PMID- 25751063
OWN - NLM
STAT- MEDLINE
DCOM- 20150702
LR  - 20240514
IS  - 1558-8238 (Electronic)
IS  - 0021-9738 (Print)
IS  - 0021-9738 (Linking)
VI  - 125
IP  - 4
DP  - 2015 Apr
TI  - A versatile modular vector system for rapid combinatorial mammalian genetics.
PG  - 1603-19
LID - 79743 [pii]
LID - 10.1172/JCI79743 [doi]
AB  - 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.
FAU - Albers, Joachim
AU  - Albers J
FAU - Danzer, Claudia
AU  - Danzer C
FAU - Rechsteiner, Markus
AU  - Rechsteiner M
FAU - Lehmann, Holger
AU  - Lehmann H
FAU - Brandt, Laura P
AU  - Brandt LP
FAU - Hejhal, Tomas
AU  - Hejhal T
FAU - Catalano, Antonella
AU  - Catalano A
FAU - Busenhart, Philipp
AU  - Busenhart P
FAU - Goncalves, Ana Filipa
AU  - Goncalves AF
FAU - Brandt, Simone
AU  - Brandt S
FAU - Bode, Peter K
AU  - Bode PK
FAU - Bode-Lesniewska, Beata
AU  - Bode-Lesniewska B
FAU - Wild, Peter J
AU  - Wild PJ
FAU - Frew, Ian J
AU  - Frew IJ
LA  - eng
GR  - 260316/ERC_/European Research Council/International
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20150309
PL  - United States
TA  - J Clin Invest
JT  - The Journal of clinical investigation
JID - 7802877
RN  - 0 (CRISPR-Associated Proteins)
RN  - 0 (Hif1a protein, mouse)
RN  - 0 (Hypoxia-Inducible Factor 1, alpha Subunit)
RN  - 0 (RNA, Small Interfering)
RN  - 0 (Retinoblastoma Protein)
RN  - 0 (Tumor Suppressor Protein p53)
RN  - EC 3.1.3.67 (PTEN Phosphohydrolase)
RN  - EC 3.1.3.67 (PTEN protein, human)
RN  - N12000U13O (Doxycycline)
SB  - IM
MH  - Animals
MH  - Apoptosis
MH  - CRISPR-Associated Proteins/*genetics
MH  - *CRISPR-Cas Systems
MH  - Cells, Cultured
MH  - Cloning, Molecular/*methods
MH  - Clustered Regularly Interspaced Short Palindromic Repeats
MH  - Doxycycline/pharmacology
MH  - Drug Resistance/genetics
MH  - Gene Deletion
MH  - Gene Knockdown Techniques
MH  - *Genetic Vectors
MH  - Humans
MH  - Hypoxia-Inducible Factor 1, alpha Subunit/physiology
MH  - Lentivirus/*genetics
MH  - Mice
MH  - Mice, SCID
MH  - PTEN Phosphohydrolase/antagonists & inhibitors/genetics
MH  - RNA, Small Interfering/genetics
MH  - Recombination, Genetic
MH  - Retinoblastoma Protein/antagonists & inhibitors/genetics
MH  - Sarcoma, Experimental/genetics/therapy
MH  - Transduction, Genetic
MH  - Tumor Suppressor Protein p53/genetics
MH  - Xenograft Model Antitumor Assays
PMC - PMC4396471
EDAT- 2015/03/10 06:00
MHDA- 2015/07/03 06:00
PMCR- 2015/07/01
CRDT- 2015/03/10 06:00
PHST- 2014/12/19 00:00 [received]
PHST- 2015/01/20 00:00 [accepted]
PHST- 2015/03/10 06:00 [entrez]
PHST- 2015/03/10 06:00 [pubmed]
PHST- 2015/07/03 06:00 [medline]
PHST- 2015/07/01 00:00 [pmc-release]
AID - 79743 [pii]
AID - 10.1172/JCI79743 [doi]
PST - ppublish
SO  - J Clin Invest. 2015 Apr;125(4):1603-19. doi: 10.1172/JCI79743. Epub 2015 Mar 9.