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Abstract Epigenetic mechanisms preside over genetic information to enable development from the fertilized, totipotent oocyte to the adult body. Methylation of histones at specific residues is one of the most important epigenetic modifications and has an essential role in both transcriptional repression and activation during embryonic development, including lineage specification and cell differentiation. The UTX (ubiquitously transcribed tetratricopeptide repeat X chromosome) and JMJD3 (jumonji domain containing 3) are identified as a histone H3 lysine 27 trimethylation (H3K27me3) demethylase, which promote muscle development, regulates metabolism and other important physiological functions. The principle of in vitro transcription system is based on DNA template by RNA polymerase in vitro synthesis of RNA. In this study, 3 pairs of primer were designed based on the mRNA sequence of JMJD3 and UTX in mouse (Musculus mus), and the in vitro transcription vectors pMD19T-T7UTX and pMD19T-T7JMJD3 were constructed, respectively. The in vitro transcription of mRNA was injected into the mouse oocytes, and the H3K27me3 was detected by immunofluorescence assay. The results showed that the in vitro transcription of UTX and JMJD3 was detected by electrophoresis, and the bands were clear and consistent with the expected size. The electrophoresis of the correct size of UTX and JMJD3 in vitro transcription products were gel recovery and purified. The use of microinjection technology taking UTX and JMJD3 gel product was injected into mouse oocytes. H3K27me3 expression was detected by laser confocal immunofluorescence technology. Result showed that the expression of H3K27me3 after injection of oocytes was significantly decreased. In this study, in vitro transcription of UTX and JMJD3 vectors were successfully constructed, respectively, using the piecewise PCR method, and proved to had very good enzyme activity at the cell level. This study successfully constructed pMD19T-T7UTX and pMD19T- T7JMJD3 vectors. At the cellular level, pMD19T-T7UTX and pMD19T- T7JMJD3 vectors could be transcribed and translated into active proteins. These vectors can be used to study the role of UTX and JMJD3 in the development and related diseases.
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Received: 28 January 2016
Published: 01 July 2016
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