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).
Pshuttle cmv vector
Manufactured by Addgene
Sourced in United States
The pShuttle-CMV vector is a plasmid used for gene expression. It contains a cytomegalovirus (CMV) promoter, which drives the expression of the inserted gene. The vector also includes features for bacterial selection and propagation.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using pshuttle cmv vector
1
Overexpression of Mouse Tie2 in Lung Microvascular Endothelial Cells
2
Adenoviral Expression of ATP2A2a S674
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We constructed adenoviral expression vector of ATP2A2a S674 according to the previously published procedure15 (link). Briefly, using pcDNA3.1-human ATP2A2a plasmid (Cat# 75187; Addgene, Watertown, MA, USA) as template, the full-length ATP2A2a S674 was obtained by point mutation using multiple PCR technology, and then cloned into pShuttle CMV vector (Cat# 16403; Add-gene). The correct direction was confirmed by enzyme restriction analysis as well as sequencing. The adenovirus scaffold vector pAdEasy-1 (Shanghai Weidi Biotechnology Co., Ltd., China), containing most of the human adenovirus serotype 5 (Ad5) genome, is deleted for the genes E1 and E3. pshuttle-ATP2A2a S674 was linearized and then recombined with pAdEasy-1 in BJ5183 cells and colonies were screened for the appropriate constructs using restriction enzyme analysis. The appropriate cosmids were linearized with restriction enzyme and transfected into HEK293A cells for packaging and amplification.
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