摘要
目的:构建SPARC基因RNAi慢病毒载体获得稳定产毒的细胞,并观察其对人MDS细胞株SKM-1细胞的转染效率及其对SPARC基因的抑制效率。方法:针对已经筛选确定的SPARC基因RNAi有效靶序列,合成靶序列的OligoDNA,退火形成双链DNA,与经AgeⅠ和EcoRⅠ双酶切后的pGCSIL-GFP载体连接产生GC-shSPARC慢病毒载体,PCR筛选阳性克隆并进行测序鉴定。用GC-shSPARC、pHelper1.0和pHelper 2.0载体共转染包装细胞293T细胞,包装产生慢病毒,以293T细胞GFP蛋白的表达水平测定病毒滴度,并将获得的重组慢病毒GC-shSPARC转染SKM-1细胞,通过荧光显微镜检测转染后GFP表达情况,测定转染效率;RT-PCR和Western blot分别验证转染后SKM-1细胞SPARC mRNA及蛋白的表达。结果:经测序证实,构建出了SPARC shRNA的慢病毒载体GC-sh SPARC。包装、浓缩病毒悬液的滴度为1×109TU/mL。荧光显微镜下能直接观察到转染组细胞的GFP表达,转染效率为70%,RT-PCR、Western blot技术分别检测到GC-shSPARC慢病毒转染SKM-1细胞SPARC mRNA、SPARC蛋白表达水平较空白组明显降低(P<0.05)。结论:成功构建SPARC基因RNAi慢病毒载体,其能高效干扰SKM-1细胞SPARC基因的表达。
AIM： To con.struct a lentiviral vector ex- pressing small-hairpin RNA（shRNA） targeting SPARC gene and investigate its silenced effect on SPARC in human myel- odysplastic syndromes（MDS） cell line SKM-1. METHODS： The targeting sequence of SPARC gene which can be effec- tively silenced in RNA interference was confirmed in our pre- vious study. The designed and synthesized single-stranded primers were annealed to double-stranded oligo sequences and subcloned into linear pGCSIL-GFP lentMral plasmid di- gested by enzyme Age I and EcoR I to produce GC- shSPARC lentiviraL vector. After being identified by PCR and sequencing, plasmids GC-shSPARC with pHelper 1.0 and pHelper 2.0 were cotransfected into 293T cells to pack- age lentiviral particles. The recombinant lentiviral vector was transfected into human SKM-1 cells, transfection efficiency was evaluated with expression of green fluorescent protein （GFP） determined by fluorescent microscope. Expression of SPARC in SKM-1 cells was detected using RT-PCR and Western blotting. RESULTS： A recombinant lentiviral vec- tor, GC-shSPARC, expressing shRNAs targeting SPARC gene was constructed and confirmed by DNA sequencing. The recombinant lentivirus was harvested from 293T cells with a viral titer of 1 × 10^9 TU/mL. GFP was observed in the70% of SKM-1 cells after transfection. Expression of SPARC mRNA and protein was significantly reduced in the GC-shSPARC transfected group than that in the control group （ P 〈 0.05 ）. CONCLUSION： The lentivirus RNAi vec- tor targeting SPARC has been successfully constructed, and can effectively inhibit the expression of SPARC in SKM- 1 cell line, which shed light on the foundation for researching the inhibition of SPARC siRNA target against human MDS cells proliferation, induction apoptosis and gene therapy.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2012年第5期466-469,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金面上项目(30971277)
重庆市自然科学基金(2009JJ0424)
教育部留学回国人员启动基金(2009年)
