ABSTRACT
<p><b>OBJECTIVE</b>To study the regulatory rolesof SIRT1 on EZH2 expression and the further effects on EZH 2’ s repression of target gene expression.</p><p><b>METHODS</b>The stable SIRT1 RNAi and Control RNAi HeLa cells were established by infection with retroviruses expressing shSIRT1 and shLuc respectively followed by puromycin selection. EZH2 protein level was detected by Western blot in either whole cell lysate or the fractional cell extract. Reverse transcription-polymerase chain reaction was performed to detect the mRNA level of EZH2. Cycloheximide was used to treat SIRT1 RNAi and Control RNAi cells for protein stability assay. Chromatin immunoprecipitation(ChIP) assay was applied to measure enrichment of SIRT1, EZH2, and trimethylated H3K27 (H3K27me3) at SATB1 promoter in SIRT1 RNAi and Control RNAi cells.</p><p><b>RESULTS</b>Western blot results showed that EZH2 protein level increased upon SIRT1 depletion. Fractional extraction results showed unchanged cytoplasmic fraction and increased chromatin fraction of EZH2 protein in SIRT1 RNAi cells. The mRNA level of EZH2 was not affected by knockdown of SIRT1. SIRT1 recruitment was not detected at the promoter regionof EZH2 gene locus. The protein stability assay showed that the protein stability of EZH2 increases upon SIRT1 knockdown. Upon SIRT1 depletion, EZH2 and H3K27me3 recruitment at SATB1 promoter increases and the mRNA level of SATB1 decreases.</p><p><b>CONCLUSIONS</b>Depletion of SIRT1 increases the protein stability of EZH2. The regulation of EZH2 protein level by SIRT1 affects the repressive effects of EZH2 on the target gene expression.</p>
Subject(s)
Humans , DNA-Binding Proteins , Chemistry , Physiology , Enhancer of Zeste Homolog 2 Protein , Gene Expression Regulation , HeLa Cells , Polycomb Repressive Complex 2 , Repressor Proteins , Physiology , Sirtuin 1 , Physiology , Transcription Factors , Chemistry , PhysiologyABSTRACT
<p><b>OBJECTIVE</b>To develop an alternative method for assessment of gene delivery systems in vivo.</p><p><b>METHODS</b>Mouse primary spleen lymphocytes were genetically modified in vitro by a retroviral vector harboring a Gaussia luciferase (Gluc) expression cassette. After implantation of these cells into recipient mice, the expression of Gluc was detected in whole blood or plasma collected.</p><p><b>RESULTS</b>As little as 10 muL whole blood drawn from the recipient mice could guarantee prompt reading of Gluc activity with a luminometer. And the reading was found in good correlation with the number of genetically modified spleen lymphocytes implanted to the mice.</p><p><b>CONCLUSIONS</b>Gluc may be useful as an in vivo reporter for gene therapy researches, and Gluc blood assay could provide an alternative method for assessment of gene delivery systems in vivo.</p>
Subject(s)
Animals , Humans , Mice , Arecaceae , Cell Line , Gene Transfer Techniques , Genes, Reporter , Luciferases , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To delete IL-11 receptor alpha chain gene from the Bacterial Artificial Chromosome (BAC) chimeric DNA by RecA protein mediated homologous recombination method and establish the transgenic mice model containing whole beta-globin gene cluster.</p><p><b>METHODS</b>Two 500 bp homologous sequences (A and B) located at the upstream and downstream of IL-11 receptor alpha chain gene respectively were cloned into the Hind III and Xba I sites of pBV vector, then the 1 kb A + B fragment was recovered from the building vector and inserted into the Sal I site of the shuttle vector pSV-RecA. After transforming the shuttle vector into the competent DH10B E. Coli containing BAC DNA, the IL-11 receptor alpha chain gene was finally deleted from the BAC DNA through chloramphenicol positive selection and fusaic acid negative selection. The new BAC clone was characterized by Pulse Field Gel Electrophoresis (PFGE). Then, we microinjected the linearized and purified BAC DNA into the mouse fertilized eggs and prepared the transgenic mice.</p><p><b>RESULTS</b>By RecA protein mediated homologous recombination method, we deleted the IL-11 receptor alpha chain gene from the BAC DNA containing the complete beta-globin Gene Cluster and established 3 respective transgenic mice lines.</p><p><b>CONCLUSION</b>Human beta-globin gene cluster in the transgenic mice mediated by new BAC expresses in a correct mode and level as compared with previous transgenic mice.</p>