RESUMO
Thromboembolic events are the main cause of mortality in BCR-ABL1-negative myeloproliferative neoplasms (MPNs) but their underlying mechanisms are largely unrecognized. The Janus kinase 2 (JAK2)V617F mutation is the most frequent genetic alteration leading to MPN. Usually found in haematopoietic progenitors and stem cells, this mutation has also been described in endothelial cells (ECs) of MPN patients. In this study, we have questioned the impact of the JAK2V617F mutation on EC phenotype and functions. We developed an induced pluripotent stem cells strategy to compare JAK2 mutant and wild-type ECs. Transcriptomic assays showed that several genes and pathways involved in inflammation, cell adhesion and thrombotic events were over-represented in JAK2V617F ECs and expression levels of von Willebrand factor and P-selectin (CD62P) proteins were increased. Finally, we found that leucocytes from MPN patients adhere more tightly to JAK2V617F ECs. Our results show that JAK2V617F ECs have a pro-inflammatory and pro-thrombotic phenotype and were functionally pro-adherent.
Assuntos
Plaquetas/fisiologia , Células Endoteliais/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Janus Quinase 2/genética , Células Progenitoras Mieloides/fisiologia , Transtornos Mieloproliferativos/genética , Trombose/genética , Adesão Celular/genética , Diferenciação Celular , Células Cultivadas , Proteínas de Fusão bcr-abl/metabolismo , Perfilação da Expressão Gênica , Humanos , Mutação/genética , Transgenes/genéticaAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/etiologia , Mielofibrose Primária/complicações , Biomarcadores , Transformação Celular Neoplásica/genética , Feminino , Humanos , Janus Quinase 2/genética , Leucemia Mieloide Aguda/diagnóstico , Pessoa de Meia-Idade , Mutação , Mielofibrose Primária/genética , Indução de RemissãoRESUMO
TALEN is one of the most widely used tools in the field of genome editing. It enables gene integration and gene inactivation in a highly efficient and specific fashion. Although very attractive, the apparent simplicity and high success rate of TALEN could be misleading for novices in the field of gene editing. Depending on the application, specific TALEN designs, activity assessments and screening strategies need to be adopted. Here we report different methods to efficiently perform TALEN-mediated gene integration and inactivation in different mammalian cell systems including induced pluripotent stem cells and delineate experimental examples associated with these approaches.
