A TALEN Genome-Editing System for Generating Human Stem Cell-Based Disease Models.
ABSTRACT Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report here the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells, the latter for which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease-dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor-neuron death, and hepatitis C infection. We found little evidence of TALEN off-target effects, but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines, we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease.
- SourceAvailable from: Mahendra Surendra Rao[Show abstract] [Hide abstract]
ABSTRACT: Targeted genome engineering to robustly express transgenes is an essential methodology for stem cell-based research and therapy. Although designer nucleases have been used to drastically enhance gene editing efficiency, targeted addition and stable expression of transgenes to date is limited at single gene/locus and mostly PPP1R12C/AAVS1 in human stem cells. Here we constructed transcription activator-like effector nucleases (TALENs) targeting the safe-harbor like gene CLYBL to mediate reporter gene integration at 38%-58% efficiency, and used both AAVS1-TALENs and CLYBL-TALENs to simultaneously knock-in multiple reporter genes at dual safe-harbor loci in human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs). The CLYBL-TALEN engineered cell lines maintained robust reporter expression during self-renewal and differentiation, and revealed that CLYBL targeting resulted in stronger transgene expression and less perturbation on local gene expression than PPP1R12C/AAVS1. TALEN-mediated CLYBL engineering provides improved transgene expression and options for multiple genetic modification in human stem cells.PLoS ONE 01/2015; 10(1):e0116032. · 3.53 Impact Factor
Conference Paper: Reliability modeling of a jet pipe electrohydraulic servo valve[Show abstract] [Hide abstract]
ABSTRACT: The hydraulic system and its components can accumulate a significant amount of contaminant after running for a period of time. The accumulated contaminant in the component will degrade its performance and even cause catastrophic failure. Traditional models are distinctly deficient when applied to analyze the performance degradation. In this paper, an improved method is proposed to trace the performance degradation during a long time operation. By means of a novel failure injection performance degradation simulation framework (FIPDS) to inject failure mechanisms into the performance model, we develop a complete reliability model of a jet pipe electrohydraulic servo valve considering multiple failure modes and mechanisms based on failure modes, mechanisms, and effects analysis (FMMEA). The performance indexes rated flow and null bias are obtained from the model simulation to evaluate the performance degradation. Simulation results demonstrate the feasibility of the method in this research.2014 Annual Reliability and Maintainability Symposium (RAMS); 01/2014
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ABSTRACT: More than 500 rare genetic bone disorders have been described, but for many of them only limited treatment options are available. Challenges for studying these bone diseases come from a lack of suitable animal models and unavailability of skeletal tissues for studies. Effectors for skeletal abnormalities of bone disorders may be abnormal bone formation directed by osteoblasts or anomalous bone resorption by osteoclasts, or both. Patient-specific induced pluripotent stem cells (iPSCs) can be generated from somatic cells of various tissue sources and in theory can be differentiated into any desired cell type. However, successful differentiation of hiPSCs into functional bone cells is still a challenge. Our group focuses on the use of human iPSCs (hiPSCs) to identify osteoclast defects in craniometaphyseal dysplasia. In this review, we describe the impact of stem cell technology on research for better treatment of such disorders, the generation of hiPSCs from patients with rare genetic bone disorders and current protocols for differentiating hiPSCs into osteoclasts.Journal of clinical medicine. 12/2014; 3(4):1490-1510.