RESUMO
A gene-trap strategy was set up in embryonic stem (ES) cells with the aim of trapping genes expressed in restricted neuronal lineages. The vector used trap genes irrespective of their activity in undifferentiated totipotent ES cells. Clones were subjected individually to differentiation in a system in which ES cells differentiated into neurons. Two ES clones in which the trapped gene was expressed in ES-derived neurons were studied in detail. The corresponding cDNAs were cloned, sequenced, and analysed by in situ hybridisation on wild-type embryo sections. Both genes are expressed in the nervous system. One gene, YR-23, encodes a large intracellular protein of unknown function. The second clone, YR-14, represents a sorting nexin (SNX14) gene whose expression in vivo coincides with that of LIM-homeodomain Islet-1 in several tissues. Sorting nexins are proteins associated with the endoplasmic reticulum (ER) and may play a role in receptor trafficking. Gene trapping followed by screening based on in vitro preselection of differentiated ES recombinant clones, therefore, has the potential to identify integration events in subsets of genes before generation of mouse mutants.
Assuntos
Proteínas de Transporte/biossíntese , Proteínas de Transporte/metabolismo , Técnicas Genéticas , Neurônios Motores/metabolismo , Animais , Sequência de Bases , Proteínas de Transporte/genética , Diferenciação Celular , Células Cultivadas , Clonagem Molecular , DNA Complementar/metabolismo , Bases de Dados Factuais , Digoxigenina/farmacologia , Eletroporação , Embrião de Mamíferos/metabolismo , Retículo Endoplasmático/metabolismo , Éxons , Galactosídeos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos , Hibridização In Situ , Indóis/metabolismo , Íntrons , Óperon Lac , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Sistema Nervoso/embriologia , Neurônios/metabolismo , Estrutura Terciária de Proteína , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Nexinas de Classificação , Células-Tronco/metabolismo , Fatores de Tempo , Proteínas de Transporte VesicularRESUMO
We have characterized different neuronal subpopulations derived from in vitro differentiation of embryonic stem (ES) cells using as markers the expression of several homeodomain transcription factors. Following treatment of embryo-like aggregates with retinoic acid (RA), Pax-6, a protein expressed by ventral central nervous system (CNS) progenitors is induced. In contrast, Pax-7 expressed in vivo by dorsal CNS progenitors, and erbB3, a gene expressed by neural crest cells and its derivatives, are almost undetectable. CNS neuronal subpopulations generated expressed combinations of markers characteristic of somatic motoneurons (Islet-1/2, Lim-3, and HB-9), cranial motoneurons (Islet-1/2 and Phox2b) and interneurons (Lim-1/2 or EN1). Molecular characterization of neuron subtypes generated from ES cells should considerably facilitate the identification of new genes expressed by restricted neuronal cell lineages.