RESUMO
The Cre/loxP site-specific recombination system combined with embryonic stem cell-mediated technologies has greatly expanded our capability to address normal and disease development in mammals using genetic approaches. The success of this emerging technology hinges on the production of Cre-expressing transgenic lines that provide cell type-, tissue-, or developmental stage-specific recombination between loxP sites placed in the genome. Here we describe and characterize the production of a double-reporter mouse line that provides a convenient and reliable readout of Cre recombinase activity. Throughout all embryonic and adult stages, the transgenic animal expresses the lacZ reporter gene before Cre-mediated excision occurs. Cre excision, however, removes the lacZ gene, allowing expression of the second reporter, the human alkaline phosphatase gene. This double-reporter transgenic line is able to indicate the occurrence of Cre excision in an extremely widespread manner from early embryonic to adult lineages. It will be a valuable reagent for the increasing number of investigators taking advantage of the powerful tools provided by the Cre/loxP site-specific recombinase system.
Assuntos
Genes Reporter , Integrases/metabolismo , Camundongos Transgênicos , Recombinação Genética , Proteínas Virais , Fosfatase Alcalina/genética , Animais , Agregação Celular , Embrião de Mamíferos/citologia , Expressão Gênica , Vetores Genéticos , Genótipo , Histocitoquímica , Óperon Lac , Camundongos , Ploidias , Deleção de Sequência , Células-Tronco , Distribuição Tecidual , TransgenesRESUMO
The groucho-related genes (Grg) of the mouse comprise at least four family members. In Drosophila, groucho is one of the neurogenic genes that participates in the Notch signalling pathway. The Groucho protein interacts with Hairy-related transcription factors to regulate segmentation, neurogenesis and sex determination. Thus, by analogy to the Drosophila proteins, murine Grg proteins may interact with mammalian Hairy and E(spl) homologues (Hes proteins) and take part in a signalling pathway downstream of murine Notch. We have isolated murine Grg4 cDNAs and examined Grg4 expression during embryogenesis. Transcripts of Grg4 were detected in proliferating epithelial tissues undergoing mesenchymal induction, overlapping with Grg3, Notch1 and Hes1 expression. Grg4 was also expressed in the central nervous system and somites, but in cells adjacent to Grg3-, Notch1-, and Hes1-expressing cells. This distinct pattern of expression suggests a role for Grg4 in later stages of cell differentiation than for the other mouse neurogenic gene homologues.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Sistema Nervoso/embriologia , Proteínas/genética , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Correpressoras , Drosophila/genética , Feminino , Camundongos , Dados de Sequência Molecular , Gravidez , Alinhamento de Sequência , Transcrição GênicaRESUMO
Herpes simplex virus (HSV) virions contain two regulatory proteins that facilitate the onset of the lytic cycle: VP16 activates transcription of the viral immediate-early genes, and vhs triggers shutoff of host protein synthesis and accelerated turnover of cellular and viral mRNAs. VP16 and vhs form a complex in infected cells, raising the possibility of a regulatory link between them. Here we show that viral protein synthesis and mRNA levels undergo a severe decline at intermediate times after infection with a VP16 null mutant, culminating in virtually complete translational arrest. This phenotype was rescued by a transcriptionally incompetent derivative of VP16 that retains vhs binding activity, and was eliminated by inactivating the vhs gene. These results indicate that VP16 dampens vhs activity, allowing HSV mRNAs to persist in infected cells. Further evidence supporting this hypothesis came from the demonstration that a stably transfected cell line expressing VP16 was resistant to host shutoff induced by superinfecting HSV virions. Thus, in addition to its well known function as a transcriptional activator, VP16 stimulates viral gene expression at a post-transcriptional level, by sparing viral mRNAs from degradation by one of the virus-induced host shutoff mechanisms.
Assuntos
Regulação Viral da Expressão Gênica , Proteína Vmw65 do Vírus do Herpes Simples/fisiologia , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Simplexvirus/fisiologia , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Animais , Sequência de Bases , Chlorocebus aethiops , Genes Precoces , Genes Virais , Substâncias Macromoleculares , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Viral/genética , Ribonucleases , Simplexvirus/genética , Transcrição Gênica , Células Vero , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
We examined the ability of binding sites for the herpes simplex virus immediate-early protein ICP4 to alter the regulation of closely linked promoters by placing strong ICP4 binding sites upstream or downstream of simple TATA promoters in the intact viral genome. We found that binding sites strongly reduced the levels of expression at early times postinfection and that this effect was partially overcome after the onset of viral DNA replication. These data confirm that DNA-bound ICP4 can inhibit the activity of a closely linked promoter and raise the possibility that ICP4 binding sites contribute to temporal regulation during infection.
