A versatile modular vector system for rapid combinatorial mammalian genetics
Joachim Albers, … , Peter J. Wild, Ian J. Frew
Joachim Albers, … , Peter J. Wild, Ian J. Frew
Published March 9, 2015
Citation Information: J Clin Invest. 2015;125(4):1603-1619. https://doi.org/10.1172/JCI79743.
View: Text | PDF
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

×

Figure 8

Combinatorial genetics using the CRISPR/Cas9 system in MuLE vectors.

Options: View larger image (or click on image) Download as PowerPoint
Combinatorial genetics using the CRISPR/Cas9 system in MuLE vectors. (A)...
(A) Schematic of MuLE vector expressing sgRNA against Trp53 and expressing H-RasG12V, hCas9, and puromycin resistance. (B) MEFs were infected with the indicated viruses expressing sgRNAs targeting Trp53 exon 7 (Ex7) or exon 8 with or without H-RasG12V expression, plated at low density 6 days after transduction, and stained with crystal violet 14 days after plating. (C) Growth of cells as tumor xenografts and images of tumors derived from the combination of Trp53 exon 7 or exon 8 sgRNAs with H-RasG12V overexpression. (D) Western blot analysis of tumor cells that were isolated from 3 separate tumors of each genotype 5 weeks after cell injection. (E) Schematic of MuLE vector simultaneously expressing sgRNAs against Trp53, Pten, and Vhl and expressing hCas9 and puromycin resistance. (F) MEFs infected with 3 independent combinations of different sgRNAs formed colonies when plated at low density 10 days after viral transduction. (G) Western blotting of cell lines (lanes 1–21) derived from colonies that formed after infection with viruses expressing the indicated combinations of sgRNAs.