Asunto(s)
Antineoplásicos/uso terapéutico , Mesilato de Imatinib/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielomonocítica Crónica/tratamiento farmacológico , Anciano , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucemia Mielomonocítica Crónica/genética , Leucemia Mielomonocítica Crónica/patología , Masculino , MutaciónRESUMEN
Recombinant therapeutic proteins have changed the face of modern medicine in the present trend and they continue to provide innovative therapies for deadly diseases. This study describes the development of a novel stable expression system for rapid amplification of genes in Chinese Hamster Ovary (CHO) cells. The expression system consists of a host CHO cell line and an expression vector (pUB-PyOri-D-C) which encodes for Polyomavirus (Py) Origin of Replication (PyOri) for amplification of integrated genes in the presence of Py Large T Antigen (PyLT) and Dihydrofolate Reductase (DHFR) selectable marker gene for selection in the presence of Methotrexate (MTX). Use of both PyOri/PyLT and DHFR can reduce the number of rounds of selection and amplification required for isolation of high producing clones. The efficiency of pUB-PyOri-D-C was compared with that of pUB-D-C plasmid using Green fluorescent protein (GFP) and Erythropoietin (EPO) as reporter proteins. Our results showed that pUB-PyOri-D-C-EPO can help development of high expressing clone in one round of selection/amplification as compared to multiple rounds of selection/amplification with pUB-D-C-EPO plasmid. CHO-DG44/EPO clone generated using pUB-PyOri-D-C-EPO gave a productivity of 119 mg/L in shake flask.
Asunto(s)
Vectores Genéticos/metabolismo , Proteínas Recombinantes/biosíntesis , Animales , Células CHO , Cricetinae , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Eritropoyetina/genética , Eritropoyetina/metabolismo , Amplificación de Genes/efectos de los fármacos , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Metotrexato/farmacología , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Tetrahidrofolato Deshidrogenasa/genética , TransfecciónRESUMEN
Chronic myelomonocytic leukemia (CMML) is a hematologic malignancy nearly confined to the elderly. Previous studies to determine incidence and prognostic significance of somatic mutations in CMML have relied on candidate gene sequencing, although an unbiased mutational search has not been conducted. As many of the genes commonly mutated in CMML were recently associated with age-related clonal hematopoiesis (ARCH) and aged hematopoiesis is characterized by a myelomonocytic differentiation bias, we hypothesized that CMML and aged hematopoiesis may be closely related. We initially established the somatic mutation landscape of CMML by whole exome sequencing followed by gene-targeted validation. Genes mutated in ⩾10% of patients were SRSF2, TET2, ASXL1, RUNX1, SETBP1, KRAS, EZH2, CBL and NRAS, as well as the novel CMML genes FAT4, ARIH1, DNAH2 and CSMD1. Most CMML patients (71%) had mutations in ⩾2 ARCH genes and 52% had ⩾7 mutations overall. Higher mutation burden was associated with shorter survival. Age-adjusted population incidence and reported ARCH mutation rates are consistent with a model in which clinical CMML ensues when a sufficient number of stochastically acquired age-related mutations has accumulated, suggesting that CMML represents the leukemic conversion of the myelomonocytic-lineage-biased aged hematopoietic system.
Asunto(s)
Biomarcadores de Tumor/genética , Hematopoyesis/genética , Leucemia Mielomonocítica Crónica/genética , Mutación/genética , Proteínas/genética , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Exoma , Femenino , Estudios de Seguimiento , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Leucemia Mielomonocítica Crónica/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Proteínas de Unión al ARN , Tasa de Supervivencia , Adulto JovenRESUMEN
The PCR—based replication assay is one of the most simple, quick and economical methods for the analysis of episomal replication. However, in spite of its advantages the method has not been able to replace the southern—based replication assay, the latter of which is a tedious and time—consuming process. This is due to the generation of spurious amplification products in the PCR—based replication assay. The replication assay is based on the use of methylation—sensitive restriction endonucleases (eg. DpnI, MboI) to distinguish bacterial replicated (adenosine methylated) and mammalian replicated plasmids (adenosine non—methylated). In this work we addressed the problem by evaluating (a) restriction enzyme digestion and (b) the minimum number of restriction sites that are required in the amplifying region. The efficiency of restriction digestion was tested by subjecting the plasmid to one and two rounds of digestion. Multiple rounds of digestions were found to be inefficient in preventing false positives when the number of DpnI sites in the amplifying region is less than 8. However, use of a minimum of 15 DpnI sites in the amplifying region was found to overcome the false positives.