Summary