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 9

Combinatorial genetic screening using MuLE vectors.

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Combinatorial genetic screening using MuLE vectors. (A) Schematic repres...
(A) Schematic representation of the workflow to generate a MuLE Entry vector shRNA library targeting the listed genes (shRNA X) and the final tricistronic lentiviral expression vector library that was used to screen for shRNAs that cause cell transformation in cooperation with oncogenic H-RasG12V overexpression. (B) Quantification of foci that were formed when WT MEFs were transduced with the indicted lentiviruses (MOI = 0.1). (C) Western blot analysis of EGFP-expressing cell clones derived from foci harboring shRNA against the indicated gene. (D) In vivo fluorescence images of mice that were subcutaneously injected with WT MEFs that had been infected with a MuLE virus expressing the identified shRNA against Cdkn2a alone plus iRFP or in combination with H-RasG12V and iRFP. (E) Tumor growth in the same mice monitored by longitudinal in vivo fluorescence imaging. All graphs depict mean ± SD. Student’s t test, n = 3. **P < 0.01; ***P < 0.001.