A versatile modular vector system for rapid combinatorial mammalian genetics
Joachim Albers, Claudia Danzer, Markus Rechsteiner, Holger Lehmann, Laura P. Brandt, Tomas Hejhal, Antonella Catalano, Philipp Busenhart, Ana Filipa Gonçalves, Simone Brandt, Peter K. Bode, Beata Bode-Lesniewska, Peter J. Wild, Ian J. Frew
Joachim Albers, Claudia Danzer, Markus Rechsteiner, Holger Lehmann, Laura P. Brandt, Tomas Hejhal, Antonella Catalano, Philipp Busenhart, Ana Filipa Gonçalves, Simone Brandt, Peter K. Bode, Beata Bode-Lesniewska, Peter J. Wild, Ian J. Frew
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Technical Advance Oncology

A versatile modular vector system for rapid combinatorial mammalian genetics

Abstract

Here, we describe the multiple lentiviral expression (MuLE) system that allows multiple genetic alterations to be introduced simultaneously into mammalian cells. We created a toolbox of MuLE vectors that constitute a flexible, modular system for the rapid engineering of complex polycistronic lentiviruses, allowing combinatorial gene overexpression, gene knockdown, Cre-mediated gene deletion, or CRISPR/Cas9-mediated (where CRISPR indicates clustered regularly interspaced short palindromic repeats) gene mutation, together with expression of fluorescent or enzymatic reporters for cellular assays and animal imaging. Examples of tumor engineering were used to illustrate the speed and versatility of performing combinatorial genetics using the MuLE system. By transducing cultured primary mouse cells with single MuLE lentiviruses, we engineered tumors containing up to 5 different genetic alterations, identified genetic dependencies of molecularly defined tumors, conducted genetic interaction screens, and induced the simultaneous CRISPR/Cas9-mediated knockout of 3 tumor-suppressor genes. Intramuscular injection of MuLE viruses expressing oncogenic H-RasG12V together with combinations of knockdowns of the tumor suppressors cyclin-dependent kinase inhibitor 2A (Cdkn2a), transformation-related protein 53 (Trp53), and phosphatase and tensin homolog (Pten) allowed the generation of 3 murine sarcoma models, demonstrating that genetically defined autochthonous tumors can be rapidly generated and quantitatively monitored via direct injection of polycistronic MuLE lentiviruses into mouse tissues. Together, our results demonstrate that the MuLE system provides genetic power for the systematic investigation of the molecular mechanisms that underlie human diseases.

Authors

Joachim Albers, Claudia Danzer, Markus Rechsteiner, Holger Lehmann, Laura P. Brandt, Tomas Hejhal, Antonella Catalano, Philipp Busenhart, Ana Filipa Gonçalves, Simone Brandt, Peter K. Bode, Beata Bode-Lesniewska, Peter J. Wild, Ian J. Frew

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Figure 1

Overview of genetic engineering experiments using MuLE vectors.

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Overview of genetic engineering experiments using MuLE vectors. (A) Rest...
(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.