The recombinant AAV vectors used to generate AAV-DJ/8-GFP (AAV-control) and AAV-DJ/8-GLUT1 (AAV-GLUT1) were constructed by subcloning the cDNA of GFP or GLUT1 (human) into the pAAV-MCS expression vector (Cell Biolabs). 293AAV cells (Cell Biolabs) were cotransfected with the recombinant AAV vector, pAAV-DJ/8 vector, and helper plasmid at a 1:1:1 ratio using polyethylenimine at the AAV core facility of Rutgers University. The recombinant AAV product was purified by the iodixanol gradient/ultracentrifugation method, and the AAV fraction was concentrated using a VIVASPIN 20 concentrator (100 kDa cutoff, Sartorius). The virus titer was determined using the Cell Biolabs AAV quantitation kit (VPK-145). To administer recombinant AAVs, doses of 2 × 1011 vector genome per mouse were injected i.v. via the jugular vein into 2- to 3-month-old male control and YAPch-KO mice, as described previously (34 (link)). Two weeks after the injection, the mice were subjected to sham or TAC surgery.
293aav cells
Manufactured by Cell Biolabs
Sourced in United States
293AAV cells are a specialized cell line derived from human embryonic kidney cells. They are designed for the production and amplification of recombinant adeno-associated virus (rAAV) particles. These cells provide a stable, high-yielding system for the generation of rAAV vectors, which are widely used in gene therapy and research applications.
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Lab products found in correlation
Quicktiter aav quantitation kit, by Cell Biolabs (4 mentions)
Sw55ti rotor, by Beckman Coulter (4 mentions)
Vivaspin 20 concentrator, by Sartorius (2 mentions)
Paav dj 8 vector, by Cell Biolabs (2 mentions)
Paav mcs expression vector, by Cell Biolabs (2 mentions)
Amicon ultra centrifugal filter, by Merck Group (2 mentions)
Phelper, by Cell Biolabs (2 mentions)
Millipore amicon ultra centrifugal filters, by Merck Group (2 mentions)
Paav dj, by Cell Biolabs (2 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Puromycin, by Apexbio (1 mentions)
Hitrap heparin column, by GE Healthcare (1 mentions)
Aav dj helper free system, by Cell Biolabs (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Miseq sequencing, by Illumina (1 mentions)
Aav quantitation kit, by Cell Biolabs (1 mentions)
11 protocols using 293aav cells
1
Construction and Delivery of Recombinant AAV Vectors
2
Construction of pAAV-CAG-hPRPF31 Rescue Vector
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To construct the pAAV-CAG-hPRPF31 rescue vector, the human PRPF31 coding sequence was obtained from NCBI, gene fragments were synthesized (Integrated DNA Technologies) and cloned into a pAAV-CAG backbone. AAV7m8 vectors carrying human PRPF31 cDNA or GFP were produced in 293AAV cells (Cell Biolabs) using a triple transfection method69 (link). Recombinant AAVs were purified by iodixanol gradient ultracentrifugation, buffer exchanged, and concentrated with Amicon Ultra-15 Centrifugal Filter Units (#UFC8100) in PBS and titered by quantitative PCR relative to a standard curve using ITR-binding primers70 (link).
3
Culturing Enzalutamide-Resistant Prostate Cancer Cell Lines
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The cell lines CWR-R1-enzalutamide resistant/luciferase+ (stably expressing a firefly luciferase), and CWR-R1-enzalutamide resistant/luciferase plus FAP (additionally expressing a human FAP receptor) were provided by the LeBeau lab from the University of Wisconsin (28 (link)). Both cell lines and 293AAV cells (Cell Biolabs) were cultured in DMEM (Gibco) supplemented with 10 % fetal bovine serum (Gibco), 4.5 g/L D-glucose, L-glutamine, 110 mg/L sodium pyruvate, and 100 U per mL penicillin/100 μg per mL streptomycin (Gibco). Media for R1-FAP cells was additionally supplemented with 3 μg/mL puromycin (ApexBio Technology). Cells were kept in a humidified incubator at 37 °C and 5 % CO2 and passaged every 2-4 days when reaching a confluency of 70-80 %.
4
Generation of Viral Vectors for Selective Expression
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Adeno-associated virus (AAV) was generated using AAV-DJ Helper Free system (Cell Biolabs) as we published previously.9 (link) To express ExSYTE selectively in the ‘Subthreshold fear conditioning’ neuronal population, the ExSYTE cassette was located in a reverse direction with two lox2272 and loxP loci (AAV-FLEX-ExSYTE) as illustrated in Figure 4D . The ExSYTE or ExSYTEH64A fused with the P2A peptide-EGFP was cloned into the modified pAAV-MCS and transfected with pAAV-DJ and pHelper into 293AAV cells (Cell Biolabs). Transfected cells were lysed, and AAVs were purified with HiTrap heparin columns (GE Health) as previously described.69 (link) AAV-EF1α-mCherry-IRES-Cre was purchased from Addgene (Cat. #55632-AAV8).
5
Recombinant AAV9 Production in 293AAV Cells
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Recombinant AAV9 was produced in 293AAV cells (Cell Biolabs). Briefly, polyethylenimine (PEI, linear, MW 25,000) was used for transfection of triple plasmids: the pAAV expression vector, pAAV9-RC (Cell Biolab) and pHelper (Cell Biolab). 72 h post transfection, cells were scrapped in their medium and centrifuged, frozen, and thawed four times by placing it alternately in dry ice or ethanol and 37°C water bath. AAV crude lysate was purified by centrifugation at 54,000 rpm for 1 h in discontinuous iodixanol gradients with a Beckman SW55Ti rotor. The virus-containing layer was extracted, and viruses were concentrated by Millipore Amicon Ultra Centrifugal Filters. Virus titers were 1.2 × 1012 GC/mL for hGFAP::Cre, hGFAP::NeuroD1-GFP and hGFAP::GFP, 1.4 × 1012 GC/mL for CAG::FLEX-NeuroD1-P2A-GFP and CAG::FLEX-NeuroD1-P2A-mCherry, and 1.6 × 1012 GC/mL for CAG::FLEX-mCherry-P2A-mCherry and CAG::FLEX-GFP-P2A-GFP, determined by QuickTiter AAV Quantitation Kit (Cell Biolabs).
6
Production and Purification of Recombinant AAV9
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Recombinant AAV stereotype 9 was produced in 293AAV cells (Cell Biolabs). Briefly, triple plasmids [pAAV expression vector, pAAV9-RC (Cell Biolab) and pHelper (Cell Biolab)] were transfected by polyethyleneimine (PEI, linear, MW 25000). Cells were scraped and centrifuged at 72 h post-transfection. Cell pellets were frozen and thawed for four times by being placed in dry ice/ethanol and 37°C water bath alternately. AAV lysate was purified by ultra-centrifugation at 54,000 rpm for 1 h in discontinuous iodixanol gradients (Beckman SW55Ti Rotor). The virus-containing layer was collected followed by concentration by Millipore Amicon Ultra Centrifugal Filters. Virus titers were initially determined by QuickTiterTM AAV Quantitation Kit (Cell Biolabs): 1.5 × 1011 – 1.2 × 1012 GC/ml for hGFAP::Cre, 1.4 × 1012 GC/ml for FLEX-NeuroD1-P2A-mCherry, 1.6 × 1012 GC/ml for FLEX-mCherry-P2A-mCherry.
7
Production and Purification of Recombinant AAVs
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AAVs carrying hGFAP::Cre and CAG::FLEx-GFP for serotype testing in human islets were as previously described.58 (link) AAV8-U6-mINS2utr5sg-EGFP-2cut (6.15 × 1013 GC/mL) was produced and purified by Penn Vector Core. AAV-DJ-U6-mINS2utr5sg-EGFP-2cut (2.92 × 1012 GC/mL), AAV-DJ-U6-hINSin1sg-CopGFP-2cut (1.83 × 1013 GC/mL), AAV-DJ-U6-hINSin1sg-CopGFP-1cut (6.02 × 1012 GC/mL), and AAV-DJ-nEF-Cas9 (3.83 × 1012 GC/mL) were produced and purified as described below. Briefly, recombinant AAVs were produced in 293 AAV cells (Cell Biolabs, San Diego, CA, USA). Polyethylenimine (PEI, linear, MW 25,000) was used for transfection of three plasmids: the pAAV vector constructs, pAAV2/8-RC (Penn Vector Core) or pAAV-DJ (Cell Biolabs), and pHelper (Cell Biolabs). At 72 h post-transfection, cells were scrapped in their medium, centrifuged, and then frozen and thawed four times by placing alternately in dry ice-ethanol and a 37°C water bath to lyse the cells and release the virus. The resulting AAV crude lysate was purified by centrifugation at 54,000 rpm for 1 h in discontinuous iodixanol gradients with a Beckman SW55Ti rotor. The virus-containing layer was extracted, and viruses were concentrated by Millipore Amicon Ultra Centrifugal Filters (Millipore-Sigma, Bedford MA, USA). Virus titers were determined by quantitative PCR (qPCR) according to Addgene protocol.
8
Directed Evolution of AAV Vectors
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AAV vectors were produced in HEK293T cells (ATCC), or 293AAV cells (Cell Biolabs) using a double (for AAV2-7mer, LoopSwap, AAV2-ErrorProne and SCHEMA libraries) or triple transfection method (Grieger et al., 2006 (link)). Short tandem repeat profiling was done by ATTC Cell Line Authentication Service and all cell lines were checked for mycoplasma using Hoechst staining. Directed evolution libraries were packaged using an empirically determined molar ratio of plasmids in the packaging cell line, such that each AAV particle contained the genome encoding its own capsid (Koerber et al., 2006 (link)). All recombinant AAVs were purified by iodixanol gradient ultracentrifugation, buffer exchanged and concentrated with Amicon Ultra-15 Centrifugal Filter Units (#UFC8100) in DPBS and titered by quantitative PCR relative to a standard curve using ITR-binding primers or by using QuickTiter AAV Quantitation Kit (Cell Biolabs). The relative titer of each variant was confirmed by Illumina MiSeq sequencing.
9
AAV Vector Production and Purification
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AAV vectors were produced in 293AAV cells (Cell Biolabs; Cat# AAV-100) using a triple transfection method (Grieger et al., 2006 (link)). Recombinant AAVs were purified by iodixanol gradient ultracentrifugation, buffer exchanged, and concentrated with Amicon Ultra-15 Centrifugal Filter Units (Cat# UFC8100) in DPBS and titered by using QuickTiter AAV Quantitation Kit (Cell Biolabs; Cat# VPK-145) and quantitative PCR relative to a standard curve.
10
Recombinant AAV Vector Production
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