RESUMO
How distinct stem cell populations originate and whether there is a clear stem cell "genetic signature" remain poorly understood. Understanding the evolution of stem cells requires molecular profiling of stem cells in an animal at a basal phylogenetic position. In this study, using transgenic Hydra polyps, we reveal for each of the three stem cell populations a specific signature set of transcriptions factors and of genes playing key roles in cell type-specific function and interlineage communication. Our data show that principal functions of stem cell genes, such as maintenance of stemness and control of stem cell self-renewal and differentiation, arose very early in metazoan evolution. They are corroborating the view that stem cell types shared common, multifunctional ancestors, which achieved complexity through a stepwise segregation of function in daughter cells.
Assuntos
Linhagem da Célula/genética , Perfilação da Expressão Gênica , Hydra/citologia , Hydra/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Evolução Biológica , Separação Celular , Regulação da Expressão Gênica , Teste de Complementação Genética , Camundongos , Filogenia , Coloração e Rotulagem , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
BACKGROUND: Stable expression of transgenes is an important technique to analyze gene function. Various drug resistance genes, such as neo, pac, hph, zeo, bsd, and hisD, have been equally used as selection markers to isolate a transfectant without considering their dose-dependent characters. RESULTS: We quantitatively measured the variation of transgene expression levels in mouse embryonic stem (mES) cells, using a series of bi-cistronic expression vectors that contain Egfp expression cassette linked to each drug resistant gene via IRES with titration of the selective drugs, and found that the transgene expression levels achieved in each system with this vector design are in order, in which pac and zeo show sharp selection of transfectants with homogenously high expression levels. We also showed the importance of the choice of the drug selection system in gene-trap or gene targeting according to this order. CONCLUSIONS: The results of the present study clearly demonstrated that an appropriate choice of the drug resistance gene(s) is critical for a proper design of the experimental strategy.
Assuntos
Células-Tronco Embrionárias/fisiologia , Marcação de Genes/métodos , Proteínas Recombinantes de Fusão/análise , Transgenes , Animais , Biotecnologia/métodos , Resistência a Medicamentos/genética , Células-Tronco Embrionárias/metabolismo , Marcadores Genéticos/genética , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Camundongos , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Seleção GenéticaRESUMO
Cancer is the leading cause of death in dogs and cats. Early diagnosis of cancer is critical for effective treatment and improving survival rates. Nematode-NOSE (N-NOSE) is a commercially available non-invasive human cancer screening test that uses the sense of smell of the nematode Caenorhabditis elegans showing a distinct chemotactic response toward the urine of an individual with cancer compared to healthy ones. 15 types of human cancer (stomach, colon-rectum, lung, breast, pancreas, liver, prostate, uterus, esophagus, gallbladder, bile duct, kidney, urinary bladder, ovary, and oropharynx cancers) can be detected by N-NOSE. A non-invasive method for accurate cancer screening is needed for pets. In this study, we evaluated the effectiveness of N-NOSE in detecting cancer using canine and feline urine samples. We found a significant difference in chemotaxis index values between healthy subjects and cancer patients in both canine (p < 0.01*) and feline (p < 0.04*) urine samples. Receiver operating characteristic (ROC) analysis highlights the good performance of the test with areas under the curve (AUC) of 0.8114 and 0.7851 for dogs and 0.7667 and 0.9000 for cats when using 2 different dilutions of urine samples. Our study suggests that N-NOSE has the potential as a simple, accurate, and low-cost cancer screening test in both dogs and cats.
RESUMO
Inhibition of glycogen synthase kinase-3 (Gsk3) supports mouse embryonic stem cells (ESCs) by modulating Tcf3, but the critical targets downstream of Tcf3 are unclear. We analyzed the intersection between genome localization and transcriptome data sets to identify genes repressed by Tcf3. Among these, manipulations of Esrrb gave distinctive phenotypes in functional assays. Knockdown and knockout eliminated response to Gsk3 inhibition, causing extinction of pluripotency markers and loss of colony forming capability. Conversely, forced expression phenocopied Gsk3 inhibition or Tcf3 deletion by suppressing differentiation and sustaining self-renewal. Thus the nuclear receptor Esrrb is necessary and sufficient to mediate self-renewal downstream of Gsk3 inhibition. Leukaemia inhibitory factor (LIF) regulates ESCs through Stat3, independently of Gsk3 inhibition. Consistent with parallel operation, ESCs in LIF accommodated Esrrb deletion and remained pluripotent. These findings highlight a key role for Esrrb in regulating the naive pluripotent state and illustrate compensation among the core pluripotency factors.