Cyclin-dependent kinase 7 （Cdk7） is the catalytic subunit of the metazoan Cdk-activating kinase （CAK）. Activation of Cdk7 requires its association with a regulatory subunit, Cyclin H. Although the Cdk7/Cyclin H co...Cyclin-dependent kinase 7 （Cdk7） is the catalytic subunit of the metazoan Cdk-activating kinase （CAK）. Activation of Cdk7 requires its association with a regulatory subunit, Cyclin H. Although the Cdk7/Cyclin H complex has been implicated in the regulation ofRNA polymerase in several species, the precise function of their orthologs in zebrafish has not been fully elucidated. In this study, we isolated from zebrafish blastula embryos two cDNAs encoding the orthologs of human Cyclin H and Cdk7, and examined the role of Cdk7/Cyclin H in zebrafish embryogenesis. Sequence analysis showed that the zebrafish Cyclin H and Cdk7 cDNAs encode proteins with 65% and 86% identity to the respective hu- man orthologs. RT-PCR and whole-mount in situ hybridization analyses of their expression in unfertilized eggs, embryos and organs of adult fish suggested that Cyclin H and Cdk7 messages are maternally loaded. Our data also showed that their transcripts were detected throughout development. Distribution of Cyclin H transcripts was found to be ubiquitous during early stages of development and become restricted to the anterior neural tube, brain, eyes, procreate tissues, liver and heart by 5 days post-fertilization. Expression of a dominant-negative form of Cyclin H delayed the onset of zygotic transcription in the early embryo, resulting in apoptosis at 5 hours post-fertilization and leading to sever defects in tis- sues normally exhibiting high levels of Cyclin H expression. These results implicate Cyclin H in the regulation of the transcriptional machinery during midblastula transition and suggest that it is an essential gene in early zebrafish larval development.展开更多
Parietal cells are one of the largest epithelium cells of the mucous membrane of the stomach that secrete hydrochloric acid. To study the function of gastric parietal cells during gastric epithelium homeostasis, we ge...Parietal cells are one of the largest epithelium cells of the mucous membrane of the stomach that secrete hydrochloric acid. To study the function of gastric parietal cells during gastric epithelium homeostasis, we generated a transgenic mouse line, namely, Atp4b-Cre, in which the expression of Cre recombinase was controlled by a 1.0 kb promoter of mouse β-subunit of H^＋-, K^＋-ATPase gene （Atp4b）. In order to test the tissue distribution and excision activity of Cre recombinase in vivo, the Atp4b-Cre transgenic mice were bred with the reporter strain ROSA26 and a mouse strain that carries Smad4 conditional alleles （Smad4Ca/Co）. Multiple-tissue PCR of Atp4b-Cre;Smad4Co/＋ mice revealed that the recombination only happened in the stomach. As indicated by LacZ staining, ROSA26;Atp4b-Cre double transgenic mice showed efficient expression of Cre recombinase within the gastric parietal cells. These results showed that this Atp4b-Cre mouse line could be used as a powerful tool to achieve conditional gene knockout in gastric parietal cells.展开更多
The piggyBac transposable element was suc-cessfully used for generation of the transgenic silkworm, Bombyx mori. The EGFP was adopted in this study as a screening marker in transgenic vector under the control of an ar...The piggyBac transposable element was suc-cessfully used for generation of the transgenic silkworm, Bombyx mori. The EGFP was adopted in this study as a screening marker in transgenic vector under the control of an artificial promoter containing Pax-6 binding sites that can drive eye-specific genes expression in various insect species including B. mori and Drosophila. 111 independent trans-genic lines were obtained among 700 fertile G0 moths. Most of the transgenic lines contained two or more chromosomal insertions of the EGFP marker, which were stably inherited over more than six generations during the time of this pro-ject. PiggyBac-mediated transposition was confirmed by identifying the characteristic TTAA duplication sequence at the insertion sites. The stable and high efficient transmission of a genetic marker into B. mori confirms the usage of this vector-marker system for industrial production and theo-retical research.展开更多
文摘Cyclin-dependent kinase 7 （Cdk7） is the catalytic subunit of the metazoan Cdk-activating kinase （CAK）. Activation of Cdk7 requires its association with a regulatory subunit, Cyclin H. Although the Cdk7/Cyclin H complex has been implicated in the regulation ofRNA polymerase in several species, the precise function of their orthologs in zebrafish has not been fully elucidated. In this study, we isolated from zebrafish blastula embryos two cDNAs encoding the orthologs of human Cyclin H and Cdk7, and examined the role of Cdk7/Cyclin H in zebrafish embryogenesis. Sequence analysis showed that the zebrafish Cyclin H and Cdk7 cDNAs encode proteins with 65% and 86% identity to the respective hu- man orthologs. RT-PCR and whole-mount in situ hybridization analyses of their expression in unfertilized eggs, embryos and organs of adult fish suggested that Cyclin H and Cdk7 messages are maternally loaded. Our data also showed that their transcripts were detected throughout development. Distribution of Cyclin H transcripts was found to be ubiquitous during early stages of development and become restricted to the anterior neural tube, brain, eyes, procreate tissues, liver and heart by 5 days post-fertilization. Expression of a dominant-negative form of Cyclin H delayed the onset of zygotic transcription in the early embryo, resulting in apoptosis at 5 hours post-fertilization and leading to sever defects in tis- sues normally exhibiting high levels of Cyclin H expression. These results implicate Cyclin H in the regulation of the transcriptional machinery during midblastula transition and suggest that it is an essential gene in early zebrafish larval development.
基金supported by Chinese National Key Program on Basic Research (Nos. 2006CB943501 and 2006BAI23B01-3)Key Project for Drug Discovery and Development in China (No. 2009ZX09501-027)+1 种基金Key Project for Infectious Diseases in China (No. 2008ZX10002-016)E-Institutes of Shanghai Municipal Education Commission (E03003)
文摘Parietal cells are one of the largest epithelium cells of the mucous membrane of the stomach that secrete hydrochloric acid. To study the function of gastric parietal cells during gastric epithelium homeostasis, we generated a transgenic mouse line, namely, Atp4b-Cre, in which the expression of Cre recombinase was controlled by a 1.0 kb promoter of mouse β-subunit of H^＋-, K^＋-ATPase gene （Atp4b）. In order to test the tissue distribution and excision activity of Cre recombinase in vivo, the Atp4b-Cre transgenic mice were bred with the reporter strain ROSA26 and a mouse strain that carries Smad4 conditional alleles （Smad4Ca/Co）. Multiple-tissue PCR of Atp4b-Cre;Smad4Co/＋ mice revealed that the recombination only happened in the stomach. As indicated by LacZ staining, ROSA26;Atp4b-Cre double transgenic mice showed efficient expression of Cre recombinase within the gastric parietal cells. These results showed that this Atp4b-Cre mouse line could be used as a powerful tool to achieve conditional gene knockout in gastric parietal cells.
文摘The piggyBac transposable element was suc-cessfully used for generation of the transgenic silkworm, Bombyx mori. The EGFP was adopted in this study as a screening marker in transgenic vector under the control of an artificial promoter containing Pax-6 binding sites that can drive eye-specific genes expression in various insect species including B. mori and Drosophila. 111 independent trans-genic lines were obtained among 700 fertile G0 moths. Most of the transgenic lines contained two or more chromosomal insertions of the EGFP marker, which were stably inherited over more than six generations during the time of this pro-ject. PiggyBac-mediated transposition was confirmed by identifying the characteristic TTAA duplication sequence at the insertion sites. The stable and high efficient transmission of a genetic marker into B. mori confirms the usage of this vector-marker system for industrial production and theo-retical research.
