ABSTRACT
The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤ 0.1% to ≈ 6%) with that of homologous gene targeting (≤ 0.1% to ≈ 15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.
Subject(s)
Chromosomal Position Effects , DNA Restriction Enzymes/metabolism , Animals , CHO Cells , Cell Line , Cricetinae , Cricetulus , DNA Restriction Enzymes/chemistry , Gene Targeting , Genetic Engineering , Genome, Human , Humans , MutagenesisABSTRACT
BACKGROUND: Improved chemical hazard management such as REACH policy objective as well as drug ADMETOX prediction, while limiting the extent of animal testing, requires the development of increasingly high throughput as well as highly pertinent in vitro toxicity assays. METHODOLOGY: This report describes a new in vitro method for toxicity testing, combining cell-based assays in nanodrop Cell-on-Chip format with the use of a genetically engineered stress sensitive hepatic cell line. We tested the behavior of a stress inducible fluorescent HepG2 model in which Heat Shock Protein promoters controlled Enhanced-Green Fluorescent Protein expression upon exposure to Cadmium Chloride (CdCl2), Sodium Arsenate (NaAsO2) and Paraquat. In agreement with previous studies based on a micro-well format, we could observe a chemical-specific response, identified through differences in dynamics and amplitude. We especially determined IC50 values for CdCl2 and NaAsO2, in agreement with published data. Individual cell identification via image-based screening allowed us to perform multiparametric analyses. CONCLUSIONS: Using pre/sub lethal cell stress instead of cell mortality, we highlighted the high significance and the superior sensitivity of both stress promoter activation reporting and cell morphology parameters in measuring the cell response to a toxicant. These results demonstrate the first generation of high-throughput and high-content assays, capable of assessing chemical hazards in vitro within the REACH policy framework.
Subject(s)
Biological Assay/methods , Nanostructures , Toxicity Tests/methods , Animals , Arsenates/pharmacology , Biological Assay/instrumentation , Cadmium Chloride/pharmacology , Cell Death/drug effects , Cell Line , Dose-Response Relationship, Drug , Environmental Pollutants/pharmacology , Environmental Pollutants/toxicity , Gene Expression Regulation , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Heat-Shock Proteins/genetics , Hepatocytes/cytology , Hepatocytes/drug effects , Hepatocytes/metabolism , Herbicides/pharmacology , High-Throughput Screening Assays/instrumentation , High-Throughput Screening Assays/methods , Humans , Microarray Analysis/instrumentation , Microarray Analysis/methods , Paraquat/pharmacology , Promoter Regions, Genetic , Toxicity Tests/instrumentationABSTRACT
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer among men in the developed world. There is a need, for both clinical and scientific reasons, to find markers to identify patients with aggressive disease as early as possible, and to understand the events leading to malignant transformation and susceptibility to metastasis. We report the first large-scale gene expression analysis of a unique HNSCC location, the hypopharynx. Four normal and 34 tumour samples were analysed with 12 600 gene microarrays. Clusters of differentially expressed genes were identified in the chromosomal regions 3q27.3, 17q21.2-q21.31, 7q11.22-q22.1 and 11q13.1-q13.3, which, interestingly, have already been identified by comparative genomic hybridization (CGH) as major regions of gene amplification. We showed that six overexpressed genes (EIF4G1, DVL3, EPHB4, MCM7, BRMS1 and SART1) located in these regions are indeed amplified. We report 119 genes that are highly differentially expressed between 'early' tumours and normal samples. Of these, we validated by quantitative PCR six novel poorly characterized genes. These genes are potential new markers of HNSCC. Comparing patients with relatively nonaggressive and aggressive tumours (without or with clinical evidence of metastasis 3 years after surgery), we identified 164 differentially expressed genes potentially involved in the acquisition of metastatic potential. This study contributes to the understanding of HNSCC, staging patients into prognostic groups and identifying high-risk patients who may benefit from more aggressive treatment.
