Production and Validation of Rap-related Lentiviruses

pcDNA3 plasmids containing Flag-Cebpa (66978), Flag-Trib1 (131156), Flag-Rap1b N17 (118322), and Flag-Rap1b V12 (118323) were purchased from Addgene. Plasmids containing Rap2a (55666) and Rap2b (55667) were also purchased from Addgene. Plasmids containing Rap2a and Rap2b genes were transferred to the pcDNA3 vector and mutated to dominant=negative N17 and constitutively active V12 using the In-fusion HD EcoDRy Cloning Kit from Takara (639690) according to the manufacturer’s protocol. Furthermore, Rap2a mutants, Cebpa, and Trib1 were cloned into pLVX for lentiviral production. Lentivirus particles were produced in HEK293T LentiX cells (ATCC) as previously described (81 (link)) and used to treat 3T3L1 and PPDIVs.

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Valentine J.M., Ahmadian M., Keinan O., Abu-Odeh M., Zhao P., Zhou X., Keller M.P., Gao H., Yu R.T., Liddle C., Downes M., Zhang J., Lusis A.J., Attie A.D., Evans R.M., Rydén M, & Saltiel A.R. (2022). β3-Adrenergic receptor downregulation leads to adipocyte catecholamine resistance in obesity. The Journal of Clinical Investigation, 132(2), e153357.

Publication 2022

Flag-Rap1b N17 Flag-Rap1b V12 In-fusion HD EcoDRy Cloning Kit

Cebpa Cells Genes Lentivirus Plasmids Rap2a Vector

Corresponding Organization :

Other organizations : University of Wisconsin–Madison, Karolinska University Hospital, Karolinska Institutet, Salk Institute for Biological Studies, Westmead Institute for Medical Research, University of Sydney, University of California, Los Angeles

Top 5 similar protocols

Variable analysis

independent variables

Flag-Cebpa
Flag-Trib1
Flag-Rap1b N17
Flag-Rap1b V12
Rap2a N17
Rap2b N17
Rap2a V12
Rap2b V12

dependent variables

Treated 3T3L1 cells
Treated PPDIVs

control variables

PcDNA3 plasmids
HEK293T LentiX cells

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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