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Padeasy system

Manufactured by Agilent Technologies
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The PAdEasy system is a laboratory equipment product manufactured by Agilent Technologies. It is designed to facilitate the cloning and expression of target genes. The core function of the PAdEasy system is to enable the construction of recombinant adenoviral vectors, which can be used for various research and applications.

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Lab products found in correlation

Quicktiter adenovirus titer immunoassay kit, by Cell Biolabs (1 mentions) Lamp1 rfp, by Addgene (1 mentions) Dsred2 peroxisomes 4, by Addgene (1 mentions) Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions) Adeno xtm rapid titer kit, by Takara Bio (1 mentions) Fk866, by Merck Group (1 mentions) Cay10591, by Cayman Chemical (1 mentions) Ex527, by Selleck Chemicals (1 mentions) Resveratrol, by Cayman Chemical (1 mentions)

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PAdEasy

5 protocols using padeasy system

1

Adenoviral SIRT6 and shRNA Production

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Adenoviruses carrying SIRT6 or GFP were generated using pAdEasy system (Agilent) while adenoviral Sirt6 (mouse) or GFP shRNAs were generated using BLOCK-iT system (Invitrogen). Adenoviruses were amplified in HEK293A cells and purified by CsCl gradient centrifugation. The viruses were titered using QuickTiter adenovirus titer immunoassay kit (Cell Biolabs) according to the manufacturer’s protocol. Generally, we used 25–50 multiplicity of infection (MOI) for overexpression and 50–100 MOI for shRNA knockdown experiments.
Xiong X., Sun X., Wang Q., Qian X., Zhang Y., Pan X, & Dong X.C. (2016). SIRT6 protects against palmitate-induced pancreatic β-cell dysfunction and apoptosis. The Journal of endocrinology, 231(2), 159-165.
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2

Cloning and Tagging of Cellular Organelles

Cited 1 time
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PCR amplification of genes of interest was performed from cDNA as previously described58 (link) using Phusion DNA polymerase (Thermo Fisher). Human ACAD11 was cloned from Hep3B cell cDNA, and mouse Psmd9 was cloned from mouse liver cDNA. Amplified open-reading frames were cloned into expression vectors using Gateway Technology, restriction enzyme digest or Gibson assembly reactions (pDEST-47 GFP, C-term 3xFlag) or pAdEasy system (Agilent). Expression vectors were sequence verified and subsequently amplified and purified by midi-prep (Promega) before use in downstream protocols. Fluorescently tagged organelle constructs were purchased from AddGene (plasmid 1817: Lamp1-RFP, 54503: DsRed2-Peroxisomes-4 and 58014: mTa-gRFP-T-Endosomes-14).
Parker B.L., calkin A.C., Seldin M.M., Keating M.F., Tarling E.J., Yang P., Moody S.C., liu Y., Zerenturk E.J., Needham E.J., Miller M.L., clifford B.L., Morand P., Watt M.J., Meex R.C., Peng K.Y., lee R., Jayawardana K., Pan C., Mellett N.A., Weir J.M., lazarus R., lusis A.J., Meikle P.J., James D.E., de Aguiar Vallim T.Q, & Drew B.G. (2019). An integrative systems genetic analysis of mammalian lipid metabolism. Nature, 567(7747), 187-193.
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3

Adenovirus-mediated NAMPT and GFP Expression

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Adenoviruses carrying NAMPT, GFP were generated using the pAdEasy system (Agilent); Sirt1 shRNA and control shRNA were generated using the BLOCK-iT system (Invitrogen). Adenoviruses were amplified in HEK293A cells and purified by CsCl gradient centrifugation. The viruses were titered using an Adeno-XTM Rapid Titer kit (Takara) according to the manufacturer’s manual.
Xiong X., Yu J., Fan R., Zhang C., Xu L., Sun X., Huang Y., Wang Q., Ruan H.B, & Qian X. (2019). NAMPT overexpression alleviates alcohol-induced hepatic steatosis in mice. PLoS ONE, 14(2), e0212523.
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4

Construction of Recombinant Ad Vectors Expressing HER3

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The human HER3 complementary DNA (cDNA) was excised from a pCMVSport6-HER3-HsIMAGE6147464 plasmid (cDNA clone MGC:88033/IMAGE:6147464 obtained from ATCC). Construction of a first-generation (E1-, E3-) Ad vector containing human full length HER3 under control of human cytomegalovirus (CMV) promoter/enhancer elements was performed using the pAdEasy system (Agilent technologies, Santa Clara, CA, USA) as previously described [22 –24 (link)]. Similar Ad-vectors containing the green fluorescence protein (GFP) or lacZ rather than HER3 was similarly generated to serve as controls.
Osada T., Hartman Z.C., Wei J., Lei G., Hobeika A.C., Gwin W.R., Diniz M.A., Spector N., Clay T.M., Chen W., Morse M.A, & Lyerly H.K. (2018). Polyfunctional anti-human epidermal growth factor receptor 3 (anti-HER3) antibodies induced by HER3 vaccines have multiple mechanisms of antitumor activity against therapy resistant and triple negative breast cancers. Breast Cancer Research : BCR, 20, 90.
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5

Adenovirus-mediated BMAL1 and CLOCK overexpression in primary hepatocytes

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Adenoviruses were generated using pAdViraPower system (Life Technologies) for Ad-Clock, pAdEasy system (Agilent) for Ad-Bmal1. Over-expression of BMAL1 or CLOCK in primary hepatocytes was achieved by transducing primary hepatocytes with the concentrated adenovirus at 1x109 pfu/mL. SIRT1 inhibitor EX527 was purchased from Selleckchem. Resveratrol and CAY10591 were from Cayman. FK866 was from Sigma.
Tong X., Zhang D., Arthurs B., Li P., Durudogan L., Gupta N, & Yin L. (2015). Palmitate Inhibits SIRT1-Dependent BMAL1/CLOCK Interaction and Disrupts Circadian Gene Oscillations in Hepatocytes. PLoS ONE, 10(6), e0130047.
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