摘要
目的构建并筛选针对小鼠胰岛素诱导基因1(insulin induced gene 1,insig1)siRNA的特异性表达质粒,建立稳定转染细胞株,并观察其对小鼠巨噬细胞RAW264.7胆固醇代谢的影响。方法根据GenBank中登录的insig1基因序列及RNA干扰原则,设计3个阳性克隆及1个阴性克隆序列,定向克隆入pGenesil-1质粒,构建insig1 shRNA真核表达质粒,测序鉴定后,脂质体法转染RAW264.7细胞,筛选干扰效果最佳的质粒,将其转染入RAW264.7细胞,经G418筛选建立稳定转染细胞株,采用RT-PCR及Western blot法检测转染细胞中insig1基因的转录水平及蛋白的表达水平,油红O染色观察转染细胞中胆固醇含量的变化。结果酶切及测序结果证实,insig1基因shRNA表达质粒构建正确,其中si-2为干扰效果最佳的质粒;在稳定转染的RAW264.7细胞中,si-2组insig1基因mRNA的转录水平和蛋白的表达水平较阴性质粒组和未转染组明显降低(P<0.05),油红O颗粒含量最多。结论成功建立了insig1 siRNA稳定表达细胞株,体外模型证明了对insig1基因的干扰可促进细胞胆固醇摄取。
Objective To construct and screen a specific expression vector for murine insulin induced gene 1(insig1) siRNA,establish a stably transfected cell strain and observe its effect on cholesterol metabolism in murine macrophage RAW264.7.Methods The gene sequences encoding three positive clones and one negative clone were designed and synthesized according to the insig1 sequence in GenBank based on RNA interference principle,and cloned into plasmid pGenesil-1.The constructed recombinant plasmid was identified by sequencing and transfected to RAW264.7 cells in mediation of liposome.The plasmid with optimal interfering effect was screened and tranfected to RAW264.7 cells,based on which a stably tranfected cell strain was established by screening with G418.The transcription level of insig1 mRNA in the established cell strain was determined by RT-PCR,while the expression level of protein by Western blot,and the change of cholesterol content was observed by oil red staining.Results Restriction analysis and sequencing proved that the expression vectors for insig1 shRNA were constructed correctly,of which si-2 was interfering plasmid.Both the transcription level of insig1 mRNA and expression level of insig1 protein in RAW264.7 cells transfected with si-2 were significantly lower(P 0.05),while the oil red stained particle content was significantly higher than those in the cells untransfected and transfected with negative control plasmid.Conclusion A cell strain for stable expression of insig1 siRNA was successfully established.Model test in vitro proved that the interference to insig1 promoted the cholesterol intake of cells.
出处
《中国生物制品学杂志》
CAS
CSCD
2011年第12期1445-1449,共5页
Chinese Journal of Biologicals
基金
重庆市卫生局医学科研计划项目(2009-2-208)