RESUMO
The CHOPWT17_TPM1KOc28 iPSC line was generated to interrogate the functions of Tropomyosin 1 (TPM1) in primary human cell development. This line was reprogrammed from a previously published wild type control iPSC line.
Assuntos
Células-Tronco Pluripotentes Induzidas , Tropomiosina , Humanos , Tropomiosina/genética , Tropomiosina/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Linhagem Celular TumoralRESUMO
The CHOPWT17_TPM1KOc28 iPSC line was generated to interrogate the functions of Tropomyosin 1 ( TPM1 ) in primary human cell development. This line was reprogrammed from a previously published wild type control iPSC line.
RESUMO
Human in vitro model systems of diabetes are critical to both study disease pathophysiology and offer a platform for drug testing. We have generated a set of tools in the human ß-cell line EndoC-ßH1 that allows the efficient and inexpensive characterization of ß-cell physiology and phenotypes driven by disruption of candidate genes. First, we generated a dual reporter line that expresses a preproinsulin-luciferase fusion protein along with GCaMP6s. This reporter line allows the quantification of insulin secretion by measuring luciferase activity and calcium flux, a critical signaling step required for insulin secretion, via fluorescence microscopy. Using these tools, we demonstrate that the generation of the reporter human ß-cell line was highly efficient and validated that luciferase activity could accurately reflect insulin secretion. Second, we used a lentiviral vector carrying the CRISPR-Cas9 system to generate candidate gene disruptions in the reporter line. We also show that we can achieve gene disruption in ~90% of cells using a CRISPR-Cas9 lentiviral system. As a proof of principle, we disrupt the ß-cell master regulator, PDX1, and show that mutant EndoC-ßH1 cells display impaired calcium responses and fail to secrete insulin when stimulated with high glucose. Furthermore, we show that PDX1 mutant EndoC-ßH1 cells exhibit decreased expression of the ß-cell-specific genes MAFA and NKX6.1 and increased GCG expression. The system presented here provides a platform to quickly and easily test ß-cell functionality in wildtype and cells lacking a gene of interest.
Assuntos
Sinalização do Cálcio , Linhagem Celular , Genes Reporter , Secreção de Insulina , Células Secretoras de Insulina , Sistemas CRISPR-Cas , Regulação para Baixo , Técnicas de Inativação de Genes , Proteínas de Homeodomínio/genética , Humanos , Transativadores/genéticaRESUMO
GATA6 is a critical regulator of pancreatic development, with heterozygous mutations in this transcription factor being the most common cause of pancreatic agenesis. To study the variability in disease phenotype among individuals harboring these mutations, a patient-induced pluripotent stem cell model was used. Interestingly, GATA6 protein expression remained depressed in pancreatic progenitor cells even after correction of the coding mutation. Screening the regulatory regions of the GATA6 gene in these patient cells and 32 additional agenesis patients revealed a higher minor allele frequency of a SNP 3' of the GATA6 coding sequence. Introduction of this minor allele SNP by genome editing confirmed its functionality in depressing GATA6 expression and the efficiency of pancreas differentiation. This work highlights a possible genetic modifier contributing to pancreatic agenesis and demonstrates the usefulness of using patient-induced pluripotent stem cells for targeted discovery and validation of non-coding gene variants affecting gene expression and disease penetrance.
Assuntos
Células-Tronco Pluripotentes Induzidas , Diferenciação Celular/genética , Fator de Transcrição GATA6/genética , Humanos , Organogênese , PâncreasRESUMO
To meet the needs of the increasingly rapid and parallelized lead optimization process, a fully integrated local compound storage and liquid handling system was designed and implemented to automate the generation of assay-ready plates directly from newly submitted and cherry-picked compounds. A key feature of the system is the ability to create project- or assay-specific compound-handling methods, which provide flexibility for any combination of plate types, layouts, and plate bar-codes. Project-specific workflows can be created by linking methods for processing new and cherry-picked compounds and control additions to produce a complete compound set for both biological testing and local storage in one uninterrupted workflow. A flexible cherry-pick approach allows for multiple, user-defined strategies to select the most appropriate replicate of a compound for retesting. Examples of custom selection parameters include available volume, compound batch, and number of freeze/thaw cycles. This adaptable and integrated combination of software and hardware provides a basis for reducing cycle time, fully automating compound processing, and ultimately increasing the rate at which accurate, biologically relevant results can be produced for compounds of interest in the lead optimization process.
Assuntos
Automação , Descoberta de Drogas , Biologia Computacional/métodos , Descoberta de Drogas/instrumentação , Descoberta de Drogas/métodos , Software , Integração de Sistemas , Interface Usuário-ComputadorRESUMO
Human monogenic diabetes, caused by mutations in genes involved in beta cell development and function, has been a challenge to study because multiple mouse models have not fully recapitulated the human disease. Here, we use genome edited human embryonic stem cells to understand the most common form of monogenic diabetes, MODY3, caused by mutations in the transcription factor HNF1A. We found that HNF1A is necessary to repress an alpha cell gene expression signature, maintain endocrine cell function, and regulate cellular metabolism. In addition, we identified the human-specific long non-coding RNA, LINKA, as an HNF1A target necessary for normal mitochondrial respiration. These findings provide a possible explanation for the species difference in disease phenotypes observed with HNF1A mutations and offer mechanistic insights into how the HNF1A gene may also influence type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Células-Tronco Embrionárias Humanas/fisiologia , Pâncreas/patologia , Respiração Celular , Células Cultivadas , Diabetes Mellitus Tipo 2/patologia , Regulação da Expressão Gênica , Fator 1-alfa Nuclear de Hepatócito/genética , Humanos , Proteínas do Leite , Mutação/genética , Pâncreas/fisiologia , Fenótipo , RNA Longo não Codificante/genéticaRESUMO
With the advancement of high-throughput biomolecular screening techniques to the lead optimization stage, there is a critical need to quality control (QC) dose-response curves generated by robotic liquid handlers to ensure accurate affinity determinations. One challenge in evaluating the performance of liquid handlers is identifying and validating a robust method for testing dispense volumes across different instruments. Although traditional automated liquid handlers are still considered the standard platform in many laboratories, nanoliter dispensers are becoming more common and pose new challenges for routine quality control procedures. For example, standard gravimetric measurements are unreliable for testing the accuracy of nanoliter liquid dispenses. However, nanoliter dispensing technology allows for the conservation of compound, reduces compound carryover from well to well through discrete dispenses, and eliminates the need for intermediate compound dilution steps to achieve a low final DMSO assay concentration. Moreover, an intermediate dilution step in aqueous solution might result in compound precipitation at high concentrations. This study compared representative automation procedures done on a variety of liquid dispensers, including manual, traditional, and nanodispense volumes. The data confirmed the importance of establishing robust QC procedures for dose-response generation in addition to accuracy and precision determinations for each instrument, and they validated the use of nanoliter pipettors for dose-response testing. The results of this study also support the requirement for thorough mixing during serial compound dilutions prepared for high-throughput lead optimization strategies using traditional liquid handlers.
Assuntos
Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/normas , Microquímica/métodos , Nanotecnologia/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Hidrazinas/análise , Hidrazinas/química , Técnicas de Diluição do Indicador , Concentração Inibidora 50 , Técnicas Analíticas Microfluídicas , Controle de Qualidade , Sensibilidade e EspecificidadeRESUMO
One high-throughput technology gaining widespread adoption in industry and academia is acoustic liquid dispensing, in which focused sound waves eject nanoliter-sized droplets from a solution into a recipient microplate. This technology allows for direct dispensing of small-molecule compounds or reagents dissolved in DMSO, while keeping a low final concentration of organic solvent in an assay. However, acoustic dispensing presents unique quality control (QC) challenges when measuring the accuracy and precision of small dispense volumes ranging from 2.5 to 100 nL. As part of an effort to develop a rapid and cost-effective QC method for acoustic dispensing of 100% DMSO, we implemented the first high-throughput photometric dual-dye-based QC protocol in the nanoliter volume range. This technical note validates the new photometric 100% DMSO QC method and highlights its cost-effectiveness when compared with conventional low-throughput fluorimetric QC methods. In addition, a potential software solution is described for the analysis, storage, and display of accumulated high-throughput QC data, called LabGauge. As the need for high-throughput QC grows, conventional low-throughput methods can no longer meet demand. Validated high-throughput techniques, such as the dual-dye photometric method, will need to be implemented.
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Dimetil Sulfóxido/química , Fluorofotometria/métodos , Ensaios de Triagem em Larga Escala , Dispositivos Lab-On-A-Chip , Som , Análise Custo-Benefício , Corantes Fluorescentes/química , Fluorofotometria/instrumentação , Humanos , Microquímica , Microfluídica/métodos , Microfluídica/tendências , Miniaturização , Nanotecnologia , Controle de Qualidade , SoftwareRESUMO
Se realizó un estudio observacional, descriptivo, transversal, con el objetivo de determinar el comportamiento clínico del cáncer de la cavidad bucal en el Hospital General Docente "Dr. Octavio de la Concepción y de la Pedraja" en el período de enero a diciembre del 2015. El universo estuvo constituido por 273 pacientes con el diagnóstico de cáncer, la muestra estuvo integrada por los 21 pacientes con diagnóstico de cáncer bucal. Se estudiaron las variables: edad, sexo, procedencia del paciente, localización anatómica, tamaño de la lesión y tipo histológico. Predominó la enfermedad en pacientes de 46 a 60 años, procedencia rural y su localización prevaleció en el labio. El carcinoma epidermoide fue la variedad histológica más frecuente(AU)
An observational, descriptive, cross-sectional study was carried out to determine the clinical behavior of oral cavity cancer in the General Teaching Hospital "Dr. Octavio de la Concepción de la Pedraja "in the period from January to December of 2015. The universe consisted of 273 patients with a diagnosis of cancer, the sample consisted of 21 patients with a diagnosis of oral cancer, the variables age, sex, patient origin, anatomical location, lesion size and histological type. The disease predominated in patients from 46 to 60 years of age, of rural origin and their location prevailed in the lip. Epidermoid carcinoma was the most frequent histological variety(AU)