AAV vectors were based on the AAV helper-free system (Agilent Technologies, CA, USA). To generate plasmids encoding shRNA driven by a U6 promoter, a 0.7-kb PvuII fragment from pSilencer 2.1-U6 NEO (Ambion Life Technologies, MA, USA) was inserted into the blunted MluI site of pAAV-hrGFP (Agilent Technologies)52 (link). The CMV promoter for hrGFP was replaced by the human synapsin I promoter. A shRNA cassette was inserted into the plasmid digested with BamHI and HindIII located directly below the U6 promoter52 (link). The shRNA target sequence for the marmoset D1R was 5′-GTCGAATGTTCTCAACCAGAA-3′, and that for the marmoset D2R was 5′-GGACAGACCTCACTACAATTA-3′. Silencing efficacy was evaluated using the Dual-Luciferase reporter assay system (Promega, WI, USA)53 (link). The shRNAs with more than 80% knockdown efficacy in vitro were identified (see Supplementary Fig. 1 ), and those having high efficacy were tested more than twice.
Paav hrgfp
Manufactured by Agilent Technologies
Sourced in United States
The PAAV-hrGFP is a lab equipment product designed for biological research applications. It functions as a plasmid-based expression vector that enables the production of recombinant human green fluorescent protein (hrGFP) in host cells. The core function of this product is to facilitate the expression and visualization of the hrGFP protein in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Aav helper free system, by Agilent Technologies (2 mentions)
Psilencer 2.1 u6 neo, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Paav mcs, by Agilent Technologies (1 mentions)
Stepone software v2, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
High pure viral nucleic acid kit, by Roche (1 mentions)
4 protocols using paav hrgfp
1
Generating Plasmids for Marmoset D1R and D2R Knockdown
2
AAV Vector Production and Purification
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The AAV Helper-Free System (Agilent Technologies, Inc., Santa Clara, CA, USA) was used to produce AAV and purify the AAV vectors43 (link). The plasmids pAAV-hSyn-FLEX-hM3Dq-mCherry, pAAV-hSyn-FLEX-mCherry and pAAV-EF1α-DIO-hChR2(E123T/T159C)-EYFP were purchased from Addgene (ID: 44361, 44362, 50459, 35509). pAAV-CMV-FLEX-hrGFP was constructed starting with the pAAV-hrGFP plasmid (Agilent Technologies), and pAAV-CMV-FLEX-DTA was constructed starting with the pAAV-MCS plasmid (Agilent Technologies), and its cell-specific ablation was confirmed as previously reported44 (link). The injection volume was 600 nl per side.
3
siRNA Plasmid Construction for HPV-16 E6/E7 Silencing
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The U6 promoter and siRNA sequences were cut out from the PvuII site of the pSilencer U6 vector (Thermo Fisher Scientific, Waltham, MA, USA) and inserted into the blunted MluI site of pAAV-hrGFP (Agilent Technologies, Santa Clara, CA, USA) to prepare pu6si-CmV-green fluorescent protein (gFp). The target site for the shRNA was the following 21-base sequence (564–582) in HPV-16 E6/E7 mRNA, taken from a previously published study (26 (link)): sense, 5′-GATCCGCATGGAGATACACCTACAttcaagagaTGTAGGTGTATCTCCATGCTTTTTTG-3′ and antisense, 5′-AATTCAAAAAAGCATGGAGATACACCTACAtctcttgaaTGTAGGTGTATCTCCATGCG-3′. The two oligomers were heated at 90°C for 3 min, annealed by slow cooling to 37°C over a 30-min period, and inserted into the multi-cloning sites of pU6-MCS-CMV-GFP and the BamHI and HindIII sites to prepare an shE6E7-targeting shRNA expression plasmid vector (pU6-shE6E7-CMV-GFP). A control vector (pU6-shNC-CMV-GFP) was prepared by inserting a 21-base sequence (Thermo Fisher scientific) known to have no influence on gene expression into pU6-shNC-CMV-GFP at the same sites.
4
Quantifying rAAV Genome Copies in Cell Lines
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Total DNA was isolated using the High Pure Viral Nucleic Acid kit (Roche Life Science) from AAV-293, Caov-3 and NIH:OVCAR-3 cell lines. The amount of DNA was quantified by 260/280/230 nm absorbance measurements using spectrophotometer Quawell Q5000 UV–Vis (Quawell). TaqMan assays for rAAV genome copy number were developed using probe and primers for the ITR region. The fluorescent probe (5′-CACTCCCTCTCTGCGCGCTCG-3′) featured 6-FAM and TAMRA. The forward primer was 5′-GGAACCCCTAGTGATGGAGTT-3′ and the reverse primer was 5′-CGGCCTCAGTGAGCGA-3′ (36 (link)). Total volume of qPCR reactions was 10 µl, contained 50 ng DNA and ran under the following conditions: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 sec and 60°C for 60 sec. qPCR was performed in StepOnePlus™ Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Absolute quantification analysis with the StepOne Software v2.3 (Thermo Fisher Scientific, Inc.) was performed to determine transcripts numbers. Data were quantified using the standard curve (37, 38). Briefly, standard curves were generated using serial dilutions of plasmid pAAV-hrGFP (Agilent Technologies) used to calculate the number of genome copies in the tested samples. The rAAV genome copy number was normalized as viral genome copy number per 50 µg of total genomic DNA.
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