关键词: Hes1基因, Ngn1基因, pCDNA3.1质粒, 神经前体细胞, 细胞分化, 反转录-聚合酶链式反应, 大鼠

Abstract: Objective To clone rat Hes1 gene cDNA, construct its eukaryotic expression vector and obtain positive neural precursor cell clones expressing Hes1 gene stably，Then to study their effects to the differentiation of neural precursor cells. Methods The total RNA was extracted from 14 days Wistar rat brain. The full-length cDNA encoding Hes1 gene was obtained using RT-PCR method and inserted into pGEM T Easy cloning vector. After the sequencing was confirmed, the gene was subcloned to pCDNA3.1 to construct recombinant eukaryotic expression vectors of pCDNA3.1-Hes1. The recombinant plasmid was transfected into neural precursor cells from cortex of 14 days Wistar rat brain by lipofectamine method and positive cell clones were screened with G418. The existence of Hes1 gene in the transfected cells genomic DNA， and the over-expression of their mRNA in the transfected cells were confirmed with Fluorescent quantitative real-time RT-PCR(FQ-PCR). Results Enzyme digestion analysis and sequencing showed that the target genes were cloned into recombinant vector. The existence and over-expression of Hes1 gene in the transfected neural precursor cells was identified with FQ-PCR and it display that HES1 mRNA have high expressed in the transfected neural precursor cells.Conclusion The eukaryotic expression plasmid containing Hes1 genes were successfully constructed. The positive neural precursor cell clones expressing Hes1 gene stably were obtained, which may be a promising cell model for studying the biological function of Hes1 genes and the role of Hes1 genes in the differentiation of neural precursor cells. The experiment manifested that the high expression of Hes1 can restrain the expression of Ngn1 gene，and enhance the neural precursor cells differentiation to

Key words: Hes1 gene, Ngn1 gene, pCDNA3.1 plasmid, Neural precursor cells, Cell differentiation, RT-PCR, Rat