Design and construction of targeted AAVP vectors for mammalian cell transduction

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Design and construction of targeted AAVP vectors for mammalian cell transduction
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  Design and construction of targeted AAVP vectors for mammalian cell transduction Amin Hajitou 1 , Roberto Rangel 1 , Martin Trepel 2 , Suren Soghomonyan 3 , Juri G Gelovani 3 , Mian M Alauddin 3 ,Renata Pasqualini 1 & Wadih Arap 1 1 Department of Genitourinary Medical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030.  2 Departmentof Hematology and Oncology and Institute for Molecular Medicine and Cell Research, University of Freiburg Medical Center, Hugstetter Strasse 55, D-79106 Freiburg,Germany.  3 Department of Experimental Diagnostic Imaging, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030.Correspondence should be addressed to R.P. (rpasqual@mdanderson.org) or W.A. (warap@mdanderson.org). Published online 15 March 2007; doi:10.1038/nprot.2007.51 Bacteriophage (phage) evolved as bacterial viruses, but can be adapted to transduce mammalian cells through ligand-directed targetingto a specific receptor. We have recently reported a new generation of hybrid prokaryotic–eukaryotic vectors, which are chimeras of genetic  cis -elements of recombinant adeno-associated virus and phage (termed AAVP). This protocol describes the design and con-struction of ligand-directed AAVP vectors, production of AAVP particles and the methodology to transduce mammalian cells  in vitro  andto target tissues  in vivo  after systemic administration. Targeted AAVP particles are made in a two-step process. First, a ligand peptide of choice is displayed on the coat protein to generate a targeted backbone phage vector. Then, a recombinant AAV carrying a mammaliantransgene cassette is inserted into an intergenomic region. High-titer suspensions ( B 10 10 –10 11 transducing units per   l l) can beproduced within 3 days after vector construction. Transgene expression by targeted AAVP usually reaches maximum levels within 1 week. INTRODUCTION The use of new genetic systems for the study of currently intractablebiological questions will require the development of ligand-directed(targeted) vectors that can be systemically delivered. Over the pastdecade, common approaches at targeted gene delivery have typically relied on ablation of the native tropism of mammalian viruses,redirection to alternative receptors or both 1–8 . Incorporation of homingpeptidesselectedfrombacteriophage(phage)displaylibrary screenings into mammalian viral vectors has been attempted, butsuch strategy has the potential to alter the structure of the capsid,affect the targeting attributes of the ligand peptides or even preventthe display within a viable viral capsid altogether 9–13 . In contrast,phage have no intrinsic tropism for mammalian cells 14,15 and canmediate modest gene expression in mammalian cells after geneticmanipulation 16–19 . In theory, phage-based vectors have some poten-tial advantages over animal viruses for mammalian cell-targeteddeliveryoftransgenes.First,therearenoknownnaturalreceptorsforphage (which have evolved as prokaryotic viruses) on mammaliancells 14,15 . However, receptor-mediated internalization by mamma-lian cells occurs if phage vectors are genetically modified to display specific ligands, such as fibroblast growth factor (FGF2), anti-ErbB2scFv F5 antibody and integrin-binding peptides 16–20 . Moreover,bacteriophage have long been administered to humans, from itsantibacterialuseintheenvironmentduringthepreantibioticera 14 tothe very recent Food and Drug Administration approval of certainphage preparations as antibacterial food additives 21 . Indeed, feasi-bilityclinical trials have shown that the selection of phage libraries incancer patients can yield ligand–receptor systems 22,23 and that seriallibraryadministrationcanbeaccomplishedwithoutmajoruntowardclinical effects 24 . Finally, unlike mammalian viruses, phage do notrequire further context modification of their capsid because thetargeting peptides are actually selected and isolated directly ashoming to specific cell-surface receptors 22,25–30 .Despite these potential advantages, phage-based vectors haveinherently been considered poor gene delivery vehicles. As aworking hypothesis, we proposed that the rate-limiting stepmight be mechanistically related to the post-targeting fate of thesingle-stranded DNA of the phage genome 1 . In an attempt toimprove phage as targeted vectors for mammalian cells, we rea-soned that the genetic incorporation of compatible  cis -elements(such as invertedterminal repeats (ITRs)) from a mammalian—yetsingle-stranded—DNA virus such as recombinant adeno-asso-ciatedvirus(AAV) wouldimprove post-targetingtransgeneexpres-sion. Thus, we have developed ligand-directed vectors as a hybridbetween AAV and phage (termed AAVP). In our targeted AAVPprototype vector 1 , the targeted phage displays an RGD-4C peptidethat binds to  a v   integrins 26,27,29,31 , with the mammalian transgenecassette flanked by full-length ITRs of AAV serotype 2. Wereported 1 that the improved mammalian transduction efficiency by targeted AAVP over conventional phage-based vectors is asso-ciated with an improved fate of the delivered transgene, throughmaintenance of the entire mammalian transgene cassette, betterpersistence of episomal DNA and formation of concatamers of thetransgene cassette 1 . Here, we detail how to insert an AAV transgenecassette into the backbone phage vector genome to generatetargetedAAVPhybrid constructs (Steps1–18) andhow to produce,purify and titrate the vector preparations (Steps 19–26). We alsodescribe a standard protocol for AAVP-mediated mammaliancell transduction, both in tissue culture and in targeted tissues in vivo  after systemic administration. Briefly, DNA olignonucleo-tide sequences encoding peptide ligands are inserted into the  Sfi Isite of the gene of the pIII minor coat protein of fUSE5-MCS(multicloning site)-based filamentous phage 1,32,33 . Phage producedin this manner display 3–5 copies of the specific peptide 32 . ThefUSE5-MCS-based filamentous phage display vector is thengenetically modified to generate the corresponding targetedAAVP vector, by inserting a recombinant AAV (carrying thedesirable promoter/transgene cassette)intoan intergenomicregionof the phage genome. This strategy also serves to construct    p  u  o  r   G   g  n   i   h  s   i   l   b  u   P   e  r  u   t  a   N    7   0   0   2   ©   n  a   t  u  r  e  p  r  o   t  o  c  o   l  s   /  m  o  c .  e  r  u   t  a  n .  w  w  w   /   /  :  p   t   t   h NATURE PROTOCOLS  |  VOL.2 NO.3  |  2007  |  523 PROTOCOL  non-targeted control vectors (either displaying no peptides ordisplaying mutant/scrambled versions of the peptide). Targetedand control AAVP particles are amplified, isolated and purified by adapting the protocols used for phage 30,32 . AAVP particles are thenresuspended in phosphate-buffered saline (PBS; pH 7.4) and recen-trifuged to remove residual bacterial debris. Next, AAVP particles insuspension are sterile-filtered through 0.45- m m pores, then titratedby infection of host bacteria for colony counting on Luria–Bertani(LB) agar plates under a double antibiotic selection and expressed asbacterial transducing units (TU). Transduction of mammalian cellsin culture is performed by incubation with the targeted AAVP for4 h in serum-free medium with a ratio of at least 10 6 TU per cell.Transgene expression begins 48–72 h later and reaches a maximumlevel by 1 week. Typically, non-targeted AAVP vectors or AAVPdisplaying mutated and/or scrambled versions of the targetingpeptide serve as negative controls for the ligand-directed (i.e.,targeting) experiments; either a corresponding version of a targetedphagevector‘‘AAV-less’’oratargetedAAVPcontainingan‘‘ITR-less’’or mutant ITR serves as a suitable control for post-internalization(i.e., integration, concatemerization) experiments.Under these conditions,  B 10–20% of cells are transduced inculture.Specificitycan bedemonstratedbyblocking theinteractionof the targeted AAVP by preincubating the target cells with thecorresponding synthetic peptide. In our prototype RGD-4C AAVP,transduction inhibition was dose-dependent, greater than 99%inhibition was observed and incubation with nonspecific negativecontrol peptides had no detectable effects 1 . This data set showcasesan example of targeted AAVP transduction of mammalian cellsexpressing a specific receptor and mediated by an establishedligand–receptor system. Transduction of a target tissue  in vivo will vary among ligand–receptors but one can attempt to useincreasing doses of targeted AAVP, starting at 10 10 TU permouse, administered intravenously (tail vein). Transgene expres-sionshouldbemonitored anditsdetectioncan be expectedstartingfrom day 3 after delivery. Depending on the specific reporter used,one can determine gene expression initiation in the target andmaximal expression timing. Evidently, different organs will requiredifferent ligand peptides and particular conditions should beanticipated for optimal temporal and spatial transduction. In anunpublished work, we have shown systemic targeted tissue-specifictransduction of the lung tissue in wild-type mice via a homingpeptide 34 that targets membrane dipeptidase in the lung endothe-lium 35 ; we have also tested a tumor-homing peptide 25 that targetsMMP-2 and MMP-9 to transduce human tumor xenografts withphage-based vectors. In addition to the suicide gene therapy strategy previously reported 1 , other targeted AAVP includingapplications for delivery of therapeutic genes (unpublished obser-vations) will follow. MATERIALS REAGENTS . Plasmids:fUSE5 1,32,33,36 phage plasmid, accession number AF218364fd-tet 1,32,33,36 phage plasmid, accession number AF217317fMCS 1,36 phage plasmid, accession number AF218733fUSE5-MCS 1 phage plasmidpAAV-GFP plasmid (Stratagene)pMOD-Luc-Sh (Luciferase reporter; InvivoGen)pCMV b  ( b -galactosidase reporter; Clontech) . Bacterial strains:XL1-Blue MR supercompetent cells (Stratagene) Escherichia coli  (MC1061 and k91Kan) (see ref. 36) . LB–tetracycline plates, LB–tetracycline–kanamycin plates and media . Terrific broth (TB) medium . SOC medium (Invitrogen) . Polyethylene glycol (PEG)/NaCl . QIAquick Nucleotide removal kit (Qiagen) . Essential restriction enzymes:  Bgl  I,  Bgl  II,  Hin dIII,  Pst  I,  Pvu II,  Sac  I,  Sfi I and  Xho I. Other restriction enzymes will be required depending on the specificsequences of the construct . Phosphatase alkaline (Roche) . T4 DNA ligase with ligase buffer (Invitrogen) . Rapid DNA ligation kit (Roche) . Agarose, electrophoresis (IscBioExpress) . E-Gel 0.8% agarose (Invitrogen) . QIAquick Gel extraction kit (Qiagen) . Phosphorylated DNA linkers (NEB) . Taq -DNA polymerase with supplied buffer (Promega) . 100 mM dNTPs (Fisher) . Oligonucleotides (Sigma-Genosys) (see  Table 1 ) . DMSO  !  CAUTION  DMSO is readily absorbed through the skin. Whenhandling DMSO, wear appropriate gloves, safety glasses and use a pipetingaid under a safety chemical hood. . Transfection reagent: Fugene (Roche) . Human embryonic kidney cells (HEK293; ATCC) . HEK293 cells are maintained in Dulbecco’s modified Eagle’s medium(Gibco), supplemented with 10% fetal bovine serum (Gibco),  L -glutamineand penicillin/streptomycin . D -Luciferin potassium salt (InvivoGen) . Immunocompetent and immunodeficient mice (Harlan–Sprague–Dawley) m CRITICAL  Mice must be used according to the national and institutionalguidelines concerning use of animals. EQUIPMENT . Fluorescent microscope (Olympus or equivalent). . Fluorescence-activated cell sorting (FACS) to analyze and sort the cells by using a BD FACS Vantage (Becton-Dickinson) . 0.22  m m filter units (Corning) . 0.45  m m filter units (Corning) . 24-well tissue culture plates . Tissue culture incubator at 5% CO 2  for HEK293 cells and other cell lines . Shaker at 37  1 C . Centrifuge Sorvall SA-600 and SLA-3000 rotors . Cell electroporator (Life Technologies) . In vivo  Bioluminescence Imaging (BLI) System 200 (IVIS200; Xenogen) . PCR machine (Eppendorf) . DNA electrophoresis equipment (Bio-Rad)    p  u  o  r   G   g  n   i   h  s   i   l   b  u   P   e  r  u   t  a   N    7   0   0   2   ©   n  a   t  u  r  e  p  r  o   t  o  c  o   l  s   /  m  o  c .  e  r  u   t  a  n .  w  w  w   /   /  :  p   t   t   h TABLE 1  |  Primer set sequences. Primer set Sequence Function A 5 ¢ -CACTCGGCCGACG-3 ¢  5 ¢ -TTCGGCCCCAGCGGC-3 ¢  Oligonucleotide conversion from single- to double-stranded DNAB 5 ¢ -TAATACGACTCACTATAGGGCAAG CTGATAAACCGATACAATT-3 ¢  Primers routinely used to amplify the oligonucleotide sequences after 5 ¢ -CCCTCATAGTTAGCGTAACGATCT-3 ¢  insertion into the construct 524  |  VOL.2 NO.3  |  2007  |  NATURE PROTOCOLS PROTOCOL  REAGENT SETUPPEG/NaCl solution stock   500 g of PEG and 584.5 g of NaCl in 2,380 ml H 2 O(double-distilled) and store at 4  1 C for up to 1 year. TB medium  9.6 g of tryptone, 19.2 g of yeast extract and 3.2 ml of glycerolin 1 liter of H 2 O. Autoclave, add 100 ml of TB supplements and 50 mg of kanamycin TB supplements  To prepare 1 liter of TB supplements, add 23.1 g of KH 2 PO 4 and 125.4 g of K 2 HPO 4  in 1 liter of H 2 O (double distilled). Filter through0.22  m m filter unit and store at 4  1 C. EQUIPMENT SETUPWhole-body BLI of luciferase  Mice are first anesthetized inside a clearplexiglass box by using an anesthetic gas admixture (2% isofluorane, 98%oxygen) and are then transferred to nose cones attached to the manifold in theimaging chamber. The imaging time is 5 min per side (dorsal/ventral),depending on the experiment. Imaging parameters are as follows: imageacquisition time, 1 min; binning, 2; no filter; f/stop (aperture size), 1. Regions of interest can be defined manually over the tumors or target tissues for measuringsignal intensities, expressed as photons s  1 cm  2 sr  1 . PROCEDUREInsertion of the targeted peptide into the pIII coat protein of fUSE5-MCS phage1|  Generate phage clones displaying targeting peptides by cloning the corresponding oligonucleotide sequences flanked by Bgl  I restrictions sites into the  Sfi  I site of the gene for the pIII coat protein of fUSE5-MCS. The fUSE5-MCS plasmid can also begenerated by replacing the 5.4 kb  Bam HI –Sac  II fragment of the fUSE5 with the 4.1 kb  Bam HI –Sac  II fragment from the fMCSplasmid that contains an MCS. 2|  Design and convert the synthetic oligonucleotide templates flanked by  Bgl  I restriction sites (500 ng) to double-strandedDNA by PCR amplification. We use the following 57 bp oligonucleotide as a template: 5 ¢ - CACTCG GCCGACG GGGCTXXXXXXXXXXXXXXXXXXXXXGGG GCCGCTGGG GCCGAA -3 ¢ . This template is flanked by the  Bgl  I restriction sites (GCCNNNNNGGC: underlined).The bold nucleotides in the above template sequence indicate the annealing of the sense and antisense primers, respectively.The nucleotide sequence encoding the displayed peptide is marked ‘‘X’’.Finally, we use primer set A (see  Table 1 ) and 2.5 U of   Taq  DNA polymerase (Promega) in 20  m l. Use the following setup:94  1 C for 2 min, followed by 35 cycles at 94  1 C for 30 s, 60  1 C for 30 s and 72  1 C for 30 s, followed by 72  1 C for 5 min, heldat 4  1 C and stored at  20  1 C until ready for Step 3. m CRITICAL STEP  For effectivePCR, it isrecommendedtoadd DMSO(2% final) toweakenhydrogenbonding and prevent formationof hairpin structures. 3|  Purify and elute the double-stranded DNA sequences containing  Bgl  I restriction sites by using a QIAquick nucleotideremoval kit. 4|  Digest oligonucleotides with  Bgl  I restriction enzyme for 4 h at 37  1 C, then inactivate  Bgl  I activity by incubation at65  1 C for 20 min. Digestion with  Bgl  I restriction enzyme generates the following sticky ends:5 ¢ - GGGCTXXXXXXXXXXXXXXXXXXXXXGGGGCCGCTG-3 ¢ 3 ¢ -TGCCCCGAXXXXXXXXXXXXXXXXXXXXXCCCCGGC-5 ¢ . 5|  Digest the fUSE5-MCS plasmid with  Sfi  I restriction enzyme for 1–2 h at 50  1 C. Run the B 9.5-kb linearized fUSE5-MCSplasmid on an agarose gel or E-Gel 0.8% agarose to confirm digestion. The fUSE 5 backbone vector contains two  Sfi  I restrictionsites 36 (GGCCNNNN/NGGCC) in the pIII gene. After digestion, the  Sfi  I restriction enzyme will generate non-identical,non-complementary three bases 3 ¢ -overhanging ends 36 . This will allow directional cloning after removal of the stuffer that lies between these sites in the phage vector. A schematic representation of the final vector sequences is shown below.fUSE5 Stuffer fUSE55 ¢ -TCGGCCGACG TGGCCTGGCCTCTG GGGCCGAA-3 ¢ 3 ¢ -AGCCGGC TGCACCGGACCGGA GACCCCGGCTT-5 ¢ ?  TROUBLESHOOTING6|  Ligate the  Sfi  I-digested fUSE5-MCS vector plasmid with the  Bgl  I-digested oligonucleotides by using the Rapid Ligation Kitfor 5–10 min at room temperature (25  1 C,  Fig. 1 ). It is recommended not to inactivate  Sfi  I and not to dephosphorylate thelinearized fUSE5-MCS plasmid, as this markedly reduces the efficiency of ligation. Ligate immediately after digestion. Also, setup a reaction with the fUSE5-MCS plasmid alone and no insert to estimate the bacterial transformation due to autoligation of the fUSE5-MCS plasmid and/or the presence of non-digested fUSE5-MCS plasmid. 7|  Use 2  m l of the ligation product to transform MC1061 or XL1-Blue MR bacteria according to the manufacturer’s instructions.Incubate transformed bacteria on LB–tetracycline plates for 24 h at 37  1 C. 8|  Verify the correct insertion and nucleotide sequence by PCR of the bacterial colonies generated. Pick single colonies (atleast ten single colonies plus colonies from vector alone as controls) in 20  m l of medium. Use the primer set B (see  Table 1 )and 2 U of   Taq  DNA polymerase (Promega) in 20  m l. Use the following setup: 94  1 C for 3 min, followed by 35 cycles at 94  1 C for 10 s, 60  1 C for 30 s and 72  1 C for 30 s. m CRITICAL STEP  It is recommended to add 2% DMSO to weaken hydrogen bonding and prevent formation of hairpin structures.    p  u  o  r   G   g  n   i   h  s   i   l   b  u   P   e  r  u   t  a   N    7   0   0   2   ©   n  a   t  u  r  e  p  r  o   t  o  c  o   l  s   /  m  o  c .  e  r  u   t  a  n .  w  w  w   /   /  :  p   t   t   h NATURE PROTOCOLS  |  VOL.2 NO.3  |  2007  |  525 PROTOCOL  9|  Run 2  m l of PCR product on a 2% agarose gel or E-Gel 0.8% agarose to verify the insertion. Include the productof an fUSE5-MCS without insert, which will be smaller in sizeon the gel. Insertion of recombinant (r) AAV into the MCS of thefUSE5-MCS plasmid displaying the targeting peptide10|  Prepare the rAAV carrying the trangene of interest.Remove and replace GFP from the pAAV-GFP plasmid with thetransgene of interest if applicable. GFP can also be replacedwith a GFP variant for maximal fluorescence such as eGFP(enhanced GFP, catalog number 6084-1 from ClonTech). Thus,the transgene of interest must be flanked by restriction sitescompatible with the MCS of the pAAV plasmid. 11|  Remove the transgene cassette flanked by the ITRs fromthe pAAV plasmid created in Step 10. Use the  Pvu II restrictionenzyme that digests adjacent to the ITRs and gel-purify thereleased transgene-ITR-segment using the gel extraction kit( Fig. 1 ). Note that expression cassettes of interest should nothave any  Pvu II site. Otherwise, alternative strategies should beused. Then, use an rAAV plasmid with a restriction map compa-tible with that of the expression cassette of interest. For instance, some AAV plasmids from Stratagene (or other commercial outfits) have convenient rare cutter restrictionenzyme sites such as  Sbf  I or   Sse 8387I adjacent to each  Pvu IIsite. Sometimes, these can be used for AAV vector genomeinsertion into the phage vector backbone if other geneticelements permit this utilization. 12|  The targeted fUSE5-MCS plasmid has a unique  Pvu IIsite in the MCS. Digest with  Pvu II to linearize and run onan agarose gel or on an E-Gel 0.8% agarose to confirmdigestion. 13|  Dephosphorylate the targeted fUSE5-MCS plasmid vector by using phosphatase alkaline according to the manufacturer’sinstructions. m CRITICAL STEP  It is recommended to dephosphorylatebefore ligation to reduce background as the fUSE5-MCSplasmid autoligates. 14|  Ligate the ITR-flanked transgene cassette into the  Pvu II-linearized fUSE5-MCS plasmid for 4 h at 23  1 C or 24 h overnightat 16  1 C using T4 DNA ligase ( Fig. 1 ). ?  TROUBLESHOOTING15|  Digestion with  Pvu II generates blunt ends. Linkers containing recognition sequences of enzymes can be added to the Pvu II-recovered ITR-flanked cassette to produce compatible cohesive ends.Linkers to  Bgl  II,  Hin dIII,  Pst  I,  Sac  I and  Xho I can be used. After determining which restriction site is most appropriate for the construction, add the corresponding restriction enzyme to digest the linkers flanking the ITR transgene cassette. Ligateinto the compatible cohesive restriction sites ( Bgl  II,  Hin dIII,  Pst  I,  Sac  I and  Xho I) of the MCS of the fUSE5-MCS plasmid. ?  TROUBLESHOOTING16|  Use 2  m l of the ligation product to transform MC1061 or XL1-Blue MR bacteria according to the manufacturer’s instructions.Plate bacteria on LB-tetracycline and grow overnight at 37  1 C for 24 h. 17|  Pick ten colonies, grow overnight in 5 ml LB and purify the plasmid DNA by using a QIAprep miniprep kit protocol for low-copy plasmids. Identify the positive clones by enzymatic restriction digestions.    p  u  o  r   G   g  n   i   h  s   i   l   b  u   P   e  r  u   t  a   N    7   0   0   2   ©   n  a   t  u  r  e  p  r  o   t  o  c  o   l  s   /  m  o  c .  e  r  u   t  a  n .  w  w  w   /   /  :  p   t   t   h ab MCS MCS  fUSE5-MCS bacteriophagefUSE5-MCS bacteriophageTargeted fUSE5-MCS bacteriophageTargeted AAVP constructITR-flanked transgene cassette: rAAV Step 1: Ligate Bgl  I-digested oligonucleotides into Sfi  I-digested fUSE5-MCSStep 2: Ligate Pvu  II-digested rAAV into Pvu  II-digested fUSE5-MCS Double-stranded Bgl  I-flanked oligonucleotides TargetingpeptideTargetingpeptideTransgenecassette MCS  5 ′ ITR5 ′ ITR3 ′ ITR3 ′ ITRCMVGFPpA Pvu  lI Pvu  lI  Pvu  lIIIIIII Sfi  l Figure 1  |  Targeted AAVP vectors. ( a ) Cloning scheme for generation of targeted AAVP and control vectors. The most convenient procedure is tofirst clone the  Bgl  I-digested oligonucleotide sequence corresponding to thetargeting peptide within the  Sfi  I site of the gene for the pIII coat protein.Next, the  Pvu II-digested ITR-flanking transgene cassette (rAAV) is clonedinto the  Puv  II site in the MCS of the targeted fUSE5-MCS plasmid or inthe cohesive restriction sites after addition of the corresponding linkers.( b ) Binding of the targeted AAVP particle to a specific cell-surface receptor in the target tissue and internalization after systemic administration.Alternative cloning approaches are discussed in the text. 526  |  VOL.2 NO.3  |  2007  |  NATURE PROTOCOLS PROTOCOL  18|  Transgene expression can be analyzed at this stage from the AAVP plasmids generated to confirm that the ITR-flankedtransgene cassette is functional when inserted into the phage genome (in the context of AAVP). Therefore, transfect humanembryonic kidney (HEK293) cells with the AAVP DNA plasmids by using the Fugene transfection reagent (Roche). Differentexperimental approaches, such as western blots or immunostainings, can be used to detect transgene expression dependingon the gene of interest. For reporter   GFP  -,  b -galactosidase - or   luciferase -containing cassettes, expression can be analyzed asdescribed in detail in Step 31. Production, purification and titration of AAVP particles19|  AAVP particles can be amplified either by growing MC1061 colonies (option A) or by infecting and growing K91Kan cells(option B). Following amplification, AAVP particles are isolated and purified from the culture supernatant following a modifiedphage purification protocol  30,32 by using the steps given below. It is important to note that option A is faster because  E  .  coli  MC1061 can be used to generate AAVP constructs. This strategy offers the advantage to directly grow positive clones to producephage particles and to skip the infection step needed for option B. However,  E  .  coli   K91Kan (option B) are pilus-positive F + bacteria and can therefore be infected by the newly produced phage particles during the overnight growth; this phenomenonwill result in higher titers compared to MC1061 (option A), which are F  bacteria. (A) Growth of MC1061 colonies (i)Amplify AAVP particles directly by growing the MC1061 colonies in LB plus tetracycline overnight at 37  1 C. (B) Infection and growth of K91Kan cells (i)Incubate 1 ml of growing K91Kan  E. coli   bacteria with 10 10 TU of AAVP for 1 h at room temperature in TB medium.Then, grow in 500 ml at 37  1 C in the presence of tetracycline and kanamycin. 20|  After overnight growth, centrifuge cultures at 6,000  g   for 20 min at 4  1 C and collect the supernatant. Repeat centrifugationto remove residual bacterial debris. 21|  Add PEG/NaCl (15% of the supernatant volume) solution to the supernatant to precipitate the AAVP phage particles.Incubate for 2 h on ice. 22|  Centrifuge suspension at 10,000  g   for 30 min at 4  1 C. A white pellet should be obtained. Discard the supernatant andcentrifuge again for 10 min. Carefully decant the supernatant. 23|  Resuspend the AAVP pellet in 10 ml of sterile PBS with agitation at 37 1 C for 30 min. 24|  Repeat the precipitation with PEG/NaCl (15% of the supernatant volume) solution for 30 min on ice. Then, centrifugeat 14,000  g   for 30 min at 4  1 C and resuspend in an adequate volume of PBS depending on the size of the pellet. 25|  Transfer the solution to an Eppendorf tube, centrifuge at B 13,000  g   for 10 min at room temperature, transfer to a newtube and recentrifuge to remove residual bacteria and debris. 26|  Filter the resulting supernatant containing the AAVP particles in suspension through a 0.45- m m filter. Then, titrate byinfection of K91Kan bacteria for 20 min at room temperature and plaque assay according to the standard protocols 30,32 . TheAAVP titers are expressed as bacterial TUs per   m l. Also, one must keep in mind that bacterial TU and multiplicity of infection areentirely different entities and should not be confused with one another. m CRITICAL STEP  E. coli   K91Kan bacteria infection for more than 20 min might generate higher titers due to the newly producedAAVP particles. When comparing two different construct versions of AAVP for transduction efficiency, it is recommended to titratethe preparations side byside toensure that same dosesare compared. Indeed, the titersmayvaryfromone AAVPversiontoanother,depending on the ligand used, the size of the transgene of interest and size of the AAV mammalian cassette. These parameterscan affect the coating of the virus in host bacteria during production. Viability of bacteria also plays a role, and it is recommendedto infect a log-phase growing bacteria with an optical density ranging between 1.6 and 2.0 at a wavelength of 600 nm (OD 600 ). ’ PAUSE POINT  AAVP titers are relatively stable and the preparations can be stored at 4  1 C for long periods of time (several months) without any significant decrease in the titers. For longer storage times, one should check the titer of the preparationbefore use. ?  TROUBLESHOOTINGTransduction of mammalian cells in culture by targeted AAVP vector and specific inhibition by using synthetic peptides—day 1: cell seeding27|  To transduce mammalian cells by targeted AAVP, it is recommended to incubate at least 10 6 AAVP TUs per cell. Transductioncan be performed in any tissue culture dish or flask; however, as an initial experiment to confirm transduction of mammaliancells, it is recommended to work with a 24-well plate to avoid the use of large amounts of AAVP. Therefore, seed 4  10 4 cellsin each well of the 24-well plate in a final volume of 0.5 ml of complete medium. Incubate overnight at 37  1 C.    p  u  o  r   G   g  n   i   h  s   i   l   b  u   P   e  r  u   t  a   N    7   0   0   2   ©   n  a   t  u  r  e  p  r  o   t  o  c  o   l  s   /  m  o  c .  e  r  u   t  a  n .  w  w  w   /   /  :  p   t   t   h NATURE PROTOCOLS  |  VOL.2 NO.3  |  2007  |  527 PROTOCOL
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