Adenovirus-Based Fluorescent Reporters

Laconic^{8 (link)}, ATeam, or FLIIPglu700 containing plasmid (Addgene: 44238, 51958, and 17866 respectively) were cloned into Ad5 adenovirus by VectorLabs for construction of Adenovirus-Laconic (Ad-Lac), Adenovirus-ATeam (ad-ATeam), or adenovirus-FLIIPglu700 (ad-Glu). Ad-LifeAct-RFP (ad-LifeAct) was from Ibidi (60122). For transduction into endothelial cells, Ad-Lac at 50:1 multiplicity of infection (MOI), ad-ATeam at 100:1 MOI, and ad-Glu at 200:1 MOI in 1:1 EGM2 media:Opti-MEM (ThermoFisher, 11058021) and 3 μL GeneJammer (Agilent, 204130) per milliliter of final volume was added for 1 hour before replacement of media. For migration and contraction experiments, ad-Lac and ad-LifeAct were co-transduced each with MOI 50:1. Endothelial cells were used 48 hours post transduction to allow for cell recovery and maximal fluorescence.

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Wu D., Harrison D.L., Szasz T., Yeh C.F., Shentu T.P., Meliton A., Huang R.T., Zhou Z., Mutlu G.M., Huang J, & Fang Y. (2021). Single-cell metabolic imaging reveals a SLC2A3-dependent glycolytic burst in motile endothelial cells. Nature metabolism, 3(5), 714-727.

Publication 2021

Opti-MEM GeneJammer

Adenovirus Cell Endothelial cells Fluorescence Genejammer Infection Plasmid

Corresponding Organization :

Other organizations : University of Chicago, National Taiwan University Hospital

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Variable analysis

independent variables

Adenovirus-Laconic (Ad-Lac)
Adenovirus-ATeam (ad-ATeam)
Adenovirus-FLIIPglu700 (ad-Glu)
Adenovirus-LifeAct-RFP (ad-LifeAct)

dependent variables

Endothelial cell migration
Endothelial cell contraction

control variables

Multiplicity of infection (MOI) for each adenovirus
Ratio of EGM2 media and Opti-MEM
GeneJammer concentration
Time after transduction (48 hours)

positive controls

None specified

negative controls

None specified

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