| 18036306 |
Yang XS, Hu YM, Xiao B, Yang QD, Xie Y: [Effects of short hair RNA on the expression of SMN1 gene in human mesenchymal stem cells]. Zhonghua Yi Xue Za Zhi. 2007 Sep 4;87(33):2369-71.
OBJECTIVE: To construct a plasmid expressing short hairpin RNA (shRNA) targeting SMN1 gene in mammalian cells and to investigate the effect of shRNA on the expression of SMN1 gene in human mesenchymal stem cells (hMSCs), thus providing experimental basis for the establishment of SMA cell model. METHODS: Five shRNAs targeting SMN1 gene was designed according to the SMN1 cDNA sequence provided by GenBank, and cloned to the downstream of U6.1 promoter in the pRNAT-U6.1/Neo vector, thus forming different recombinant plasmids: pshRNA-SMN1, pshRNA-SMN2, pshRNA-SMN3, pshRNA-SMN4, and pshRNA-SMN5 expressing specific shRNAs targeting SMN1 gene. The plasmid psh-RNA-0 was used as controls. The recombinant plasmid pshRNA-SMN1 was transfected into human mesenchymal stem cells (hMSCs) by Lipofectomine (TM) 2000, and its effects on the mRNA expression and protein expression of fl-SMN were observed by RT-PCR and Western blotting. RESULTS: The successful construction of recombinant plasmid pshRNA-SMN was identified by enzyme cutting and DNA sequencing. After transfection, the expression levels of fl-SMN mRNA and protein in hMSCs of the pshRNA-SMN1-1 group and pshRNA-SMN1-4 group were significantly lower than that of the control group (both P < 0.05) with an interfering efficiency of 64.05% and 61.04% in mRNA level, and 52.97% and 61.57% in protein level respectively. However, the expression of Delta7-SMN mRNA was not effected. CONCLUSION: The constructed recombinant plasmid called pshRNA-SMN1 inhibits the expression of fl-SMN mRNA and protein in hMSCs efficiently. |
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