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 10

Generation of 3 autochthonous mouse models of undifferentiated sarcoma using MuLE vectors.

Options: View larger image (or click on image) Download as PowerPoint
Generation of 3 autochthonous mouse models of undifferentiated sarcoma u...
(A) Intramuscular injection of ROSA26-lox-STOP-lox-tdTomato mice with control or Cre-expressing virus. Bottom left panel shows infected myofibers, and small cells adjacent to myofibers are seen at higher magnification (arrowheads, bottom right panel). (B) Bioluminescence imaging 3 and 31 days after injection of 3 × 105 functional viral particles into each gastrocnemius muscle of 18-day-old SCID/beige mice with MuLE-luciferase viruses expressing combinations of shRNA against Cdkn2a, Trp53, and Pten with or without expression of H-RasG12V. (C) Quantification (mean ± SD) of luminescent signal intensities over time after injection. †Sacrifice of all mice in these cohorts by this time point. (D) A tumor (arrow) in a mouse injected with the shCdkn2a plus H-RasG12V MuLE virus only in the right gastrocnemius muscle. (E) Histological image of the tumor (T) from D surrounded by muscle tissue (M). (F–K) Representative histology of tumors derived from injection of shCdkn2a plus H-RasG12V (F and G), shTrp53 plus H-RasG12V (H and I), and shTrp53 plus shPTEN plus H-RasG12V (J and K) viruses. Arrowheads in G, I, and K highlight pleomorphic rhabdoid cells. (L) EM showing an example of a tumor cell with sarcomere formation; M and N show higher magnification of the regions in L marked with an arrowhead and arrows, respectively, showing Z-bands or irregular masses of Z-band material with converging filaments. (O) Western blot analysis of independent cell lines (lanes 1–8) derived from independent tumors of the indicated genotypes. MEFs, muscle tissue, and C2C12 myoblast cells served as controls. Scale bars: 50 μm (A and F–K); 10 μm (L); 500 nm (M and N).