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Phelper

Manufactured by Agilent Technologies
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PHelper is a versatile laboratory equipment designed to assist researchers and technicians in various scientific applications. Its core function is to provide a stable and controlled environment for precise and reliable measurements. PHelper is a compact and user-friendly device that can be utilized across multiple laboratory settings.

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Lab products found in correlation

Benzonase, by Merck Group (4 mentions) Lightcycler 480 sybr green 1 master, by Roche (3 mentions) Aav 293 cells, by Agilent Technologies (3 mentions) Paav mcs, by Agilent Technologies (3 mentions) Prep2 cap2, by Agilent Technologies (2 mentions) Paav rc, by Agilent Technologies (2 mentions) Iodixanal, by Merck Group (2 mentions) Aavpro titration kit, by Takara Bio (2 mentions) Amicon ultra 15 centrifugal filter devices 100 k, by Merck Group (2 mentions) Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions) Paav dj8, by Cell Biolabs (1 mentions) Paav dj, by Cell Biolabs (1 mentions) Faststart universal sybr green master mix, by Roche (1 mentions) Optipreptm, by Axis-Shield (1 mentions) Vpk 420 dj, by Cell Biolabs (1 mentions) Nebuilder hifi, by New England Biolabs (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions) Dulbecco s phosphate buffered saline dpbs, by Merck Group (1 mentions) Optipreptm density gradient medium, by Merck Group (1 mentions) 2v6.11 cells, by American Type Culture Collection (1 mentions)

37 protocols using phelper

1

Production and Purification of AAV Vectors

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Expression cassettes for the IgG or the scFv-IgG were subcloned into an AAV2-ITR containing plasmid, with A1AT stuffer DNA retained as needed to maintain the AAV genome size for proper packaging. In the case of the dual promoter IgG ITR plasmid, no stuffer DNA was included as the cassette was already the maximum size permitted for efficient packaging. AAV-Empty vector consisted of the CBA promoter, Tbgh polyA, and A1AT stuffer DNA. AAV2/1 virus was produced via transient transfection. In brief, HEK293 cells were transfected using PEI (polyethyleneimine) with a 1:1:1 ratio of three plasmids (containing the ITR, AAV rep/cap and Ad helper). The Ad helper plasmid (pHelper) was obtained from Stratagene/Agilent Technologies (Santa Clara, CA). Purification was performed using column chromatography, as previously described [47 (link)]. Virus was titered using qPCR against the polyA sequence, and AAVs were stored in 180mM Sodium Chloride, 10mM Sodium Phosphate (5mM Monobasic + 5mM Dibasic), 0.001% F68, pH 7.3 at -80C until use.
Elmer B.M., Swanson K.A., Bangari D.S., Piepenhagen P.A., Roberts E., Taksir T., Guo L., Obinu M.C., Barneoud P., Ryan S., Zhang B., Pradier L., Yang Z.Y, & Nabel G.J. (2019). Gene delivery of a modified antibody to Aβ reduces progression of murine Alzheimer’s disease. PLoS ONE, 14(12), e0226245.
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2

AAV Production Protocols

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AAV production was performed as previously described.29 (link) In brief, an AAV vector plasmid (pAAV-CAG-mCherry, pAAV-CAG-Kitl or pAAV-CAG-Cre), an adenovirus helper plasmid (pHelper; Agilent Technologies, Santa Clara, CA), and an AAV helper plasmid (pAAV-RC; Agilent Technologies, pAAV1, pAAV6.2, pAAV9; gift from Penn Vector Core [University of Pennsylvania, PA], pAAV-DJ, pAAV-DJ8; Cell biolabs, San Diego, CA, and pAAV-7M8; Addgene, Boston, MA) were transiently transfected into 293T cells. Viral titers (in vector genomes (VG)/mL) were determined by real-time PCR using FastStart Universal SYBR Green Master Mix (Roche Diagnostic GmbH, Penzberg, Germany) and specific primers, as described previously (Watanabe et al., 2018). For screening, the titer of the virus was 1 × 1012 vector genomes/mL. In some experiments, we also used AxCAN-Egfp (RIKEN BRC), CSII-EF1-IRES-Venus (RIKEN BRC) and CSII-EF1-Kitl. The titer of AxCAN-Egfp was 2.0 × 108 plaque-forming units/mL. The titers of CSII-EF1-IRES-Venus and CSII-EF1-Kitl were 1.5 × 109 transducing units (TU)/mL and 2.8 × 109 TU/mL, respectively.
Kanatsu-Shinohara M., Lee J., Miyazaki T., Morimoto H, & Shinohara T. (2022). Adeno-associated-virus-mediated gene delivery to ovaries restores fertility in congenital infertile mice. Cell Reports Medicine, 3(5), 100606.
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3

In Vivo Delivery of AAV-PQBP1 Vector

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The AAV vector plasmids contained an expression cassette, consisting of a human cytomegalovirus immediate-early promoter (CMV promoter); followed by cDNA encoding human PQBP1 or human PQBP1-EGFP; and a simian virus 40 polyadenylation signal sequence (SV40 poly (A)) between the inverted terminal repeats of AAV 3 genome. The recombinant AAV vectors were produced by transient transfection of HEK293 cells using the vector plasmid, an AAV2 rep and AAV1 vp expression plasmid, and an adenoviral helper plasmid, pHelper (Agilent Technologies). The recombinant viruses were purified by isolation form 2 sequential CsCl gradients, and the viral titers were determined by qRT-PCR. For in vivo administration of AAV vectors, C57BL/6J pregnant mice (E10) were injected with AAV-PQBP1 vector (2.0×1011 genome copies) by intraperitoneal administration.
Ito H., Shiwaku H., Yoshida C., Homma H., Luo H., Chen X., Fujita K., Musante L., Fischer U., Frints S.G., Romano C., Ikeuchi Y., Shimamura T., Imoto S., Miyano S., Muramatsu S.I., Kawauchi T., Hoshino M., Sudol M., Arumughan A., Wanker E.E., Rich T., Schwartz C., Matsuzaki F., Bonni A., Kalscheuer V.M, & Okazawa H. (2014). In utero gene therapy rescues microcephaly caused by Pqbp1-hypofunction in neural stem progenitor cells. Molecular Psychiatry, 20(4), 459-471.
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4

AAV-Mediated PQBP1 Gene Delivery in Mice

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The AAV vector plasmids contained an expression cassette, consisting of a human cytomegalovirus immediate-early promoter (CMV promoter), followed by cDNA encoding human PQBP1 or human PQBP1-EGFP, and a simian virus 40 polyadenylation signal sequence (SV40 poly (A)) between the inverted terminal repeats of AAV 3 genome. The recombinant AAV vectors were produced by transient transfection of HEK293 cells using the vector plasmid, an AAV2 rep and AAV1 vp expression plasmid, and an adenoviral helper plasmid, pHelper (Agilent Technologies, Santa Clara, CA, USA). The recombinant viruses were purified by isolation from two sequential CsCl gradients, and the viral titers were determined by qRT-PCR. For in vivo administration of AAV vectors, C57BL/6J pregnant mice (E10) were injected with AAV-PQBP1 vector (2.0 × 1011 genome copies) by intraperitoneal administration.
Ito H., Shiwaku H., Yoshida C., Homma H., Luo H., Chen X., Fujita K., Musante L., Fischer U., Frints S.G., Romano C., Ikeuchi Y., Shimamura T., Imoto S., Miyano S., Muramatsu S.I., Kawauchi T., Hoshino M., Sudol M., Arumughan A., Wanker E.E., Rich T., Schwartz C., Matsuzaki F., Bonni A., Kalscheuer V.M, & Okazawa H. (2014). In utero gene therapy rescues microcephaly caused by Pqbp1-hypofunction in neural stem progenitor cells. Molecular Psychiatry, 20(4), 459-471.
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5

Neuron-Specific GLUT1 Expression via AAV

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The AAV vector plasmids contained an expression cassette consisting of the neuron-specific synapsin I promoter, followed by human SLC2A1, sharing high homology with mouse GLUT1 (96% identical at the amino acid level) with or without myc-DDK (FLAG®), and a simian virus 40 polyadenylation signal sequence between the inverted terminal repeats of the AAV3 genome (Fig. 1). We synthesized the AAV9 vp cDNA as described previously [20] (link) with the substitution of thymidine for adenine 1337, which introduced an amino acid change from tyrosine to phenylalanine at position 446. Recombinant AAV vectors were produced by transient transfection into HEK293 cells using the vector plasmid, an AAV3 rep and AAV9 vp expression plasmid, and the adenoviral helper plasmid pHelper (Agilent Technologies, Santa Clara, CA). We purified the recombinant viruses by isolation from two sequential continuous CsCl gradients, and viral titers were determined by quantitative PCR.
Nakamura S., Osaka H., Muramatsu S.I., Takino N., Ito M., Aoki S., Jimbo E.F., Shimazaki K., Onaka T., Ohtsuki S., Terasaki T, & Yamagata T. (2017). Gene therapy for a mouse model of glucose transporter-1 deficiency syndrome. Molecular Genetics and Metabolism Reports, 10, 67-74.
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6

Production and Characterization of Engineered AAV Serotypes

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Recombinant AAV9, AAVMYO, MyoAAV4A and 2 A were generated by the molecular biology and virus facility at IGBMC, by a triple transfection of HEK293T/17 cell line with the expression plasmid pAAV-CMV-luc-IRES-GFP-SV40pA (Addgene #105,533, Massachusetts, US) and the auxiliary plasmids pHelper (Agilent, California, US) and the capsid plasmids pAAV2/9 P0008 (Penn Vector Core, Pennsylvania, US), pAAVMYO, pMyoAAV2A or pMyoAAV4A (all created by modifying the AAV2/9 capsid by insertion of peptide sequences made available in the literature [27 (link), 28 (link)]). Recombinant adeno-associated viruses (rAAV) were harvested 48 h after transfection from cell lysates treated with 100U/mL Benzonase (Merck, New jersey, US). All AAV serotypes were purified by Iodixanol gradient ultracentrifugation (OptiprepTM, Axis Shield) followed by dialysis and concentration against Dulbecco’s PBS containing 0.5mM MgCl2 using centrifugal filters (Amicon Ultra-15 Centrifugal Filter Device 100 K). Viral titers were determined either by qPCR using the LightCycler480 SYBR Green I Master (Roche, Switzerland) and primers targeting eGFP sequence or by digital droplet PCR (ddPCR) [37 (link)]. Viruses were stored at -80 °C until use.
Ji J., Lefebvre E, & Laporte J. (2024). Comparative in vivo characterization of newly discovered myotropic adeno-associated vectors. Skeletal Muscle, 14, 9.
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7

Molecular Cloning Techniques for AAV Production

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Standard molecular cloning techniques were used to generate DNA constructs in this study. Double-stranded DNA was synthesized by Integrated DNA Technologies and inserted into pAAV backbones with NEBuilder HIFI (New England Biolabs, E2621). sgRNA sequences were synthesized as overlapping single-stranded DNA oligos (Integrated DNA Technologies) that were then annealed together and ligated into sgRNA expression cassettes using T4 DNA ligase (New England Biolabs, M0202).
pUCmini-iCAP-AAV-PHP.eB12 (link) (Addgene #175004), pUCmini-iCAP-AAV.CAP-B1014 (link) (Addgene #103005), pUCmini-iCAP-AAV.MaCPNS215 (link) (Addgene #185137), AAV-DJ rep-cap (Cell Biolabs, VPK-420-DJ), AAV6 rep-cap (Cell Biolabs, VPK-420-DJ), and pHelper (Agilent, #240071) plasmids were used for production of AAVs. Prior to use, all plasmids were sequence verified via whole-plasmid sequencing (Primordium Labs).
Coughlin G.M., Borsos M., Appling N., Barcelona B.H., Mayfield A.M., Mackey E.D., Eser R.A., Chen X., Kumar S.R, & Gradinaru V. (2023). Spatial genomics of AAVs reveals mechanism of transcriptional crosstalk that enables targeted delivery of large genetic cargo. bioRxiv.
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8

AAV-mediated Gene Delivery in Mice

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AAV293 cells (Agilent Technologies) were cultured in DMEM (Mediatech) supplemented with 10% FBS and 1× antibiotic antimycotic solution (Mediatech). Cells were cultured in a humidified incubator with 5% CO2 and 95% air at 37°C, confirmed to be mycoplasma free, and authenticated by short tandem repeat DNA profiling. The cells were transfected with three plasmids [AAV transgene, pHelper (Agilent Technologies), and AAV2/9] for viral production. Viral particles were purified using a discontinuous iodixanol (Sigma) gradient and resuspended in PBS with 10% glycerol and stored at −80°C. Viral titer or vector genome number was determined by quantitative PCR using custom TaqMan assays specific for woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequences. Standard curves for WPRE were obtained from serial dilutions over a 6 log range of the corresponding plasmids. AAV-mediated gene delivery provides a means to achieve long-lasting transgene expression without the inflammatory responses that are commonly associated with other viral vectors. When introduced into adult mice, sustained expression of up to 1 year has been observed. The major site of transgene expression is the hepatocytes.
Zhou M., Learned R.M., Rossi S.J., Tian H., DePaoli A.M, & Ling L. (2019). Therapeutic FGF19 promotes HDL biogenesis and transhepatic cholesterol efflux to prevent atherosclerosis. Journal of Lipid Research, 60(3), 550-565.
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9

Recombinant AAV2/2 Virus Production

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AAV-ophNdi1 (patent no. 10220102), AAV-Ndi1 [39 (link)] and AAV-CAG-EGFP (AAV-EGFP; [47 (link)]) recombinant AAV2/2 viruses were generated as previously described [50 (link)]. Briefly, HEK293 cells were transfected with pAAV-ophNdi1, pAAV-Ndi1 or pAAV-EGFP, pRep2/Cap2 and pHelper (Agilent Technologies, Santa Clara, CA, USA) at a ratio of 1:1:2, using polyethylenimine. At 72 h post-transfection, AAV particles were purified from the clarified lysate by triple caesium gradient ultracentrifugation and dialysed against PBS supplemented with Pluronic F68 (0.001%; [51 (link)]). Genomic titres (vg/mL) were determined by quantitative real-time PCR [52 (link)].
Chadderton N., Palfi A., Maloney D.M., Carrigan M., Finnegan L.K., Hanlon K.S., Shortall C., O’Reilly M., Humphries P., Cassidy L., Kenna P.F., Millington-Ward S, & Farrar G.J. (2023). Optimisation of AAV-NDI1 Significantly Enhances Its Therapeutic Value for Correcting Retinal Mitochondrial Dysfunction. Pharmaceutics, 15(2), 322.
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10

AAV Serotype 2 Packaging Protocol

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To package the AAV genome into the AAV serotype 2 capsid, HEK-293A cells were cotransfected with an AAV-ITR plasmid, pHelper (Agilent), and pAAV-RC (Agilent) using calcium phosphate precipitation. Two days later, HEK-293A cells were harvested, centrifuged, and suspended in artificial cerebrospinal fluid (aCSF). The collected cells were lysed by freeze-thaw cycles following the vortexing. To isolate AAV vectors, cell debris was removed by centrifugation, and the supernatant was subjected to DNase/RNase-treatment. The titer of isolated AAV was >1012 genomic copies/ml, which was determined by quantitative PCR.
Nakatsuka D., Kanda T., Sato M., Ishikawa Y., Cherasse Y, & Yanagisawa M. (2024). A novel GABAergic population in the medial vestibular nucleus maintains wakefulness and gates rapid eye movement sleep. iScience, 27(3), 109289.
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