RESUMO
We have generated mice carrying a germline mutation in the tyrosine kinase catalytic domain of the trkB gene. This mutation eliminates expression of gp145trkB, a protein-tyrosine kinase that serves as the signaling receptor for two members of the nerve growth factor family of neurotrophins, brain-derived neurotrophic factor and neurotrophin-4. Mice homozygous for this mutation, trkBTK(-/-), develop to birth. However, these animals do not display feeding activity, and most die by P1. Neuroanatomical examination of trkBTK (-/-) mice revealed neuronal deficiencies in the central (facial motor nucleus and spinal cord) and peripheral (trigeminal and dorsal root ganglia) nervous systems. These findings illustrate the role of the gp145trkB protein-tyrosine kinase receptor in the ontogeny of the mammalian nervous system.
Assuntos
Proteínas de Membrana/genética , Proteínas Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/genética , Receptores de Fator de Crescimento Neural/genética , Animais , Animais Recém-Nascidos , Sequência de Bases , Blastocisto/fisiologia , Southern Blotting , Primers do DNA , Embrião de Mamíferos , Feminino , Gânglios Espinais/anormalidades , Cor de Cabelo/genética , Heterozigoto , Homozigoto , Íntrons , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Neurônios/citologia , Reação em Cadeia da Polimerase , Receptor do Fator Neutrófico Ciliar , Receptor trkB , Medula Espinal/anormalidades , Células-Tronco/fisiologia , TransfecçãoRESUMO
We have confirmed that the gene trap vector pGT4.5 creates spliced fusion transcripts with endogenous genes and prevents the synthesis of normal transcripts at the site of integration. cDNA was prepared to the lacZ fusion transcript in three ES cell lines to recover endogenous exon sequences upstream of lacZ. Each of the clones detected a unique-sized endogenous transcript, as well as the fusion transcript in the ES cell line from which the clone was derived. Sequence analysis of these clones and larger clones isolated from a random-primed cDNA library showed that the splice acceptor was used properly. For two insertions, the expression patterns of the lacZ reporter and the associated endogenous gene were compared in situ at three embryonic stages and were found to be similar. Three gene trap insertions were transmitted into the germ line, and abnormalities were observed with two of the three insertions in the homozygous state. RNA obtained from mice homozygous for the two mutant gene trap insertions was analyzed for normal endogenous transcripts and negligible amounts were detected, indicating that little splicing around the gene trap insertion occurred. This work demonstrates the capacity of the gene trap vector to generate lacZ fusion transcripts, to accurately report endogenous gene expression, and to mutate the endogenous gene at the site of integration.
Assuntos
Vetores Genéticos/genética , Mutagênese Insercional/genética , Splicing de RNA/genética , Proteínas Recombinantes de Fusão/genética , Células-Tronco/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Quimera , Clonagem Molecular , Expressão Gênica/genética , Óperon Lac , Camundongos , Dados de Sequência Molecular , Mutação/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The two mouse genes, En-1 and En-2, that are homologs of the Drosophila segmentation gene engrailed, show overlapping spatially restricted patterns of expression in the neural tube during embryogenesis, suggestive of a role in regional specification. Mice homozygous for a targeted mutation that deletes the homeobox were viable and showed no obvious defects in embryonic development. This may be due to functional redundancy of En-2 and the related En-1 gene product during embryogenesis. Consistent with this hypothesis, the mutant mice showed abnormal foliation in the adult cerebellum, where En-2, and not En-1, is normally expressed.
