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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Nat Protoc. Author manuscript; available in PMC Aug 9, 2013.
Published in final edited form as:
PMCID: PMC3739714
NIHMSID: NIHMS476366

AAV-mediated gene targeting methods for human cells

Abstract

Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types, with targeting frequencies ranging from 10−5 to 10−2 per infected cell. these targeting frequencies are 1–4 logs higher than those obtained by conventional transfection or electroporation approaches. a wide variety of different types of mutations can be introduced into chromosomal loci with high fidelity and without genotoxicity. Here we provide a detailed protocol for gene targeting in human cells with AAV vectors. We describe methods for vector design, stock preparation and titration. optimized transduction protocols are provided for human pluripotent stem cells, mesenchymal stem cells, fibroblasts and transformed cell lines, as well as a method for identifying targeted clones by southern blots. this protocol (from vector design through a single round of targeting and screening) can be completed in ~10 weeks; each subsequent round of targeting and screening should take an additional 7 weeks.

INTRODUCTION

Gene targeting has provided important insights into fundamental biological processes and the potential to cure genetic diseases. Although the process of homologous recombination (HR) works efficiently in bacteria and yeast, the maximum targeting efficiency achievable in mammalian cells is usually 1 per 106 transfected cells13. In addition, the targeting vector DNA can integrate randomly by nonhomologous end joining. The preparation of standard targeting constructs is also complicated, requiring several kilobase pairs of genomic DNA for homology arms, which can be expensive and time consuming.

Efforts to improve targeting frequencies have led to the exploitation of double-strand breaks to stimulate recombination. Customizable nucleases such as zinc finger nucleases (ZFNs) have improved the HR rates by logs, such that >1% of cells undergo targeting in some settings46. However, genotoxicity due to both on- and off-target cleavage by ZFNs is a potential problem7, as is the technical expertise required to design and test custom nucleases. Moreover, repair of the ZFN target-site break by nonhomologous end joining can lead to frequent mutations4,8, as was observed in up to 28% of target alleles in human embryonic stem cells (ESCs) in one study4. Moreover, nonhomologous integration of the targeting construct or ZFN expression cassette has also been reported6. Assessment of the in vivo cleavage specificity of ZFNs in a complex genome remains an unresolved issue that limits their use.

Adeno-associated virus (AAV) vectors have single-stranded DNA genomes, are efficiently delivered to the nucleus and can be constructed with simple PCR-based methods, making them an attractive alternative for gene targeting studies. Recombinant AAV vectors can efficiently target homologous chromosomal loci and introduce defined sequence changes with high fidelity. Several kinds of changes, including point mutations, insertions, deletions and selectable cassettes, have been introduced in a diverse variety of human cells933. Table 1 summarizes many of the various human cell lines and genes targeted by AAV vectors to date. Most of these experiments were conducted on transformed cell lines to elucidate the effect of specific genes involved in signaling pathways1113. However, AAV has also been successfully used to target primary human cells such as fibroblasts, keratinocytes, mesenchymal stem cells (MSCs), ESCs and induced pluripotent stem cells (iPSCs) at multiple loci including the genes COL1A1, COLIA2, HMGA1, HPRT1, NANOG and KRT14 (refs. 1518,32). More recently, AAV vectors have also been used for epitope tagging in a variety of human cell lines34,35, allowing proteins to be studied when expressed from their normal chromosomal locations.

TABLE 1
A summary of some human genes targeted with AAV vectors.

Using the methods described in this protocol, AAV-mediated gene targeting frequencies can vary from 10 to 10−2. Although a range of targeting frequencies is expected from any technology, there are a number of factors that can affect the targeting frequency of AAV vectors. These include the length of the homology arms36, the nature of the sequence change being introduced (deletion vectors target at lower frequencies)20, the position of the change being introduced within the homology arms36, the multiplicity of infection used19, and the proliferative status of the infected cells37. Other factors that may affect targeting frequencies but that have not been demonstrated experimentally include the transcriptional state of the target locus, the presence of nonisogenic DNA in the homology arms and the use of unpurified vector stocks. Finally, it should be noted that many experiments require expression of a selectable marker to measure targeting frequencies; if targeted alleles fail to express the selectable marker at adequate levels the apparent targeting frequencies will be reduced.

AAV-mediated gene targeting does have certain limitations, such as the small packaging capacity of AAV (~4.7 kb), the relative inefficiency of targeting silent loci38,39, the inability to target nondividing cells37 and a potential for random integration40. Conventional targeting approaches suffer from some of these same limitations, including random integration41,4244, and poor targeting in non-dividing cells45 and at silent loci44. It should be noted that the chances of a cell having both random and targeted integrations are minimal when using AAV vectors; in published reports, over 250 targeted clones were screened and only 7 also contained random integrants1517,19,32,46.

Experimental design

Vector design and preparation

Specific guidelines for vector design depend on the intended application. However, there are some general principles to follow. The packaging limit of AAV vectors is 4.7 kb; therefore, the targeting cassette has to be efficiently designed with the smallest possible genetic elements, and within this constraint the length of the homology arms should be maximized. For example, targeting frequencies were two- to fivefold higher with 3.0 kb of total homology, as compared with 1.7 kb of homology36. In addition, the mutation to be introduced should be centrally positioned with respect to the homology arms. Targeting frequencies drop precipitously when one of the homology arms is less than ~100 bp36. An advantage of the short homology arms used in AAV vectors is that, unlike the longer arms required for plasmid-based targeting approaches, they can be easily prepared by PCR of genomic DNA. A simplified technique to assemble an AAV targeting vector plasmid using three-way fusion PCR with homology arms and a selection cassette has been described24. The protocol described here involves independently amplifying each homology arm and selection cassette. The homology arms and the AAV backbone are assembled in a three-way ligation step followed by ligation of the selection cassette.

Although a multitude of mutations have been introduced using AAV vectors (see Table 1), insertions are clearly favored over deletions. Although the mechanism for this preference is unknown, the effect can be significant, with insertion vectors targeting at more than tenfold higher frequencies over a range of insertion/deletion sizes from 1 bp to more than 1 kb (ref. 20). As combined insertion-deletion vectors (those designed to both remove genomic DNA and insert foreign sequences) target at intermediate frequencies, this design can be preferable when introducing deletions.

Several typical vector designs are shown in Figure 1. Many applications require that a gene be knocked out so that it no longer expresses a functional protein. This can be done most easily by inserting a selectable marker and polyadenylation (pA) signal into a coding exon in a way predicted to inactivate the protein (Fig. 1a). If the gene is expressed at low levels or not at all, the selectable marker cassette must include a promoter. AAV vectors with this design have used promoters such as phosphoglycerate kinase, thymidine kinase and others22,23. Expressed loci can be targeted with a promoter-trap vector such that the chromosomal promoter drives expression of the selectable marker, and an internal ribosome entry site (IRES) element can be placed upstream of the selectable marker to initiate translation23. The promoter-trap design has the advantage that most random integrants will not express the selectable marker, increasing the percentage of drug-resistant colonies that are targeted. Typically, the random integration frequency is about tenfold higher than the gene targeting frequency19,23, and this ratio can be higher in silent loci38,39.

Figure 1
Vector design strategies. Maps of vector designs and target loci are shown. (a) For knockout vectors, an exon is disrupted with a promoter (P) or an IRES driving a selectable marker (SM) and flanked by loxP sites (L). Stop codons in all three reading ...

There are several points to consider in these designs. First, it is preferable to target a downstream exon if possible (not exon 1), because sequences immediately upstream of exon 1 contain the chromosomal target gene promoter and could increase gene expression at random locations when included in a homology arm. Second, even though the selectable marker cassette disrupts a coding region of an exon, it can still fail to disrupt protein function. This can happen when a non-essential peptide is encoded by that exon, and exon skipping occurs with splicing out of the transgene cassette15. It can be avoided by targeting an exon with a sequence that is not a multiple of 3 bp, such that exon-skipping will produce a frameshift in the coding sequence after splicing. Third, in many instances it may be desirable to remove the selectable marker cassette after targeting. This can be done by flanking the cassette with loxP sites and introducing Cre recombinase to delete sequences between these sites47. In this case, the pA site as well as a sequence with a stop codon in all three reading frames should be placed outside the loxP sites (Fig. 1a), so that these elements continue to disrupt protein function after Cre-mediated transgene deletion15. Finally, in some cases it may be possible to knockout a gene by directly inserting a selectable marker reading frame at the chromosomal initiation codon (ATG-trap variant of the promoter trap; Fig. 1b). The advantage of this approach is that it is extremely rare for random integrants to express the selectable marker, as this requires integration in a transcribed gene upstream of the first coding exon. An AAV vector designed to target the HMGA1 gene by an ATG trap resulted in > 95% of drug-resistant colonies being targeted17. It is best to use this strategy when there are 5′ untranslated exons to avoid including chromosomal promoter sequences in the homology arms.

Specific knock-in mutations can also be introduced with AAV vectors. In this case, a loxP-flanked selectable marker cassette is placed next to the mutation being introduced so that both are incorporated into the target locus (Fig. 1c). Cre-mediated recombination can then remove the selectable marker (including the pA site) and leave behind the desired mutation. It is important to confirm that the mutation was introduced with the selectable marker, as a recombination crossover can theoretically occur between these elements. To minimize the chance of this, the loxP-flanked cassette should be placed as close as possible to the mutation being introduced, keeping in mind that a single loxP site will remain after Cre-mediated deletion, and this could potentially affect gene function if placed too close to a splice junction or other genetic elements. As with knockout vectors, the selectable marker can include a promoter when targeting an unexpressed locus, or use a splice acceptor-IRES element in a promoter-trap strategy when targeting an expressed locus. Both approaches have been used with AAV vectors21,22.

In some cases, it may be possible to perform targeting without introducing a selectable marker. This type of vector design is simple, with homology arms flanking the mutation being introduced (Fig. 1d). As targeting occurs in up to 0.1–1% of the entire cell population17,19, in principle, one can screen ~100–1,000 colonies to identify a targeting event without using a selectable marker. However, this labor-intensive approach is not usually necessary, given the versatility of selectable marker strategies. Instead, this strategy is effective when correcting mutations, and has been used to target several reporter constructs (lacZ48, neo49, GFP46 and alkaline phosphatase9), as well as chromosomal genes (GusB (ref. 48) and HPRT1 (ref. 9)). HPRT1 is an especially versatile target as it is an X-linked gene present as a single copy in diploid male cells, and drug selections are available for both HPRT1 and HPRT1+ cells. In a recent study, the KRT14 gene was successfully targeted in 0.6% of the total cells transduced32. Mutant enhanced GFP has been corrected using AAV vectors under nonselective conditions at an efficiency of 0.1% of the entire population46, although lower frequencies have been reported in the absence of double-strand break–induced AAV targeting50,51.

Single-nucleotide polymorphisms (SNPs) occur at a frequency of 1 per 500–1,000 bp in humans; therefore, they are likely to be present in the homology arms of targeting vectors. Conventional gene targeting studies in mice have shown that the use of isogenic DNA improves targeting frequencies by 20-fold, suggesting that mismatches between the target and targeting construct inhibit recombination52. In the case of human cells, a deleterious effect of SNPs on targeting frequencies has not been demonstrated. Several studies have shown that non-isogenic DNA can be used for targeting different human cell lines53, including when AAV vectors are used17. However, to date no one has directly compared the targeting frequencies of vectors with and without known SNPs in the homology arms; therefore, it is possible that SNPs have an effect. To avoid possible SNP effects, one can amplify the homology arms from the same cell line to be targeted. In addition, to avoid the introduction of PCR errors, we amplify homology arms in three independent PCR reactions, and subclone and sequence three independent products. When the exact same sequence is recovered at least twice, it must be present at a minimum of one target locus allele. Novel SNPs can sometimes be identified in this way and distinguished from PCR errors.

AAV stock production

Wild-type AAV is a defective, non-pathogenic parvovirus that requires coinfection with a helper virus such as adenovirus for establishing a productive infection. Recombinant AAV vectors are derived by replacing the rep and cap genes of wild-type AAV with foreign DNA, which is flanked by the AAV inverted terminal repeats, the only obligatory viral element. The entire vector genome is prepared in a plasmid form along with a bacterial origin and an antibiotic resistance gene, and transfected into human cells during vector production. Stocks are produced by co-transfecting this vector plasmid with additional helper plasmid(s) containing the AAV rep and cap genes and specific adenovirus helper genes into human cells, which then replicate the vector genome and package it into virions. We use helper plasmids pDG (ref. 54) to produce serotype 2 vectors and pDGM3B (ref. 55) to produce serotype 3 vectors (which replaces the AAV2 capsid gene in pDG with that from AAV3B). These plasmids combine the AAV and adenovirus helper functions. Excellent protocols for the preparation of AAV stocks by both iodixanol and CsCl methods are provided by Grieger et al.56. They describe a three-plasmid transfection method to generate AAV stocks. Our approach uses a dual-plasmid transfection system, in which both the adenoviral and AAV genes required are included on a single helper plasmid54, and the serotype 2 stocks are purified by heparin affinity column; this is followed by desalting57.

Serotype 2 was the original serotype developed for AAV vectors. However, over 100 different serotypes of AAV have since been isolated from various animal species58, and several of these have been used to produce vectors. These serotypes have distinct capsids that use different cellular receptors to transduce specific cell types at markedly different rates. For most of the experiments conducted on cultured cells the AAV2 serotype works well. One notable exception is that the AAV3 serotype transduces human pluripotent stem cells (PSCs) at twofold higher frequencies than the AAV2 serotype18, and might be preferred for these applications. Experiments done in our laboratory have also shown a two- to fourfold higher transduction efficiency of ESCs by AAV3 compared with AAV2 serotypes (R.K.H. and D.W.R., unpublished data). Both AAV2 and AAV3 vector serotypes are produced in the same way, except that distinct helper plasmids containing serotype-specific cap genes are used.

Purification of AAV based on CsCl2 density gradients59 was originally used to isolate AAV vector particles, and it is still popular. However, CsCl2 is toxic to cells and must be completely removed before using the stock. We recommend using nontoxic iodixanol density gradients instead57, as for many applications the vector can be delivered directly to cells while in iodixanol. It is important to use some form of density gradient purification so that empty virions can be removed from the stock (these have a different density than packaged virions), as these empty virions could bind to cellular receptors and inhibit transduction. Additional methods can be used to further purify vector particles, but these may be serotype specific. In the case of AAV2, a heparin affinity column works well, as the closely related heparan sulfate molecule is the natural cell surface receptor for AAV2. Extensive vector purification is not always necessary, and some investigators have successfully used crude stocks (transfected cell lysates) for gene targeting22. Although this saves time, the presence of cellular debris and low titers of crude stocks can decrease transduction frequencies.

Vector stocks can be titered in several ways. The most consistent methods are based on quantifying the number of vector genomes by using Southern blots, dot blots or quantitative PCR. The dot blot and quantitative PCR methods are quicker, but they will not provide any information on the integrity of the vector genomes, which may undergo rearrangements during vector production. Alkaline gel Southern blots are an especially informative way to titer vector stocks60, as the genomes are analyzed as denatured single strands. However, in our experience this method can result in degradation and smearing of hybridized bands. Instead, we routinely titer AAV vector stocks by Southern blots of neutral gels, taking into account that some vector genomes may pair with each other to create double-stranded molecules.

Transduction of cells

AAV vectors transduce many types of cultured cells, and all that is necessary is to add vector directly to the cell culture medium. We typically plate cells the day before infection to ensure a consistent cell number and density. Vector dose is determined by the number of genome-containing particles per cell, which can range from hundreds to many thousands. If using an antibiotic resistance gene, the antibiotic selection should not be commenced earlier than 24 h after infection, and typically at least 48 h later, to allow the transgene to be adequately expressed. The minimum lethal dose for the antibiotic varies depending on the cell type and should be empirically determined using a kill curve. A negative control of untransduced cells should always be included in parallel. In addition, a dish with 0.5% (somatic cells) or 5% (ESCs/iPSCs) of infected cells should be grown without selection, to determine the total number of colony-forming units (CFUs) plated. A special consideration for PSCs is their poor plating efficiency, particularly when passaged as single cells. Because of this, more transduced colonies can be obtained if cultures are not manipulated after infection until colonies are visible and picked. Somatic cells can be passaged as early as 1 d after infection, and replated to the appropriate size dishes for colony selection. This allows one to infect in a smaller volume, which results in more effective vector delivery.

Once drug-resistant colonies appear, these can be counted and/or isolated for further expansion and analysis. The transduction frequency equals the total number of drug-resistant CFUs obtained from a single infection divided by the total number of unselected CFUs from the same infection. Total CFU numbers can be obtained from colony counts by correcting for the fraction of cells plated with and without selection. Importantly, the drug-resistant colonies may include both targeted clones and clones with randomly integrated vector genomes. Therefore, to determine the targeting frequency, the fraction of drug-resistant colonies that are targeted must also be established (see below). The absolute targeting frequency can then be expressed as the total number of targeted CFUs per infection divided by the total number of unselected CFUs. The targeting frequency can also be expressed as the fraction of drug-resistant colonies that are targeted; however, this number is highly dependent on vector design and does not accurately assess the proportion of cells undergoing HR.

Targeting can be demonstrated conclusively by analyzing genomic DNA from expanded, transduced colonies. The most definitive proof is a Southern blot using a restriction enzyme with at least one recognition site outside the homology region and a genomic probe outside the homology region as well (example in Fig. 2). This ensures that only the chromosomal target locus is detected and not random integrants. The probe should be 500–1,000 bp in size and free of repeated sequences, as determined by RepeatMasker. A separate vector-specific probe such as the selectable marker gene should be used to confirm targeting and detect random integration events (typically < 5% of targeted clones contain additional random integrants, reflecting the frequency of random integration in the total cell population). DNA from the parental, untargeted cell line should always be included as a control in Southern blots. Concatamerized vector multimers can integrate at the target locus16; accordingly, one should assay both sides of the targeted locus with specific restriction enzymes and/or probes for a complete characterization. PCR can also be used to identify targeted clones and with appropriate controls can be quite reliable. Typically, one primer lies in the vector selectable marker and the other in flanking chromosomal DNA outside the homology region. However, once targeted clones are identified, we recommend confirming PCR results with Southern blots. When analyzing fewer than 50 clones, it is often quicker to just perform Southern blots.

Figure 2
Detecting homologous recombination with a Southern blot. (a) Schematic representation of the AAV targeting vector, AAV2-HMGA1-Neo-pA shown above the wild type (WT) HMGA1 locus and the locus created by targeting. Targeting homologies are indicated as gray ...

MATERIALS

REAGENTS

  • 293T cells61 (ATCC, cat. no. CRL-1573)
  • Agarose, Genepure LE (ISC Bioexpress, cat. no. E-3120-500)
  • Ampicillin (Shelton Scientific, cat. no. IB02040)
  • Amphotericin B (Invitrogen, cat. no. 04195780 D)
  • Bactoagar (Difco Laboratories, cat. no. 214010)
  • Basic fibroblast growth factor (bFGF; Invitrogen, cat. no. 13256-029)
  • Benzonase (Novagen, cat. no. 70664-3)
  • β-Mercaptoethanol (Sigma, cat. no. M7522) An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0001.jpg It is toxic, avoid inhalation, ingestion and skin contact. Use gloves, safety glasses and good ventilation.
  • BSA (Sigma, cat. no. A3912)
  • Bromophenol blue (Sigma, cat. no. B-5525)
  • CaCl2·2H2O (Sigma, cat. no. C3306-500G)
  • Potassium acetate (C2H3KO2, Sigma, cat. no. P1190-500G)
  • Calf intestinal alkaline phosphatase (CIP, New England Biolabs, cat. no. M0290S)
  • DH10B electrocompetent cells (Invitrogen, cat. no. 18290015)
  • Disodium EDTA·2H2O (Sigma, cat. no. E5134-500G)
  • Dispase (Invitrogen, cat. no. 17105-041)
  • DMEM/F12 (Invitrogen, cat. no. 11320-033)
  • DMEM high glucose (Invitrogen, cat. no. 10566-016)
  • DMEM low glucose (Invitrogen, cat. no. 11885-084)
  • DMSO (Sigma, cat. no. D4540) An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0002.jpg It is light sensitive.
  • DR4 mouse embryonic fibroblasts (MEFs)62
  • Dow Corning High-Vacuum Grease (Ellsworth Adhesives, cat. no. 1597418)
  • Easytides deoxycytidine 5′ triphosphate {α32P} (PerkinElmer) An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0003.jpg It is radioactive. Avoid contact with skin. Handle inside a fume hood; wear protective clothing including gloves, plastic shield and safety glasses.
  • Ethanol (Decon Labs, cat. no. 2716)
  • FBS (Hyclone, cat. no. SH30071.03)
  • Gelatin from porcine skin (Sigma, cat. no. G-1890)
  • Glacial acetic acid (JT Baker, cat. no. 9508-05)
  • Glycerol (Fisher, cat. no. BP229-4)
  • HCl (JT Baker, cat. no. 9535-02) An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0004.jpg It is toxic, avoid inhalation, ingestion and skin contact: use gloves, safety glasses and good ventilation.
  • HEPES (Fisher, cat. no. BP310-100) An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0005.jpg It causes eye, skin and respiratory tract irritation. Avoid inhalation, ingestion and skin contact during use.
  • Iodixanol (Greiner America, cat. no. 103-0061; 60% (wt/vol) Optiprep (Nycomed))
  • Isopropanol (Fisher, cat. no. A416-500)
  • KCl (Fisher, cat. no. P217-500)
  • Knockout serum replacement (KSR, Invitrogen, cat. no. 10828-028)
  • λDNA/HindIII digest (Invitrogen, cat. no. 15612-013)
  • LB-ampicillin plates containing 100 μg ml−1 ampicillin
  • l-Glutamine (Invitrogen, cat. no. 25030-081)
  • Methanol (Fisher, cat. no. A412-4)
  • MgCl2·6H2O (Sigma, cat. no. M2393-500G)
  • Mycoalert mycoplasma detection kit (Lonza Cologne AG, cat. no. LT07-218)
  • Sodium citrate (Na3C6H5O7, Sigma, C8532-1KG)
  • NaCl (Fisher, cat. no., BP358-212)
  • NaH2PO4 (Sigma, cat. no. S3139-500G)
  • Na2HPO4 (Fisher, cat. no. BP393-3)
  • NaOH (JT Baker, cat. no. 3722-01)
  • Nonessential amino acids (Invitrogen, cat. no. 11140-050)
  • pAAV-MCS, AAV vector plasmid (Agilent Technologies, cat. no. 240071) An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0006.jpg Prepare in advance using Qiagen Plasmid Maxi kit.
  • PBS (1×; Invitrogen, cat. no. 14190-144)
  • pDG, AAV2 helper plasmid54 (Aldevron, cat. no. 5503-1). The plasmid sequence can be accessed at https://www.lablife.org/ An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0007.jpg Prepare the plasmid in advance using Qiagen Plasmid Maxi kit.
  • pDGM3B, AAV3 helper plasmid55 (similar to pDG but the AAV2 capsid gene is replaced with that from AAV3B). The plasmid and its sequence can be requested from Dr. David Russell) An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0008.jpg Prepare the plasmid in advance using Qiagen Plasmid Maxi kit.
  • Penicillin/streptomycin (Invitrogen, cat. no. 15070-630)
  • Pfu Turbo DNA polymerase (Stratagene, cat. no. 600250)
  • pGEM-T Easy Vector System I (Promega, cat. no. A1360)
  • Phenol red (Sigma, cat. no. P0290)
  • Plugged Cassou straws (Veterinary concepts, cat. no. 04170)
  • QIAGEN Plasmid Maxi Kit (Qiagen, cat. no. 12163)
  • QIAprep Spin Miniprep Kit (Qiagen, cat. no. 27104)
  • QIAquick Gel Extraction Kit (Qiagen, cat. no. 28704)
  • Rediprime II Random Prime Labeling System (Amersham, cat. no. RPN1633)
  • Restriction enzymes (New England Biolabs)
  • RNAse A solution (Sigma, cat. no. R6513)
  • Salmon sperm DNA (DNA sodium salt from Salmon testes; Sigma-Aldrich, cat. no. D1626)
  • SDS (Fisher, cat. no. BP166-500) An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0009.jpg It is toxic, avoid inhalation, ingestion and skin contact; use gloves and safety glasses and maintain good ventilation.
  • Sodium pyruvate (Invitrogen, cat. no. 11360-070)
  • T4 DNA ligase (New England Biolabs, cat. no. M0202T)
  • Tryptone (granulated, EMD Biosciences, cat. no. 1.07213.2500)
  • Tris base (Sigma, cat. no. T1503-500G)
  • Trypsin-EDTA solution (Invitrogen, cat. no. 15400-054)
  • Ultrapure phenol/chloroform/isoamylalcohol (25:24:1 (vol/vol/vol); Invitrogen, cat. no. 15593-031) An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0010.jpg The mixture is toxic, avoid inhalation, ingestion and skin contact; use gloves and safety glasses and maintain good ventilation. It is also light sensitive.
  • Xylene cyanol (Sigma, cat. no. X-4126)
  • Yeast extract (EMD Biosciences, cat. no. 1.03753.0500)
  • Ethidium bromide (Sigma-Aldrich, cat no. E1510)
  • Distilled water (dH2O)

EQUIPMENT

  • Amersham Hybond-XL (GE Healthcare, cat. no. RPN 203S)
  • Automated cell counter (Nucleo Counter)
  • Calibrated pipettes (50 μl; VWR International, cat. no. 53432-783)
  • Centrifuge tube (50 ml, for pelleting and freeze thawing AAV stock; Corning, cat. no. OPP 430522)
  • Centrifuge tubes (Beckman OptiSeal Tubes, Beckman, cat. no. 361625)
  • Chromatography paper 3 MM CHR (Whatman International, cat. no. 3030-866)
  • Clamp stand
  • Cloning cylinders, borosilicate glass (Bellco Glass, cat. no. 2090-00608)
  • Cryotube vials (Nunc, cat. no. 377224)
  • Dissecting forceps (sterilize before use)
  • Dissecting scissors (sterilize before use)
  • Dry ice (~2 lb)
  • Electrophoresis apparatus (Horizontal gel system SGU-014T-IPL, CBS Scientific)
  • Electroporation system (Micro Pulser, Bio-Rad)
  • Fluorometer (Hoefer DyNA Quant 200, Amersham)
  • G-50 Micro columns, Illutra ProbeQuant (GE Healthcare, cat. no. 28-9034-08)
  • Gene Pulser cuvettes (Bio-Rad, cat. no. 165-2089)
  • Glass Pasteur pipettes (sterile, cotton plugged)
  • Hemocytometer (Brightline Hemacytometer, Hausser Scientific)
  • High-speed centrifuge (RC 5B Plus, Sorvall)
  • HiTrap Desalting column (GE Healthcare, cat. no. 17-1408-01)
  • HiTrap heparin affinity column (1.0 ml; GE Healthcare, cat. no. 17-0406-01)
  • H2O bath at 37 °C (183, Precision Scientific)
  • Hybridization cylinders (Robbins Scientific)
  • Hybridization incubator (400, Robbins Scientific)
  • Inverted microscope (DMLS, Leica)
  • Irradiator with a Cesium-137 source (J.L. Shepherd and Associates, model no. 143-5A)
  • Kodak 480 RA processor (X-OMAT, KODAK)
  • Liquid nitrogen tank (CryoMed, Forma Scientific)
  • Luer adaptor, Union Luer female (GE Healthcare, cat. no. 18-1112-51)
  • Microcentrifuge (5415C, Eppendorf)
  • Millex GS filter unit (0.22 μm; Millipore, cat. no. SLGS033SS)
  • Mouth pipette (supplied along with the calibrated pipette except for the 0.2-μm Millex GS filter unit; VWR International, cat. no. 53432-783)
  • Needles (18- and 20-gauge)
  • PhosphorImager (Storm 820, Amersham)
  • Rotating shaker (Labquaker shaker 400110, Barnstead/Thermolyne)
  • Rubber policeman
  • Screw-cap micro tubes (Sarstedt, cat. no. 72.692.005)
  • Spinal needle with blunt end (1.27 mm × 89 mm)
  • Stereomicroscope (MZ6, Leica)
  • Stericup and Steritop vacuum-driven filtration system (0.22-μm pore, GP Millipore Express PLUS membrane; Millipore, cat. no. SCGPT01RE)
  • Syringe pump (Legato 200, KD Scientific)
  • Syringes (10 ml)
  • Tabletop centrifuge, ventilated and refrigerated (model no. 5677, Forma Scientific)
  • Tissue culture incubator (37 °C with 5% CO2 atmosphere; Steri-Cult 200, Forma Scientific)
  • Ultracentrifuge (XL-80, Beckman)
  • UV Stratalinker (2400, Stratagene)
  • UV transilluminator (FBT1V-88, Fisher)
  • Water bath shaker (C76, New Brunswick Scientific)
  • RepeatMasker software (http://www.repeatmasker.org/)
  • ImageQuant software (GE Healthcare)
  • Kimwipes

REAGENT SETUP

An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0011.jpg Unless stated otherwise, reagents can be stored indefinitely at the specified temperature.

CaCl2 (2.0 M) Dissolve 29.4 g of CaCl2·6H2O in a final volume of 100 ml dH2O. Sterilize by passing through a 0.22-μm filter and store in 2-ml aliquots at 4 °C.

Cell lysis buffer (for AAV stock preparation) Add 3 ml of 5 M NaCl and 5 ml of 1 M Tris-HCl (pH 8.5) to 80 ml of dH2O. Adjust the pH to 8.5 with NaOH and adjust the volume to 100 ml with dH2O. Sterilize by passing through a 0.22-μm filter and store at 4 °C.

Church buffer Combine 500 ml of 1 M phosphate buffer (pH 7.2), 2 ml of 0.5 M EDTA, 10 g BSA and 70 g SDS in a final volume of 1 liter with dH2O. Store at room temperature (25 °C).

Coomassie blue stain Combine 1.5 g Coomassie Brilliant Blue G, 300 ml methanol and 100 ml glacial acetic acid in a final volume of 1 liter with dH2O. Store at room temperature.

D10 (DMEM with 10% FBS (vol/vol)) Combine 500 ml of DMEM high glucose with 50 ml of FBS, 5 ml of penicillin/streptomycin and 2.5 ml of amphotericin B (final concentration, 1.25 μg ml−1). There is no need to filter-sterilize the solution as all components are sterile. Store at 4 °C and use within 1 month.

Dispase solution (0.1% (wt/vol)) Dissolve 100 mg of Dispase in 100 ml of ESC wash medium. Sterilize by passing through a 0.22-μm filter and store at 4 °C. Use within 1 week.

EDTA solution 0.5 M (pH 8.0) Dissolve 186.1 g of disodium EDTA·2H2O in 700 ml of H2O. Adjust the pH to 8.0 with NaOH and adjust volume to 1 liter with dH2O. Sterilize by autoclaving and store at room temperature.

ESC medium Combine 500 ml of F12/DMEM, 100 ml of KSR, 6 ml of penicillin/streptomycin, 6 ml of sodium pyruvate, 6 ml of nonessential amino acids, 0.6 ml of 0.1 M β-mercaptoethanol and 0.3 ml of a 4-μg ml−1 bFGF solution. Sterilize by passing through a 0.22-μm filter and store at 4 °C. Use within 2 weeks.

ESC wash medium Add 50 ml of KSR to 500 ml of DMEM high glucose. There is no need to filter sterilize as both components are sterile. Store at 4 °C for up to 2 months.

Gelatin (0.1% (wt/vol)) Dissolve 5 g of gelatin in 1 liter of dH2O to prepare 0.5% stock solution, sterilize by autoclaving and store at room temperature. Prepare a 0.1% gelatin working solution as a 1:5 dilution of the 0.5% stock in sterile dH2O. Store at room temperature.

Genomic DNA lysis solution Combine 1 ml of 1 M Tris-HCl (pH 8.0), 0.2 ml of 0.5 M EDTA (pH 8.0) and 0.1 g of SDS in a final volume of 100 ml with dH2O. Store at room temperature.

HEPES-saline (2×) Dissolve 8.18 g of NaCl and 5.96 g of HEPES in 400 ml dH2O. Adjust pH to 7.1 with 0.5 N NaOH. Adjust the volume to 500 ml with dH2O. Sterilize by passing through a 0.22-μm filter and store in aliquots at 4 °C.

Irradiated MEFs MEFs are used as feeder cells for the culture of ESCs/iPSCs. DR4 MEFs are used because they are resistant to G418, 6-thioguanine, puromycin and hygromycin, and can be used in experiments that use these selective agents. An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0012.jpg Prepare the irradiated MEFs in advance as described in Box 1.

Box 1

PREPARATION OF IRRADIATED MEFS

  1. Euthanize a 13.5-d pregnant mouse by cervical dislocation.
    An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0046.jpg Experimental animals should be handled in accordance with institutional regulations.
  2. Thoroughly wet the mouse with 70% (vol/vol) ethanol and place the mouse on an underpad with the abdomen facing up. Cut a parabolic-shaped incision with scissors on the abdominal wall near the posterior end.
  3. With scissors, cut the uterine horns free from the connective membranous tissue while holding the uterus with forceps.
  4. Once the uterine horns are free, cut the cervix to remove the uterus and place the uterus in a 10-cm dish containing 10 ml of 1× PBS.
  5. Cut through the uterine wall near an embryo and move the embryo out of the uterus with forceps.
  6. Pierce the amniotic sac and tease the embryo out, freeing it from the placenta and umbilical cord.
  7. Place the embryo in a second 10-cm dish containing 1× PBS. Repeat for each embryo in the uterus.
  8. Remove and discard the head from each embryo with scissors. Remove and discard the heart and liver (red tissue) from each embryo.
  9. Transfer the headless embryos to a fresh 10-cm dish containing 10 ml of 1× PBS.
  10. Take the embryos to the tissue culture hood.
  11. Place a syringe tip cap on a 10-ml syringe, remove the plunger from the syringe and back-load the embryos into the syringe with forceps. Add 7 ml of MEF medium and reinsert the plunger.
  12. Replace the syringe tip cap with an 18-gauge needle on the syringe and push the embryos through into a 50-ml tube.
    An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0047.jpg Discard syringe and needle to a sharps container.
  13. Place a syringe tip cap on the 10-ml syringe again. Back-load the tissue suspension into the syringe.
  14. Use 2–3 ml of MEF medium to rinse the 50-ml tube and add to the syringe. Reinsert the plunger. Replace the syringe tip cap with a 20-gauge needle and push the tissue suspension as before into a new 50-ml tube.
  15. Centrifuge the cell suspension at 250g for 5 min at 20 °C in a tabletop centrifuge.
  16. Aspirate the medium from the conical tube and resuspend the pellet in MEF medium and plate 1 ml into 10-cm dishes (passage 1, p1). The amount of MEF medium used for resuspension is dependent on the number of embryos harvested with 1 ml of MEF medium per two to three embryos. Incubate in a 37 °C incubator for 2–3 d.
  17. After 2–3 d, harvest cells from each 10-cm dish using trypsin-EDTA. Centrifuge and resuspend in MEF medium (2.5 ml per 10-cm dish harvested) and plate 0.5 ml of cell suspension to each 10-cm dish (p2).
    An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0048.jpg Plate a small fraction of the cells to one 6-cm dish to use for a mycoplasma test. The next day change the medium on the 6-cm dish to MEF medium without penicillin/streptomycin. Perform mycoplasma test using the Mycoalert mycoplasma detection kit according to the manufacturer’s instructions 1 d later.
  18. Three days after plating cells, harvest all 10 cm dishes with trypsin-EDTA, centrifuge at 250g for 5 min at 20 °C and resuspend in MEF freezing medium (1 ml per 10-cm dish).
  19. Aliquot 1 ml per cryotube vial and store at −80 °C overnight. Transfer the p2 MEFs to liquid nitrogen the following day.
    An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0049.jpg p2 MEFs can be stored in liquid nitrogen indefinitely.
  20. Thaw one vial of p2 MEFs (approximately 5 × 106 cells) to a 15-ml conical tube in 5 ml of MEF medium.
  21. Pipette up and down gently to mix.
  22. Centrifuge at 250g for 5 min at 20 °C in a tabletop centrifuge.
  23. Aspirate the medium and resuspend the pellet in 5 ml of MEF medium.
  24. Transfer 0.5 ml of the cell suspension into ten 10-cm dishes (p3), each containing 10 ml of MEF medium. Incubate in a 37 °C incubator with 5% CO2 atmosphere for 3 d.
    An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0050.jpg After transferring all the cell suspension, mix the cells evenly by shaking the dish up and down, as well as left and right to ensure that the cells are evenly distributed.
  25. After 3 d, harvest the p3 MEFs using trypsin-EDTA solution, centrifuge at 250g for 5 min at 20 °C and discard the supernatant.
  26. Resuspend the pellet in 30 ml of MEF medium and plate 0.5 ml of cell suspension in 60 10-cm dishes, each containing 10 ml of MEF medium.
  27. Mix well to ensure that cells are evenly distributed. Incubate in a 37 °C incubator for 3 d.
  28. After 3 d, prepare 15 ml of MEF freezing medium.
  29. Harvest five 10-cm dishes at a time, using 2 ml of trypsin-EDTA and neutralizing with 4 ml of MEF medium per dish. Centrifuge to pellet down the cells at 250g for 5 min at 20 °C in a tabletop centrifuge. The same tube can be used for pelleting cells from all 60 dishes sequentially.
  30. Resuspend the final pellet from the 60 10-cm dishes in 30 ml of MEF medium.
  31. Irradiate the cells with 40 Gy using an irradiator with a Cesium-137 radiation source.
  32. Remove a 10-μl aliquot from the 30-ml cell suspension and count cells with a hemocytometer.
  33. Centrifuge the cells at 250g for 5 min at 20 °C in a tabletop centrifuge.
  34. Remove the supernatant and resuspend the cells at a concentration of 2 × 107 cells per ml in MEF freezing medium. In this way, you can prepare one straw with 200 μl of freezing medium containing 4 × 106 MEFs, or one cryotube vial with 800 μl of freezing medium containing 1.6 × 107 cells.
  35. Load Cassou straws (option A) or prepare cryotube vials (option B) as described below.

(A) Freezing MEFs in Cassou straws

  1. Attach a sterile, plugged Cassou straw to a 1-ml syringe.
  2. Aspirate ~½ inch of freezing medium without cells to the top of the straw, and suck up 1/8 to 1/4 inch of an air bubble.
  3. Suck up the cells in the freezing medium (~0.2 ml) leaving ½ inch empty at the bottom.
    An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0051.jpg Mix cells in the tube before loading straws.
  4. Pull the straw out of medium, then suck up last ½ inch with air at the bottom.
  5. Place back in sterile package. Repeat steps (i–iv) for all the straws.
  6. Flame-seal all the straws (melt, blacken and then finger squeeze), first the bottom then the top.
  7. Place at −80 °C overnight and then transfer to liquid nitrogen the next day.
    An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0052.jpg p4 MEFs can be stored in liquid nitrogen indefinitely.

(B) Freezing MEFs in cryotube vials

  1. Aliquot 800 μl of cell suspension in freezing medium to each vial.
  2. Place cryotube vials at −80 °C overnight. Transfer to liquid nitrogen the next day.
    An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0053.jpg p4 MEFs can be stored in liquid nitrogen indefinitely.

KCl (250 mM) Dissolve 1.86 g of KCl in dH2O in a final volume of 1 liter. Sterilize by autoclaving and store at room temperature.

Loading dye (6×) Combine 5 ml of glycerol, 25 mg of bromophenol blue and 25 mg of xylene cyanol in sterile dH2O in a final volume of 10 ml. Store at 4 °C in 1-ml aliquots.

LB medium (Luria-Bertani medium) Dissolve 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl in dH2O in a final volume of 1 liter. Adjust pH to 7.0 with 5 N NaOH. Autoclave and store at room temperature for up to 3 months.

MEF freezing medium To prepare 15 ml of freezing medium, combine 1.5 ml of FBS, 1.5 ml of DMSO and 12 ml of MEF medium. Prepare fresh before use.

MEF medium Mix 500 ml of DMEM high glucose with 50 ml of FBS and 5.6 ml of penicillin/streptomycin. There is no need to filter-sterilize the medium, as all components are sterile. Store at 4 °C and use within 1 month.

MgCl2 (2 M) Dissolve 19 g of MgCl2 in dH2O in a final volume of 100 ml. Sterilize by autoclaving and store at room temperature.

MSC medium Combine 500 ml of DMEM low glucose with 50 ml of FBS, 5.6 ml of l-glutamine and 5.6 ml of penicillin/streptomycin. There is no need to filter-sterilize the medium as all components are sterile. Store at 4 °C and use within 1 month.

NaCl/PBS-MK buffer (1 M) Dissolve 5.84 g of NaCl, 26.3 mg of MgCl2 and 14.91 mg of KCl in 1× PBS in a final volume of 100 ml. Sterilize by passing through a 0.22-μm filter and store at 4 °C.

PBS-MK buffer Dissolve 26.3 mg of MgCl2 and 14.91 mg of KCl in 1× PBS in a final volume of 100 ml. Sterilize by passing through a 0.22-μm filter and store at 4 °C.

Phosphate buffer (0.15 M) Add 4.95 ml of 1.0 M NaH2PO4 and 10.05 ml of 1.0 M Na2HPO4 to dH2O in a final volume of 100 ml. Sterilize by passing through a 0.22-μm filter and store in aliquots at 4 °C.

Protein precipitation solution Combine 60 ml of 5 M C2H3KO2 and 11.5 ml of glacial acetic acid in a final volume of 100 ml with dH2O. Store at room temperature.

Salmon sperm DNA Dissolve salmon sperm DNA in dH2O at a concentration of 10 mg ml−1. To dissolve the DNA, stir the solution for at least 2–4 h at room temperature. Adjust the concentration of NaCl to 0.1 M and extract the solution once with phenol and once with phenol/chloroform. Recover the aqueous phase and shear the DNA by passing the solution rapidly 12 times through a 17-gauge needle. Precipitate using ethanol, centrifuge and dissolve at a concentration of 10 mg ml−1. Denature DNA by boiling for 10 min and store at − 20 °C in 1-ml aliquots. Boil for 5 min and snap-chill on ice before use in hybridization.

Super optimal broth (SOB) medium Dissolve 20 g of tryptone, 5 g of yeast extract and 0.5 g of NaCl in 900 ml of dH2O. Add 10 ml of 250 mM KCl and adjust pH to 7.0 with 5 N NaOH. Adjust the volume to 1 liter with dH2O and sterilize by autoclaving on a liquid cycle. Store at room temperature for up to 3 months. Add 5 ml of sterile 2 M MgCl2 before use.

Southern wash solution 1 Add 10 ml of 20× SSC and 1 ml of 10% (wt/vol) SDS to dH2O in a final volume of 100 ml. Store at room temperature.

Southern wash solution 2 Add 1.5 ml of 20× SSC and 15 ml of 10% (wt/vol) SDS to dH2O in a final volume of 150 ml. Store at room temperature.

SSC (20×) Dissolve 175.3 g of NaCl and 88.2 g of Na3C6H5O7 in 600 ml dH2O and adjust pH to 7.0 with 10 M NaOH. Adjust the volume to 1 liter with dH2O. Sterilize by autoclaving and store at room temperature.

TAE buffer (50×) Combine 242 g of Tris base, 57.1 ml of glacial acetic acid and 100 ml of 0.5 M EDTA in a final volume of 1 liter with dH2O. Store at room temperature.

Tris-EDTA (TE, pH 8.0) Combine 1 ml of 1 M Tris-HCl (pH 8.0) with 0.2 ml of 0.5 M EDTA solution in a final volume of 100 ml with dH2O. All components should be sterile. Store at room temperature.

Tris-HCl (1 M, pH 8.5) Dissolve 121.1 g of Tris base in 800 ml of dH2O. Adjust the pH to 8.5 with HCl. Adjust the volume to 1 liter with dH2O. Sterilize by autoclaving and store at room temperature.

EQUIPMENT SETUP

Preparation of cloning cylinders Prepare cloning cylinders by layering them on top of Dow Corning High-Vacuum Grease in a glass Petri dish; sterilize by autoclaving.

PROCEDURE

AAV stock production An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0013.jpg 9 d

1| Thaw a vial of 293T cells (each vial contains ~3 × 106 cells) and plate cells in a 10-cm dish containing 10 ml D10. Maintain in a 37 °C incubator with 5% CO2 atmosphere.

2| After 3 d, aspirate the medium and wash the dish with 2 ml of 1× PBS. Add 2 ml of trypsin-EDTA solution and incubate at 37 °C for 2 min or until cells detach from culture dish.

3| Add 4 ml of D10 to neutralize trypsin-EDTA solution and transfer the cell suspension to a 15-ml conical tube.

4| Centrifuge at 250g for 5 min at 20 °C in a tabletop centrifuge.

5| Resuspend the cells in 1 ml of D10. Take a 10-μl aliquot and count the cells using a hemocytometer.

6| Adjust cell concentration to 106 cells per ml with D10 and plate 1 ml of cell suspension in eight 10-cm dishes, each containing 10 ml D10. Culture for 72 h in a 37 °C incubator with 5% CO2 atmosphere.

7| After the 3-d incubation, harvest cells as described in Steps 2–4. Resuspend in 5 ml of D10 and take a 10-μl aliquot for counting with a hemocytometer. Adjust cell concentration to 4 × 106 cells per ml and plate 1 ml of cell suspension in 30 10-cm dishes, each containing 10 ml D10. Incubate in a 37 °C incubator overnight.

8| The next day, prepare solution A by combining 15.5 ml of 2× HEPES-saline and 155 μl of 0.15 M phosphate buffer in a 50-ml tube and, mixing well.

9| In another 50-ml tube, prepare solution B by combining 1.938 ml of 2 M CaCl2, 387.5 μg of plasmid pDG, 387.5 μg of AAV vector plasmid (prepared as described in Box 2) and water to a final volume of 15.5 ml. Mix well.

Box 2

AAV VECTOR PLASMID CONSTRUCTION

A protocol for the construction of AAV targeting vector plasmids is also mentioned in Rago et al65. The method described below differs from theirs in several ways, including sequential ligation reactions for the homology arms and selection cassette, instructions for PCR amplification of the homology arms that avoid amplification-induced errors and suggestions for screening plasmids and quantifying the inverted terminal repeat (ITR) deletions that inevitably occur. The vector plasmid sequence and status of its ITRs may influence the function of the vector stocks that are produced with it. The protocol below describes the assembly of a vector plasmid designed to insert a loxP-flanked selection cassette and produce a functional knockout of the target gene.

  1. Prepare the AAV vector backbone by digesting 5 μg of plasmid pAAV-MCS with NotI restriction enzyme and gel-purifying the backbone fragment with the QIAquick Gel Extraction Kit according to the manufacturer’s instructions.
  2. Dephosphorylate the digested backbone using 0.5 U CIP per μg of DNA at 37 °C for 1 h.
  3. The vector backbone is extracted using phenol-chloroform, precipitated using ethanol and resuspended in 50 μl of TE.
  4. Amplify the selection marker cassette using 5′ and 3′ oligos designed to include loxP sites and restriction sites compatible with the homology arms. A polyadenylation signal should be included outside the loxP site on the reverse oligo, and a sequence with a stop codon in all three reading frames should be placed outside the loxP sites on the forward oligo.
  5. Run the amplified selection cassette on 0.8% (wt/vol) agarose gel in 1× TAE buffer and extract the desired fragment using QIAquick Gel Extraction Kit.
  6. Design primers for the amplification of homology arms. We amplify the 5′ and 3′ homology arms in separate PCR reactions from the cell line to be used in the targeting experiment. The forward oligo for the 5′ homology arm and the reverse oligo for the 3′ homology arm are designed to have a NotI site for insertion into the AAV vector backbone. The reverse oligo for the 5′ homology arm and the forward oligo for the 3′ homology arm should have appropriate restriction sites for ligating to the selection marker fragment.
  7. Set up three independent PCR reactions for the amplification of each homology arm using TURBO Taq polymerase, as described below. This is to avoid PCR errors.
    ComponentAmount (μl)Final
    PCR reaction buffer (10×)5
    dNTPs (2.5 mM)40.2 mM
    Forward oligo (10 μM)2.50.5 μM
    Reverse oligo (10 μM)2.50.5 μM
    Turbo DNA polymerase (2.5 U μl−1 Pfu)10.05 U μl−1
    Template DNA (100–500 ng)12–10 ng μl−1
    dH2O34
    Total volume50
  8. Perform PCR as follows:
    Cycle numberDenatureAnnealExtend
    195 °C, 2 min
    2–3595 °C, 30 s5 °C below Tm, 30 s72 °C, 1 min per kb
    3672 °C, 10 min
  9. Run the products from the three independent PCR reactions separately on 0.8% (wt/vol) agarose gel in 1× TAE buffer and gel-extract the homology arms with the QIAquick Gel Extraction Kit.
  10. Clone the gel-eluted PCR products from each of the three separate PCR reactions into pGEM-T Easy Vector using the pGEM-T Easy Vector system I kit according to the manufacturer’s instructions.
  11. Sequence at least one clone from each independent PCR reaction. When the exact same sequence is recovered at least twice, PCR errors can be ruled out and this sequence must be present at a minimum of one target locus allele. Choose this sequence for incorporation into the vector plasmid. New SNPs can sometimes be identified this way.
  12. Digest the plasmids containing cloned homology arms with the appropriate restriction enzymes for vector construction and gel-purify these fragments.
  13. Set up a three-way ligation reaction with the AAV vector backbone (30 ng) and the 5′ and 3′ homology arms using T4 DNA ligase according to the manufacturer’s instructions. A control ligation with only the AAV vector backbone should also be set up. The vector backbone and homology arm DNAs should be in a 1:3:3 molar ratio in the ligation reaction.
    An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0054.jpg The homology arms and the vector backbone should have compatible sticky ends for the ligation to be successful.
  14. Electroporate 2 μl of the ligation mix as well as the control mix separately into 50 μl of electrocompetent DH10B cells. Immediately add 500 μl of SOB medium to the electroporation cuvette, transfer to a 14-ml tube and incubate at 37 °C for 1 h while shaking (200 r.p.m.) in a water bath.
  15. After 1 h, take out the 14-ml tube and plate 100 μl of the cells on LB-ampicillin plates from both the vector and control ligations. Incubate the plates overnight at 37 °C.
  16. The next day, count the colonies on the vector and control plates. There should be significantly more colonies in the vector plate. Inoculate ten colonies from the vector plate in 2 ml of LB-ampicillin broth and incubate overnight at 37 °C with shaking (200 r.p.m.).
  17. The next day, isolate DNA with the QIAprep Spin Miniprep Kit according to manufacturer’s instructions and perform diagnostic digests with restriction enzymes to confirm the presence of ligated fragments.
  18. Digest this plasmid with a restriction enzyme cutting in between the homology arms. In addition, digest the selection marker cassette with the same restriction enzyme (from Step 5).
  19. Set up a ligation reaction with the vector backbone (with homology arms inserted) and the selection cassette using T4 DNA ligase according to the manufacturer’s instructions. A 1:3 vector/insert ratio should be maintained using 30 ng of the backbone. Set up a control ligation (no insert) in parallel.
  20. Perform Steps 14–17 as described above.
  21. Perform diagnostic restriction enzyme digestions on the plasmids to confirm the presence of the ligated fragments, the junction restriction sites, as well as the AhdI and BglI sites present in the hairpin regions of the ITRs, which can be deleted during bacterial culture.
  22. When the plasmid is confirmed, streak out the miniprep culture of the correct plasmid onto a fresh LB-ampicillin plate and incubate overnight at 37 °C.
  23. The next day, pick a single colony, inoculate a 2-ml culture of LB-ampicillin broth and incubate overnight at 37 °C with shaking (200 r.p.m.).
  24. The next day, inoculate this starter culture into 500 ml of LB-ampicillin broth, and incubate overnight at 37 °C with shaking (200 r.p.m.).
  25. The next day, isolate plasmid DNA using the Qiagen Maxiprep kit.
  26. Further purify the plasmid DNA by phenol/chloroform/isoamyl extraction and ethanol precipitation. Resuspend the DNA in 500 μl of TE and check with restriction enzymes, including AhdI and BglI.
    An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0055.jpg When AAV constructs are grown in bacterial cultures, there is a tendency for the inverted terminal repeats (ITRs) to acquire deletions. The simplest way to check for ITR deletions is by restriction digestion of the vector plasmid with AhdI and BglI, which cut within the viral ITRs. If the ITRs are deleted or rearranged, then these sites are lost and the plasmid appears to be partially digested at these sites. Plasmids with a significant percentage of ITR-deleted forms (>10%) should not be used, as these forms do not replicate and package properly. Poor packaging could adversely affect vector function and possibly reduce targeting frequencies.
  27. Incubate at 70 °C for 10 min and cool at room temperature to ensure sterility before transfection.

10| Add solution B to solution A, mix well and quickly add 1.0 ml of the transfection mix to each of the 30 dishes of 293T cells. An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0014.jpg Use a 10-ml pipette and tilt the dish so that the 1.0 ml of transfection mix is added to a greater depth of medium to prevent ‘blasting’ cells off the dish.

11| Agitate the dishes gently to evenly distribute the precipitate and return the dishes to the 37 °C incubator.

12| Observe a dish 1 h later under a microscope. The precipitate should be visible as small grains about the size of bacteria. Return the dishes to the 37 °C incubator.

13| After 48 h, prepare dry ice/ethanol bath by placing a centrifuge tube rack in a styrofoam container. Add ~2 lbs of dry ice, pour 2 liters of ethanol over it and cover the container.

14| Collect the cells and medium by scraping the cells off the dish with a rubber policeman and transferring it to a 50-ml conical tube. We use Corning 50-ml conical tubes, as they are more resistant to the subsequent freeze-thaw cycles. Rinse dishes with 5 ml of 1× PBS and transfer it to the same conical tube. Harvest four dishes at a time into the same 50-ml tube.

15| Centrifuge at 250g for 5 min at 20 °C in a tabletop centrifuge.

16| Aspirate the medium from the conical tube and repeat Steps 14–15 until cells are pelleted from all 30 dishes. The same tube can be used for pelleting cells from all the dishes.

17| Resuspend the final pellet from the 30 10-cm dishes in 8 ml of cell lysis buffer.

18| Freeze the pellet in the dry ice/ethanol bath and thaw in a 37 °C water bath.

19| Repeat Step 18 twice and store at −80 °C. An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0015.jpg The pellet can be stored at −80 °C indefinitely and thawed at your convenience.

20| Thaw cell lysate in a 37 °C water bath.

21| Add Benzonase to the cell lysate at a final concentration of 50 U ml−1. Benzonase is added to eliminate cellular DNAs/RNAs as well as excess plasmid DNAs present during transfection.

22| Incubate at 37 °C in a water bath for 30 min. Prepare iodixanol gradient (refer to Box 3) during the incubation.

Box 3

PREPARATION AND LOADING OF IODIXANOL GRADIENT

Preformed iodixanol step gradients are used to expedite the centrifugation step. The 40% and 25% iodixanol steps are used to remove contaminants with lower densities including empty capsids, and a 60% step serves as a cushion for genome-containing recombinant AAV virions. To clearly see the steps, phenol red is included in the upper 25% and lower 60% steps. The first step (15%) has 1 M NaCl to destabilize ionic interactions between macromolecules. The following volumes are for two gradients using OptiSeal tubes. The 1.27 mm × 89 mm spinal needle holds ~0.4 ml.

1. Prepare the following iodixanol solutions:

15% Iodixanol step: Mix 4.5 ml of 60% iodixanol and 13.5 ml of 1 M NaCl/PBS-MK buffer.

25% Iodixanol step: Mix 5 ml of 60% iodixanol, 7 ml of PBS-MK buffer and 30 μl of Phenol red.

40% Iodixanol step: Mix 6.7 ml of 60% iodixanol and 3.3 ml of PBS-MK buffer.

60% Iodixanol step: Mix 10 ml of 60% iodixanol and 45 μl of Phenol red.

2. Overlay each centrifuge tube with these solutions in the order below using a 10-ml syringe and 1.27 mm × 89 mm spinal needle, taking care to avoid bubbles. The same needle can be used for loading all steps.

5 ml of 60% iodixanol step

5 ml of 40% iodixanol step

6 ml of 25% iodixanol step

9 ml of 15% iodixanol step

3. Continue from Step 25 of PROCEDURE.

23| Centrifuge at 5,000g for 30 min at 4 °C in a Sorvall high-speed centrifuge using an HS-4 rotor.

24| Collect vector-containing supernatant. The volume of the supernatant is approximately 5 ml.

25| Load the 5 ml of vector-containing supernatant over the iodixanol density gradient prepared in Step 22. Top off the tube with cell lysis buffer. This density gradient purification step can remove contaminants that may influence transduction and targeting frequencies.

26| Centrifuge at 461,300g for 1 h at 18 °C with maximum acceleration and deceleration in a Beckman Ti70 rotor on a Beckman ultracentrifuge; use proper spacers for tubes.

27| Puncture the tube on the side slightly below (3–5 mm) the 60–40% interface with an 18-gauge needle (bevel up) attached to a 10 ml syringe.

28| Collect 3–4 ml from each centrifuge tube by aspiration using the same needle. An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0016.jpg Avoid the proteinaceous material near the 40–25% interface.

29| To use the vector without removing iodixanol, proceed directly with Step 30; iodixanol is inert and non-toxic to cells. Alternatively, further purify Serotype 2 vector stocks over a HiTrap heparin affinity column, followed by desalting over a HiTrap desalting column as described in Box 4, before proceeding with Step 30.

Box 4

PURIFICATION OF AAV2 STOCKS BY HEPARIN AFFINITY COLUMN FOLLOWED BY DESALTING

  1. Remove the top cap from the HiTrap heparin affinity column and add a few drops (~200 μl) of PBS-MK buffer to the top of the column to avoid air bubbles.
  2. Connect the HiTrap Luer adaptor to the top of the column. Some of the buffer will overflow; absorb this with Kimwipes.
  3. Add a few drops (~100 μl) of PBS-MK buffer to the top Luer adaptor.
  4. Place the 10 ml syringe with 10 ml PBS-MK buffer into the Luer adaptor.
  5. Remove the bottom and snap it off.
  6. Wash the column with PBS-MK buffer. It takes ~8 min to deliver 10 ml PBS-MK.
    An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0056.jpg Air bubbles must be removed from the liquid in the syringe at all steps before pushing it through the column, as they tend to impede flow. Do not exceed a flow rate of 4 ml min−1.
    An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0057.jpg Each time a step is completed, replace the bottom cap before you remove the syringe from the column, then remove the syringe and add 100 μl of PBS-MK buffer to the Luer adaptor. When the syringe is placed in the Luer adaptor for the next step, remove the bottom cap.
  7. Load the column using the 10-ml syringe containing the iodixanol fraction (from Step 29 of PROCEDURE). Use a syringe pump and rate of 20 ml h−1. Reserve approximately 25–30 μl before loading on the column for quantitation.
  8. Collect the flow-through in a 14-ml tube.
  9. Wash column with 5 ml of PBS-MK buffer using a syringe pump at the rate of 20 ml h−1.
  10. Collect the flow-through in a 14-ml tube.
  11. Elute vector from the column with 2.4 ml of 1 M NaCl/PBS-MK buffer.
  12. Collect the first 0.4 ml in a microcentrifuge tube. This is the ‘void volume’.
  13. After this initial 0.4-ml void volume, collect the following fractions: fraction 1 is 1 ml, fraction 2 is 0.5 ml and fraction 3 is 0.5 ml. The vector should come off the column in the first 1.5 ml (fractions 1 and 2).
  14. Desalt after the heparin affinity column purification with a HiTrap desalting column. Wash the HiTrap desalting column with 25 ml of DMEM (or other medium/buffer) in a 30-ml syringe. An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0058.jpg Do not exceed a flow rate of 5 ml min−1.
  15. Prepare the following:
    A 3-ml syringe containing 1.5 ml of fractions 1 and 2.
    A 3-ml syringe containing 3 ml of DMEM high glucose (or other medium/buffer to be used when transducing cells).
  16. Position the column to collect fractions into microcentrifuge tubes: the first tube for the 1.5 ml of void flow-through, then ‘exchange’ fractions X1–X3 (1 ml each).
  17. Load the 1.5 ml of vector into the 3-ml syringe.
  18. Quickly load the 3 ml of DMEM high glucose or other medium in the second 3-ml syringe.
  19. The vector should be in fractions X1 and X2. Pool fractions X1 and X2 and make 10 aliquots of 200 μl each (or other aliquot sizes) in microcentrifuge tubes. Prepare 1:10 and 1:100 dilutions of the AAV stock in TE buffer for determining titer later.
    An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0059.jpg The vector stock and dilutions can be stored at −80 °C indefinitely. If necessary, store stocks in small aliquots and avoid repeated freezing and thawing.

AAV titering using neutral gel electrophoresis and Southern blotting An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0017.jpg 4 d

30| To prepare plasmid standards for quantitation, digest the same vector plasmid used to prepare the stock, such that the vector genome is released from the surrounding inverted terminal repeats.

Set up the restriction digest by adding the following to a microcentrifuge tube: restriction buffer (10×, 2 μl); plasmid DNA (1,000 ng); restriction enzyme (5 U); and dH2O to a final volume of 20 μl.

31| Incubate the restriction digest in a 37 °C water bath for 1 h. Stop the reaction by adding 2 μl of 0.5 M EDTA.

32| Run 2 μl of the restriction digest on a 1% (wt/vol) agarose gel to confirm that the plasmid is completely digested.

33| Accurately quantify the digested DNA content by using a fluorometer and make serial dilutions as follows: 100, 30, 10 and 3 pg μl−1.

34| Prepare 1% (wt/vol) agarose gel in 1× TAE. Allow it to set at room temperature for 1 h.

35| Label four microcentrifuge tubes as 1,000, 300, 100 and 30 pg. Pipette out 10 μl from each standard dilution into the appropriate tube.

36| Take the AAV stock out of the −80 °C freezer and thaw at room temperature.

37| Prepare two microcentrifuge tubes, each containing 3 μl of TE and 1 μl of 6× loading dye. Add 2 μl of the 1:10 vector stock dilution to the first tube and 2 μl of the 1:100 stock dilution to the other (dilution preparation described in step 19, Box 4).

38| Clip the AAV stock tubes tightly.

39| Boil them for 2.5 min, snap-cool quickly on ice. An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0018.jpg The vector is boiled to disrupt its capsid and to release the single-stranded DNA genome before loading it onto a neutral gel.

40| Load the standards and AAV stock dilutions along with λ/HindIII marker on the 1% (wt/vol) agarose gel and run at 120 V for 4 h.

41| Stain the gel in 1× TAE containing 0.5 μg ml−1 of ethidium bromide for 30 min. The gel can be destained in dH2O for 20 min if required. Photograph on a UV transilluminator with a fluorescent ruler by the side.

42| Transfer the gel to a tray and acid-depurinate with 500 ml of 0.25 M HCl for 10 min, twice. Rock the gel gently.

43| Remove the HCl solution and neutralize with 500 ml of 0.4 M NaOH for 15 min, twice. Rock the gel gently.

44| Transfer to a Hybond-XL membrane with 1 liter of 0.4 M NaOH overnight using standard Southern blot wicking methods63.

45| The next day, briefly rinse the membrane in 2× SSC.

46| UV crosslink the membrane on a UV Stratalinker (auto-crosslink setting). An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0019.jpg The membrane can be stored at 4 °C, wrapped in plastic wrap, for several months.

47| Prehybridize the membrane with 14 ml of Church buffer containing 150 μg ml−1 of salmon sperm DNA (boiled for 5 min and snap-chilled on ice) for 3–4 h in a hybridization cylinder rotating in a hybridization incubator at 65 °C.

48| Synthesize a radiolabeled probe by random priming with a Rediprime II kit according to the manufacturer’s instructions. An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0020.jpg Follow all relevant institutional guidelines while working with radioactivity. Radioactive probe must be prepared in a fume hood. Wear protective clothing including gloves, plastic shield and safety glasses.

49| Purify the radiolabeled probe with a ProbeQuant G-50 microcolumn. Denature it by boiling for 5 min and snap-cool on ice. An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0021.jpg Use screw cap tubes for boiling to prevent leakage of radioactive probe.

50| Add the denatured probe to the prehybridization solution and incubate at 65 °C overnight in the hybridization oven.

51| The following day, remove the radioactive hybridization solution and dispose of it properly. Wash the hybridized membrane twice with 45 ml of Southern wash solution 1 for 15 min at 65 °C in a hybridization cylinder. An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0022.jpg Follow institutional guidelines while working with radioactivity. The radioactive waste must be discarded in the designated radioactive waste disposal container. Wear protective clothing including gloves, plastic shield and safety glasses.

52| Wash the membrane twice with 45 ml of Southern wash solution 2 for 15 min at 65 °C in a hybridization cylinder.

53| Expose the membrane to X-ray film at −80 °C overnight. Develop the X-ray film on a Kodak 480 RA processor. This provides a visual record of the stock titer.

54| Measure the radioactive signal by placing the membrane in a PhosphorImager cassette for 30–60 min and analyze on a PhosphorImager using ImageQUANT densitometry software.

55| Generate a standard curve based on the radioactive signal of the standard dilutions and determine the vector titer. The radioactive signal from the vector sample(s) should fall within the standard curve and can be used to calculate the number of genome-containing vector particles per ml. Accurate titering of the vector stock is critical for performing infections at controlled multiplicity of infections (MOIs), which is known to influence targeting frequencies. An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0023.jpg

Transduction of cells with AAV vector stock

56| To transduce somatic cell lines (MSCs, fibroblasts, transformed cell lines) follow option A; the protocol works efficiently for primary or transformed cell lines but MSCs have been used here as an example. To transduce pluripotent cells, follow option B.

(A) Transduction of somatic cell lines An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0024.jpg 14 d

  1. Harvest MSCs by trypsin treatment, as described in Steps 2–4, except use MSC medium instead of D10.
  2. Resuspend the cell pellet in 1 ml of MSC medium. Take a 10-μl aliquot and determine the cell number using a hemocytometer.
  3. Plate 2.5 to 5 × 104 cells per well of a 24-well plate in 0.5 ml of MSC medium. Culture in a 37 °C incubator overnight.
  4. The following day, thaw a vial of AAV vector stock quickly in a 37 °C water bath. Pipette up and down vigorously with a pipette, vortex for 30 s, and spin in a microcentrifuge for 30 s at maximum speed to suspend the vector and pellet debris.
  5. Add the desired amount of vector stock. Typically, an MOI of 2,000–20,000 genome-containing vector particles per cell is adequate. When a particular AAV stock is being used for the first time, cells should be transduced at three MOIs, each differing by a log, to determine the optimal dose for transduction.
  6. Expose cells to the vector overnight. Leave one well untransduced as a control for selection.
  7. The following day, harvest the cells by trypsin treatment and plate three 10 cm dishes, each with 33% of cells (other dilutions can be plated instead, in order to obtain the desired number of transduced colonies per dish). In addition, plate 0.5% of the cells to calculate plating efficiency in a 10-cm dish. The untransduced cells should also be plated the same way to serve as a control for selection.
  8. Add antibiotic to the MSC medium at the appropriate concentration. The concentration should be empirically determined beforehand using a kill curve. Add antibiotic-containing medium 48 h post transduction to all dishes, except to the 0.5% plating dish reserved for determining plating efficiency.
  9. Change the medium every third day.
  10. Maintain the dishes in selection medium for the next 8–10 d or until the cells in the uninfected control dish are dead. An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0025.jpg
  11. If clones are not to be picked, stain the dishes with Coomassie blue. Stain the unselected dish as well. Otherwise, proceed to Step 57A. An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0026.jpg

(B) Transduction of PSCs An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0027.jpg 20 d

  1. Add 3.5 ml of 0.1% (wt/vol) gelatin to a 6 cm dish. Place the dish in a 37 °C incubator for 30 min.
  2. Aspirate the gelatin and seed the dish with 3.3 × 105 irradiated (mitotically inactivated) MEF cells in MEF medium (prepared as described in Box 1). An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0028.jpg As selection is used in gene targeting experiments, we use irradiated MEFs prepared from the DR-4 mouse strain bearing resistance genes for four different drugs: G418, 6-thioguanine, puromycin and hygromycin62.
  3. Culture in a 37 °C incubator with 5% CO2 atmosphere overnight.
  4. The following day, wash the MEF dish with 1× PBS and replenish with 3 ml of ESC medium. Return the dish to the 37 °C incubator.
  5. Fill a 15-ml conical tube with 7 ml of prewarmed ESC wash medium.
  6. Take a frozen straw of ESCs/iPSCs from liquid N2 and thaw in a beaker of lukewarm tap water. We freeze ESCs and iPSCs in Cassou straws64, with about 2 to 4 × 105 cells per straw; one straw can be used to seed one 6-cm dish preseeded with MEFs. To thaw, cut the bottom off the straw with sterile scissors over a waste bin.
  7. Cut the top off the straw over the 15-ml conical tube and let the cells drain into the tube.
  8. Centrifuge the cells at 250g for 3 min at 20 °C, resuspend in 1 ml of ESC medium and plate in the pre-seeded MEF dish containing 3 ml ESC medium (from Step 56B(iv)). Culture in a 37 °C incubator with 5% CO2.
  9. After 3 d, prepare a six-well plate and a 6-cm dish of irradiated MEFs. Briefly, a six-well plate is coated with 1.5 ml of 0.1% (wt/vol) gelatin per well and a 6-cm dish with 3.5 ml for 30 min at 37 °C. The gelatin is aspirated and the six-well plate is seeded with 1.8 × 105 MEFs per well and the 6-cm dish with 3.3 × 105 MEFs in MEF medium. Incubate in a 37 °C incubator overnight.
  10. Four days after thawing ESC/iPSCs, remove the medium from the dish. Add 2 ml of 0.1% (wt/vol) Dispase solution to the 6 cm dish to enzymatically disaggregate the cells.
  11. Incubate at room temperature for 3 min. The dish should be observed under an inverted microscope. The ESC/iPSC colonies should begin to curl up along their edges.
  12. Aspirate 0.1% (wt/vol) Dispase solution.
  13. Add 2 ml of ESC wash media to the dish and aspirate it.
  14. Add 2 ml of ESC wash medium again and pipette with a plugged Pasteur pipette to remove cells, working from the bottom of the dish up.
  15. Collect all the cells into a 15-ml conical tube containing 5 ml of ESC wash medium.
  16. Rinse the dish with 3 ml of ESC wash medium and transfer into the 15-ml conical tube.
  17. Centrifuge at 250g for 3 min at 20 °C in a tabletop centrifuge.
  18. Discard the supernatant and resuspend the pellet in 3 ml of ESC medium.
  19. Take a 100-μl aliquot for counting cells on an automated cell counter (Nucleo Counter) according to the manufacturer’s instructions. The cell counts can also be determined using a hemocytometer after disaggregating the 100-μl aliquot into a single cell suspension with trypsin-EDTA solution; however, the cells are very small and difficult to observe.
  20. Plate 105 cells per well of a six-well plate preseeded with irradiated MEFs (from Step 56B(ix)) and place in a 37 °C incubator with 5% CO2. In addition, plate 5 × 103 cells over a 6-cm dish (from Step 56B(ix)) for calculating the plating efficiency.
  21. The following day, change the medium with fresh ESC medium. Infect the cells with the AAV stock by adding stock directly to the dish at an MOI of 2,000–20,000 genome-containing particles per ESC/iPSC cell, after resuspending vector particles as described in Step 56A(iv). Leave one well untransduced as a control for selection. Place in a 37 °C incubator overnight.
  22. The next day, change the medium with ESC medium. Place in a 37 °C incubator overnight.
  23. After 48 h, add ESC medium containing the appropriate antibiotic in all wells except in the plating efficiency dish, which should not receive the antibiotic.
  24. Change the medium every alternate day. At 7–8 d after plating, stain the 6-cm plating efficiency dish with Coomassie blue.
  25. On the seventh day, spike the dishes with irradiated MEFs. Briefly, thaw irradiated MEFs in 7 ml of ESC wash medium, centrifuge at 250g for 5 min at 20 °C in a tabletop centrifuge at room temperature. An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0029.jpg
  26. As the cells are spinning, remove the medium from the six-well plate, wash once with 1× PBS and replenish with 1 ml of fresh ESC medium per well.
  27. Aspirate the medium from the conical tube, resuspend the MEFs in 6 ml of ESC medium and add 1 ml per well of the six-well plate. When spiking, we plate half the number of MEFs plated initially. For example, for spiking the six-well plate, 5.5 × 105 (9.1 × 104 per well) MEFs are required. Place in a 37 °C incubator.
  28. Maintain the dishes in selection media for the next 8–10 d or until the cells in the uninfected control well are dead. The medium should be changed daily. Proceed to step 57B for picking drug-resistant foci.

Picking drug-resistant clones

57| Follow option A to pick somatic cell clones; the protocol works efficiently for primary or transformed cell lines. MSCs have been used here as an example. Follow option B to pick pluripotent cell clones.

(A) Picking drug-resistant clones: somatic cell clones An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0030.jpg 0.5 h
  1. Identify drug-resistant colonies by scanning the dishes (from Step 56A(xi)) with an inverted microscope and circle them using a permanent ink pen on the bottom of the dish.
  2. Prepare a 24-well plate with 0.5 ml of MSC medium per well planning on one well per colony to be picked.
  3. Aspirate medium from the dish containing drug-resistant colonies and wash once with 1× PBS.
  4. With forceps, place cloning cylinders (see EQUIPMENT SETUP) over all the foci to be picked. Ensure that the cloning cylinders are firmly attached to the dish and that they encircle the colonies.
  5. Pipette 20 μl of trypsin-EDTA solution into the cloning ring and incubate for 2 min at 37 °C. Add 30 μl of MSC medium to neutralize the trypsin-EDTA.
  6. Collect the detached cells from the cloning cylinder with a pipette and plate each colony into one well of the 24-well plate prepared in Step 57A(ii).
  7. After picking the clones, stain the dishes with Coomassie blue to determine the number of CFUs.
  8. (viii) If required, further culture and passage the clones to bigger dishes for freezing and harvesting DNA. We generally transfer somatic cells to 10-cm dishes for DNA extraction.

(B) Picking drug-resistant clones: PSCs An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0031.jpg 1 h
  1. Seed a 24-well plate with 3.7 × 104 irradiated MEFs per well, 1 d before picking resistant colonies. Place in a 37 °C incubator overnight.
  2. The next day, wash the 24-well plate with 1× PBS and add 0.5 ml of ESC medium.
  3. Identify drug-resistant colonies by scanning the dishes (from Step 56B(xxviii)) with an inverted microscope and circle the resistant colonies underneath the dish with a permanent marker.
  4. Prepare calibrated pipettes by pulling them over a flame to refine the tip. The new ends should be shaped in the form of a small needle with an opening.
  5. Set up a stereomicroscope in the tissue culture hood. It can be wiped with a 70% (vol/vol) ethanol solution to improve sterility.
  6. Assemble the mouth pipette by attaching rubber tubing on either side of a Millex GS filter unit (0.22 μm). Attach the small red mouthpiece to one end (the rubber tubing and the mouthpiece are supplied along with the calibrated pipettes).
  7. Spray the calibrated pipette with 70% (vol/vol) ethanol and wipe off with a Kimwipe.
  8. Attach the calibrated pipette to the other end of the mouth pipette.
  9. While viewing with the dissecting microscope, break up each colony into tiny fragments with the calibrated pipette and aspirate the fragments by suction.
  10. Plate the fragments from each colony into a separate well of the 24-well MEF plate prepared in Step 57B(i). An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0032.jpg Use a separate calibrated pipette for each clone to avoid contamination.
  11. After picking the clones, stain the dishes with Coomassie blue to determine the number of CFUs. This will identify the colonies that were not picked.
  12. If required, carry out further passaging and expansion of the clones for freezing and DNA extraction using 0.1% (wt/vol) Dispase solution, as described in Step 56B(x–xviii), using larger wells and dishes as needed. ESCs/iPSCs should be passaged twice a week to prevent them from differentiating. We typically passage ESCs/iPSCs to a 6-cm dish for DNA extraction.

Genomic DNA isolation An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0033.jpg Overnight incubation and 2 h

58| Remove the medium from the tissue culture dishes from Steps 57A(viii) or 57B(xii), rinse once with 1× PBS and aspirate. Let the dishes air-dry. One dense 6-cm ESC/iPSC dish containing approximately 1 × 106 cells yields ~20 μg of genomic DNA. A 10-cm MSC dish containing approximately 1 × 106 yields up to 20 μg of DNA.

An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0034.jpg The dishes can be wrapped in laboratory film and frozen at −20 °C for up to 6 months, if desired. Dishes should be thawed at room temperature on a bench before use.

59| Add 750 μl of genomic DNA lysis buffer per dish. Incubate for 10 min with gentle, occasional swirling.

60| Scrape cells with a rubber policeman, pour into a 2-ml microcentrifuge tube. The solution should be very viscous and mostly clear.

61| Incubate at room temperature overnight on a rotating shaker (ten rotations per min).

62| The next day, add 4.5 μl of RNAse A (4 mg ml−1 stock) once the DNA is in solution. Mix samples by inverting 25 times and incubate at 37 °C for 60 min.

63| Cool samples to room temperature. Add 250 μl of protein precipitation solution to the RNAse-treated cell lysate.

64| Vortex to mix the cell lysate with the protein precipitation solution.

65| Centrifuge at 17,900g (typically full speed) for 10 min at room temperature in a microcentrifuge. The precipitated proteins will form a tight pellet.

66| Gently pour the supernatant into a fresh microcentrifuge tube. If the protein pellet is not tight, then vortex, incubate on ice for 3 min and centrifuge again at 17,900g for 5 min at room temperature in a microcentrifuge.

67| Pour the supernatant into a fresh microcentrifuge tube containing 750 μl of 100% isopropanol, leaving behind the precipitated protein. Mix the samples by inverting gently 50 times.

68| Centrifuge at 17,900g for 5 min at room temperature in a microcentrifuge. The DNA will be visible as a small white pellet.

69| Add 500 μl of 70% (vol/vol) ethanol and invert the tube several times to wash the pellet.

70| Centrifuge at 17,900g for 5 min in a microcentrifuge at room temperature. Pour off the ethanol carefully.

71| Invert and drain the tube on a clean absorbent paper and allow to air-dry for 10 min. An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0035.jpg The pellet may not be very tight; drain the ethanol gently, taking care not to disturb the pellet at the bottom.

72| Add 100 μl of TE buffer. Hydrate the DNA by incubating at 65 °C for 1 h and overnight at room temperature. An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0036.jpg For long-term storage, store at −20 °C.

Screening for targeted clones by Southern blotting An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0037.jpg 5 d

73| Digest 5–10 μg of genomic DNA samples with 50 U (5 U μg−1 of DNA) of restriction enzyme for 8 h. Half of the restriction enzyme can be added at the beginning and the other half can be added after 4 h. An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0038.jpg The restriction digests can be stored at −80 °C and heated to 56 °C for 3 min before loading.

74| Pour an 0.8% (wt/vol) agarose gel in 1× TAE, and load 5 μg of the digested samples after mixing with the loading dye. An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0039.jpg High-molecular-weight genomic DNA is sometimes not uniformly distributed in TE, which can lead to inaccurate measurement before digestion. Thus, the digested DNA samples need to be requantitated on a fluorometer before loading onto the gel. λ/HindIII DNA can be used as size markers.

75| Run the gel at 120 V for 4–5 h in 1× TAE buffer.

76| Perform Southern blotting as described in Steps 41–54. A representative Southern blot is shown in Figure 2. An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0040.jpg

An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0041.jpg Troubleshooting advice can be found in Table 2.

TABLE 2
Troubleshooting table.

An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0042.jpg

An external file that holds a picture, illustration, etc.
Object name is nihms-476366-ig0043.jpg

Steps 1–29, AAV stock production: 9 d

Steps 30–55, AAV titering using neutral gel: 4 d

Step 56A, Obtaining transduced colonies of MSCs/fibroblasts/transformed cell lines: 14 d

Step 56B, Obtaining transduced colonies of PSCs: 20 d

Step 57A, Picking drug-resistant clones (MSCs/fibroblasts/transformed cell lines): 0.5 h

Step 57B, Picking drug-resistant clones PSCs: 1 h

Steps 58–72, Genomic DNA isolation: overnight incubation and 2 h

Steps 73–76, Screening for targeted clones by Southern blotting: 5 d

ANTICIPATED RESULTS

These protocols typically produce AAV2 stocks with yields of 1012 viral particles or more using the iodixanol and heparin affinity column protocol. The stocks can also be used directly without purifying them over a heparin affinity column, which can be especially useful when other serotypes are used that cannot be purified on a heparin affinity column. With optimal vector design (e.g., an insertion vector) and when targeting an expressed locus, targeting frequencies are ~10−3 (1 targeted cell per 103 infected cells). For example, when targeting COL1A2 in MSCs in which this gene is expressed, 0.23% of infected cells were targeted15. Transduction with AAV vectors containing promoter-driven selection markers results in ~1 targeted clone per 5–20 drug-resistant clones analyzed when targeting the HPRT1 locus17,19,23, which is expressed at moderate levels in all cells. This ratio can be significantly lower at other loci38,39. With promoter-trap or IRES vectors, >80% of drug-resistant clones can be targeted1517.

ACKNOWLEDGMENTS

This work was supported by grants DK55759, HL53750 AR48328 and GM086497 to D.W.R. from the US National Institutes of Health. We thank D.R. Deyle for helpful suggestions.

Footnotes

AUTHOR CONTRIBUTIONS I.F.K., R.K.H. and D.W.R. wrote the manuscript.

COMPETING FINANCIAL INTERESTS The authors declare competing financial interests (see the HTML version of this article for details).

Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/.

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