Journal: The Journal of Clinical Investigation

Article Title: A versatile modular vector system for rapid combinatorial mammalian genetics

doi: 10.1172/JCI79743

Figure Lengend Snippet: (A) Restriction enzyme cloning is used to generate MuLE Entry vectors with a desired genetic insert cloned downstream of a desired promoter (P), with the entire promoter-insert element being surrounded by appropriate attL-attR sites. (B) Schematic overview of the MultiSite Gateway–based recombination cloning of 2, 3, or 4 MuLE Entry vectors into lentiviral destination vectors to generate multicistronic MuLE lentiviral expression vectors. The specific attL-attR sites that mediate each recombination are depicted. CMR, chloramphenicol resistance gene; ccdB, ccdB toxin gene. (C) Transfection of 293T cells with a MuLE expression vector plasmid together with a lentiviral packaging vector (psPAX2) and a vector encoding either amphotropic (MD2G) or ecotropic (pEco) envelope proteins generates MuLE lentiviruses for transduction of cultured cells or for in vivo injection into mouse tissues.

Article Snippet: For a 10-cm dish, lentiviral vector (8 μg) was cotransfected with the lentiviral packaging vector psPAX2 (Addgene, no. 12260) and either the ecotropic envelope (pCMV-Eco, Cellbiolabs, no. RV-112) or the amphotropic envelope (pMD2.G, Addgene, no. 12259).

Techniques: Clone Assay, Expressing, Transfection, Plasmid Preparation, Transduction, Cell Culture, In Vivo, Injection

Journal: The Journal of Clinical Investigation

Article Title: A versatile modular vector system for rapid combinatorial mammalian genetics

doi: 10.1172/JCI79743

Figure Lengend Snippet: (A) Luminescent imaging of various human and mouse cultured cells after infection with ecotropic MuLE viruses expressing an empty cassette (Ctrl) or luciferase (Luc). The human kidney cell lines 293T and 786-0 were not infected by these viruses, but various primary mouse cells and cell lines were infected, including MEFs, embryonic stem cells (ES), kidney epithelial cells (KEC), endometrial epithelial cells (EEC), aortic endothelial cells (AEC), hepatocytes (Hep), myoblasts (C2C12), melanoma cells (B16), lung carcinoma cells (LLC-1), and colorectal carcinoma cells (MC-38). (B) Schematic of MuLE vectors with or without the kidney epithelium–specific Ksp1.3 promoter cloned upstream of a cDNA encoding EGFP. (C) Representative bright field (BF) and green fluorescence (EGFP) images of primary mouse kidney epithelial cells (PKCs) and primary MEFs transduced with the lentiviral vectors shown in B. Original magnification, ×1 (A); ×10 (C).

Article Snippet: For a 10-cm dish, lentiviral vector (8 μg) was cotransfected with the lentiviral packaging vector psPAX2 (Addgene, no. 12260) and either the ecotropic envelope (pCMV-Eco, Cellbiolabs, no. RV-112) or the amphotropic envelope (pMD2.G, Addgene, no. 12259).

Techniques: Imaging, Cell Culture, Infection, Expressing, Luciferase, Clone Assay, Fluorescence, Transduction