Paav mcs expression vector

Manufactured by Cell Biolabs

The PAAV-MCS expression vector is a plasmid designed for gene expression studies. It contains a multiple cloning site (MCS) for the insertion of a gene of interest, allowing for the expression of the encoded protein. The vector also includes necessary elements for replication and selection in bacterial and mammalian cell lines.

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Lab products found in correlation

Paav dj 8 vector, by Cell Biolabs (2 mentions) Vivaspin 20 concentrator, by Sartorius (2 mentions) 293aav cells, by Cell Biolabs (2 mentions) Pureyield plasmid maxiprep system, by Promega (1 mentions) In fusion hd cloning plus kit, by Takara Bio (1 mentions) Helper plasmid, by Agilent Technologies (1 mentions) Paav gfp control plasmid, by Cell Biolabs (1 mentions) Nebuilder hifi dna assembly polymerase, by New England Biolabs (1 mentions) Cloneamp hifi polymerase, by Takara Bio (1 mentions) C1000 touch thermal cycler, by Bio-Rad (1 mentions) Aavpro 293t cells, by Takara Bio (1 mentions) Aav quantitation kit, by Cell Biolabs (1 mentions)

Spelling variants of this product

PAAV-MCS (9 citations) PAAV-MCS vector (7 citations) PAAV-MCS Promoterless Expression Vector (3 citations)

5 protocols using paav mcs expression vector

Construction and Delivery of Recombinant AAV Vectors

Cited 1 time

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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.

Kashihara T., Mukai R., Oka S.I., Zhai P., Nakada Y., Yang Z., Mizushima W., Nakahara T., Warren J.S., Abdellatif M, & Sadoshima J. (2022). YAP mediates compensatory cardiac hypertrophy through aerobic glycolysis in response to pressure overload. The Journal of Clinical Investigation, 132(6), e150595.

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Viral Delivery of Myogenic Factors

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The cDNA for MyoD (Addgene, plasmid # 14710) and Mist1 (accession # BC094061) were subcloned individually into the pAAV-MCS Expression Vector (Cell Biolabs Inc., # VPK-410) with the In-Fusion HD Cloning Plus Kit (Clontech, # 638910). The insertion of the cDNAs was confirmed by sequencing and the plasmids were amplified overnight in DH5α Escherichia coli (ThermoFisher, # 18265017), purified using the PureYield Plasmid Maxiprep System (Promega, #A2392) and were sent to our in-house viral core for packaging and large scale purification. A third virus was made using pAAV-MCS and served as an empty vector control (AAV-Ctrl). Adult C57BL/6 and Sgcd^−/− mice were administered either AAV9-MyoD, AAV-Mist1 or AAV-Ctrl via an intramuscular injection into the TA muscle at a concentration of 1 × 10¹² viral particles in 40 µl of sterile PBS under inhaled isoflurane anesthesia to effect. Prior to injection, mice were treated with a hair removal product (Nair) at the site of injection.

Boyer J.G., Huo J., Han S., Havens J.R., Prasad V., Lin B.L., Kass D.A., Song T., Sadayappan S., Khairallah R.J., Ward C.W, & Molkentin J.D. (2022). Depletion of skeletal muscle satellite cells attenuates pathology in muscular dystrophy. Nature Communications, 13, 2940.

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Overexpressing Mdm2 and Mdmx in Neurons

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Mice Mdm2 and Mdmx were cloned in our lab and inserted into pAAV-MCS Expression vector (CELL BIOLABS INC, San Diego, CA) using flanking BamHI and XhoI restriction sites. Briefly, AAV 293 were transfected with rAAV (pUCmini-iCAP-PHP.eB (addgene plasmid #103005, Watertown, MA) and rAAV-ITR Mdmx or Mdm2 expression plasmid (single stranded genome) or pAAV-GFP Control Plasmid (CELL BIOLABS INC, San Diego, CA) and helper plasmid (Agilent Technologies, Santa Clara, CA) by the calcium phosphate method. 120 h after transfection cells were harvested and vector purified using a standard iodixanol density gradient and ultracentrifugation protocol. Iodixanol was removed and vector concentrated in PBS by diafiltration using Amicon Ultra 100 kDa MWCO centrifugal devices (Millipore, Billerica, MA). Vector was stored at − 80 °C until use. rAAV titers were determined by quantitative PCR and expressed as genome copies per ml (gc/ml). They AAV particles were applied to the primary neuronal culture at MOI of 5 × 10⁴ at 3 days in vitro and incubated for 3 days.

Yan H., Sasaki T., Kanki H., Hirata Y., Nishiyama K., Hisada S., Matsumura S., Nagaoka Y., Sumiyoshi T., Nagano S., Nakata A., Yoshida M., Uesato S, & Mochizuki H. (2022). MDMX elevation by a novel Mdmx–p53 interaction inhibitor mitigates neuronal damage after ischemic stroke. Scientific Reports, 12, 21110.

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Recombinant MyoAAV-PDK4 Virus Production

Cited 2 times

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Pdk4 cDNA was cloned from genomic cDNA generated from mouse heart tissue using CloneAmp HiFi polymerase (Clontech Laboratories). The cDNA was then cloned into the pAAV-MCS expression vector (Cell Biolabs Inc.) utilizing the NEBuilder HiFi DNA Assembly polymerase (New England BioLabs) (for primers, see key resources table). MyoAAV-PDK4 virus was produced in-house using the MyoAAV 1A capsid as previously described.^{43 ,44 (link)} Briefly, AAV Pro 293T cells (Takara) were plated in 15 cm dishes at a density of 2 ×10⁷ cells/dish. The next day, each plate was transfected with 10.5 μg helper plasmid, 5.25 μg of the Rep/Cap plasmid, and 5.25 μg of the PDK4 MyoAAV-1A plasmid. Recombinant virus was harvested from the cells and media, and purified by ultracentrifuge using an iodixanol gradient.⁴³ AAV titers were quantified by Taqman-based qPCR with C1000 Touch Thermal Cycler (Bio-Rad Laboratories). MyoAAV-luciferase was used as the control virus. For delivery, virus was delivered 4xE10^{13 (link)} vg/Kg of mice via retro-orbital injection at 2-month of age.

Huo J., Prasad V., Grimes K.M., Vanhoutte D., Scott Blair N., Lin S.C., Bround M.J., Bers D.M, & Molkentin J.D. (2023). MCUb is an inducible regulator of calcium-dependent mitochondrial metabolism and substrate utilization in muscle. Cell reports, 42(11), 113465.

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Recombinant AAV Vector Production

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The recombinant adeno-associated virus (AAV) vectors used to generate AAV-DJ/8-PPARα-WT or S280D were constructed by cloning the cDNA of PPARα-WT or S280D (mouse) from the pDC316-YFP-PPARα-WT or S280D vector into the pAAV-MCS expression vector (Cell Biolabs, Inc, #VPK-410) downstream of the CMV promoter with BamHI and SalI. 293AAVcells (Cell Biolabs, Inc.) were co-transfected with the recombinant AAV vectors, pAAV-DJ/8 vector, and helper plasmid in a 1:1:1 ratio using polyethylenimine (PEI) at the AAV core, Department of Cell Biology and Molecular Medicine, New Jersey Medical School, Rutgers University, Newark, NJ (Grimm et al., 2008 (link)). The recombinant AAV produced was purified by the iodixanol gradient/ultra-centrifugation method, and the AAV fraction was concentrated using a VIVASPIN 20 concentrator (100 kDa cut-off, Sartorius, Germany). The virus titer was determined using the Cell Biolabs AAV quantitation kit (Cat. # VPK-145). To administer recombinant AAVs, doses of 2×10¹¹ vector genome per mouse were injected intravenously via the jugular vein of C57BL/6J wild-type mice.

Nakamura M., Liu T., Husain S., Zhai P., Warren J.S., Hsu C.P., Matsuda T., Phiel C.J., Cox J.E., Tian B., Li H, & Sadoshima J. (2019). Glycogen Synthase Kinase-3α Promotes Fatty Acid Uptake and Lipotoxic Cardiomyopathy. Cell metabolism, 29(5), 1119-1134.e12.

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