摘要
构建含有Zic3基因的真核表达载体pEGFP-Zic3,并转染小梅山猪骨髓间充质干细胞(BMSC),观察BMSC中Zic3的表达情况。用RT-PCR技术获得Zic3基因,并将其连接到真核表达载体pEGFP-C1中,获得真核表达载体质粒pEGFP-Zic3,采用双酶切法和DNA测序鉴定构建的载体是否正确。体外培养小梅山猪BMSC,通过免疫细胞化学法检测小梅山猪BMSC表面标志。采用脂质体法将pEGFP-Zic3转染BMSC,通过荧光显微镜观察确定重组质粒在小梅山猪BMSC内的表达和定位,用RT-PCR法检测Zic3的表达情况,流式细胞仪检测细胞周期和细胞凋亡情况。双酶切和测序证实已成功构建含目的基因Zic3的真核表达载体pEGFP-Zic3;小梅山猪BMSC体外培养状态良好,且表达CD29,不表达CD34和CD45;pEGFP-Zic3转染BMSC后可见大量绿色荧光,与对照组相比,转Zic3基因BMSC增殖能力提高,但细胞凋亡率未发生明显变化。结论:成功构建了含目的基因Zic3的真核表达载体pEGFP-Zic3,并高效转染BMSC,Zic3表达增加,且Zic3可提高细胞增殖能力。
To construct pEGFP-Zic3 eukaryotic expression vector carrying Zic3 gene, the vector was transferred into Xiaomeishan swines bone marrow mesenchymal stem cells（BMSC） to observe the Zic3 expression. The Zic3 gene was amplified by RT-RCR and then subcloned into eukaryotic express vector pEGFP-C1 to generate recombinant expression vector pEGFP-Zic3. The Xiaomeishan swines BMSC was cultured in vitro, and BMSC surface marker was detected by immunocytochemistry method. Furthermore,we trans- fected pEGFP-Zic3 plasmid into Xiaomeishan swines bone marrow mesenchymal stem cells （BMSC） by liposome, its expression was identified by observation of fluorescence microscopy and RT-PCR, and its cell cycle and apoptosis were detected through flow cytometry. The plasmid pEGFP-Zic3 was successfully constructed by double digestion and sequencing. CD29 could express themselves in BMSC, but CD34 and CD45 could not. Then plasmid pEGFP-Zic3 was cotransfected into Xiaomeishan swines BMSC, and green fluo- rescence was observed confirming successful transfection. The ability of cell proliferation in Xiaomeishan swines BMSC transfected with pEGFP-Zic3 was significantly increased, and the apoptosis rate showed no significant difference compared to the control group. Conclusions：The pEGFP-Zic3 eukaryotic expression vector carrying Zic3 gene was constructed successfully. The pEGFP-Zic3 vector was transfected into BMSC successfully to increase the expression of target gene and improve the ability of cell proliferation.
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2014年第5期111-116,共6页
Journal of Nanjing Agricultural University
基金
国家自然科学基金青年基金项目(31201802)
关键词
Zic3基因
小梅山猪
真核表达
骨髓间充质干细胞
Zic3 gene
Xiaomeishan swines
eukaryotic expression
bone marrow mesenchymal stem cells （BMSC）
作者简介
樊懿萱，助教，主要从事转基因和动物胚胎工程研究，E-mail：fanyixuan@njau．edu．cn。
通信作者：张艳丽，副教授，硕导，主要从事动物胚胎工程研究，E-mail：zhangyanli@njau．edu．cn。
