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Padeasy 1 vector

Manufactured by Agilent Technologies
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The PAdEasy-1 vector is a plasmid used for cloning and expressing genes in bacterial and mammalian cells. It contains essential elements for DNA replication, selection, and expression.

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Lab products found in correlation

293a cells, by Thermo Fisher Scientific (2 mentions) Pgem t vector, by Promega (2 mentions) Bj5183 competent cells, by Agilent Technologies (1 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions) Pshuttle cmv vector, by Addgene (1 mentions) Pmd19 t, by Takara Bio (1 mentions)

Close spelling variants of this product

PAdEasy vector PAdEasy-1 PAdEasy

5 protocols using padeasy 1 vector

1

Adenoviral Expression of IRF6

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Adenovirus production followed the AdEasy protocol56 (link). Briefly, human IRF6 cDNA from pCMV-SPORT6-IRF6 plasmid was cloned into pAdTrack-CMV vector (Agilent Technologies) at Xho I and Xba I restriction enzyme sites. The AdIRF6 adenovirus expression plasmid was generated from recombination between pAdEasy-1 vector and pAdTrack-CMV-IRF6 in BJ5183 competent cells (Agilent Technologies). AdIRF6 plasmid (40 μg) was linearized with Pac I restriction enzyme and subsequently transfected into AD293 cells using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol for adenovirus packaging. Adenoviruses were harvested at 14 to 20 days after transfection.
Ke C.Y., Xiao W.L., Chen C.M., Lo L.J, & Wong F.H. (2015). IRF6 is the mediator of TGFβ3 during regulation of the epithelial mesenchymal transition and palatal fusion. Scientific Reports, 5, 12791.
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2

Adenovirus-Mediated Overexpression of miR-182

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Control adenovirus and adenovirus expressing human SREBP-2 were purchased (Eton Biosciences) and amplified in 293A cells (Life Technologies). Adenovirus expressing the precursor form of mmu-miR-182 was generated using the AdEasy XL Adenoviral Vector System. The DNA sequence from 157-bp upstream through 224-bp downstream of the mouse pri-miR-182 sequence was amplified by PCR with primers containing NheI and XhoI restriction sites on either end. This fragment was cloned into the pGEM-T vector (Promega), sequenced, transferred into the pShuttle vector and subsequently cloned into the pAd-Easy-1 vector (Agilent).
Miao J., Ling A.V., Manthena P.V., Gearing M.E., Graham M.J., Crooke R.M., Croce K.J., Esquejo R.M., Clish C.B., Vicent D, & Biddinger S.B. (2015). Flavin-containing monooxygenase 3 as a potential player in diabetes-associated atherosclerosis. Nature Communications, 6, 6498.
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3

Adenoviral Vector Construction for miR-182

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Control adenovirus and adenovirus expressing human SREBP-2 were purchased (Eton Biosciences) and amplified in 293A cells (Life Technologies). Adenovirus expressing the precursor form of mmu-miR-182 was generated using the AdEasy™ XL Adenoviral Vector System. The DNA sequence from 157-bp upstream through 224-bp downstream of the mouse pri-miR-182 sequence was amplified by PCR with primers containing NheI and XhoI restriction sites on either end. This fragment was cloned into the pGEM-T vector (Promega), sequenced, transferred into the pShuttle vector and subsequently cloned into the pAd-Easy-1 vector (Agilent).
Miao J., Ling A.V., Manthena P.V., Gearing M.E., Graham M.J., Crooke R.M., Croce K.J., Esquejo R.M., Clish C.B., Vicent D, & Biddinger S.B. (2015). Flavin-containing monooxygenase 3 as a potential player in diabetes-associated atherosclerosis. Nature Communications, 6, 6498.
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4

Overexpression of Mouse Tie2 in Lung Microvascular Endothelial Cells

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Mouse Tie2 (mTie2) cDNA plasmids was purchased from Sino Biological Inc. (# MG51087-G). To produce the overexpression adenovirus, the mTie2 cDNA was sub-cloned into pShuttle-CMV vector (a gift from Bert Vogelstein51 (link) (Addgene plasmid # 16403)) and subsequently recombined into pAdEasy-1 vector (Agilent Technologies) by co-transforming with the BJ5183 cells. After the recombinant Ad plasmid was transformed with XL10-Gold cells, the virus was packaged and amplified in HEK-293AD cells. The harvested mTie2 adenovirus was used to infect Tie2−/− HLMVECs as previously described52 (link) and outlined in the AdEasyAdenoviral Vector System User Manual (Agilent Technologies #240009-12).
Gong H., Liu M., Klomp J., Merrill B.J., Rehman J, & Malik A.B. (2017). Method for Dual Viral Vector Mediated CRISPR-Cas9 Gene Disruption in Primary Human Endothelial Cells. Scientific Reports, 7, 42127.
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5

Adenoviral Overexpression of miR-200c

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The recombinant adenoviral vector for overexpression of miR200c, Ad-miR200c, was constructed according to the protocol previously reported (30 (link)). Briefly, the pri-miR200c sequence was amplified from 293 cell genomic DNA. PCR products were cloned into plasmid pMD19-T (Takara Biotechnology Co., Ltd.), verified by sequencing and subcloned into shuttle plasmid pAdTrack-CMV to construct pAdTrack-miR200c. pAdTrack-miR200c, linearized with PmeI, was used to transform BJ5183-AD-1 cells harboring the adenoviral pAdeasy-1 vector (Stratagene; Agilent Technologies, Inc.) for homologous recombination. Colonies were screened by plasmid miniprep and PacI restriction analysis to obtain clones with recombinant miR200c (designated ‘pAdeasy-miR200c’). PacI-linearized pAdeasy-miR200c was used to transfect 293 cells to obtain packaged recombinant miR200c adenovirus (designated Ad-miR200c). Ad-miR200c was amplified by repeated infection and verified by PCR. The pAdTrack-CMV empty vector was used as control (designated ‘Ad-control’). The titers and the multiplicity of infection were determined according to the manufacturer's protocols. After transfection for 48 h, green fluorescence was observed using an inverted fluorescence microscope (1X71; Olympus Corporation).
Xie H., Nie L., Zhang M., Su Z., Chen X., Xu M., Gong J., Chen N, & Zhou Q. (2019). Suppression of α-methylacyl-coenzyme A racemase by miR200c inhibits prostate adenocarcinoma cell proliferation and migration. Experimental and Therapeutic Medicine, 19(3), 1806-1816.
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