LMNA突变体HEK293、C2C12模型的构建及其核纤层蛋白A/C亚细胞定位

目的构建A型核纤层蛋白基因(LMNA)野生型、突变体的融合蛋白真核表达载体及LMNA突变体慢病毒载体,研究其在HEK293、C2C12中表达、核纤层蛋白A/C亚细胞定位及细胞核改变。方法将野生型全长LMNA cDNA克隆入pEGFP-N1质粒,构建LMNA野生型质粒pEGFP-N1-LMNA,以野生型质粒为模板构建c.1117A>G定点突变质粒pEGFP-N1-LMNA-I373V。将构建的2种质粒分别转染HEK293、C2C12,用G418筛选转染的C2C12,荧光显微镜下观察细胞核形态及GFP标记的核纤层蛋白A/C亚细胞定位。以pEGFP-N1-LMNA-I373V为模板,构建pHBLV-h-LMNA-I373V-3*flag-GFP-PURO慢病毒,对慢病毒进行包装与滴度测定。pHBLV-GFP-PURO、pHBLV-LMNA-C1117-3*flag-GFP-PURO分别转染C2C12,免疫荧光染色法观察转染后细胞核形态及核纤层蛋白A/C亚细胞定位的改变。结果构建的pEGFP-N1-LMNA、pEGFP-N1-LMNA-I373V及pHBLV-h-LMNA-I373V-3*flag-GFP-PURO测序与目的基因序列完全一致。pEGFP-N1-LMNA转染的HEK293、C2C12核纤层蛋白A/C均匀表达于核膜下,pEGFP-N1-LMNA-I373V转染的HEK293、C2C12内核纤层蛋白A/C核内异常聚集,呈散点样分布;与HEK293相比,C2C12细胞转染效率明显降低。慢病毒转染C2C12的转染率高,突变体慢病毒转染的细胞核形态异常及核纤层蛋白A/C核内分布异常。结论成功构建2种LMNA突变体真核表达载体及突变体转染的HEK293、C2C12模型,为LMNA突变体导致疾病的机制研究奠定科学基础。

Construction of HEK293 and C2C12 cell models transfectedby LMNA mutant and related intracellular sublocalization of lamin A/C

Objective To construct an expression vector of fusion protein from wild type LMNA and mutant and the lentivirus vector of LMNA mutant,to study the expression and sublocalization of lamin A/C and the change of nucleus in HEK293 and C2C12 cells.Methods The wild type LMNA was cloned into pEGFP-N1 plasmid to construct pEGFP-N1-LMNA,then pEGFP-N1-LMNA-I373V plasmid for c.1117A>G site-directed mutant was constructed.The pEGFP-N1-LMNA and pEGFP-N1-LMNA-I373V were transfected respectively into HEK293 and C2C12 cells.Then C2C12 cells were selected with G418 and the sublocalization of GFP-labeled lamin A/C and nuclear morphology were observed under fluorescent microscope.pHBLV-h-LMNA-I373V-3*flag-GFP-PURO was constructed by using pEGFP-N1-LMNA-I373V plasmid as template.After packaging and detecting the titer of pHBLV-GFP-PURO and pHBLV-LMNA-C1117-3*flag-GFP-PURO,C2C12 cells were transfected by the lentivirus.The sublocalization of lamin A/C and abnormal nuclear morphology were examined by immunofluorescence staining.Results The sequences of pEGFP-N1-LMNA,pEGFP-N1-LMNA-I373V and pHBLV-LMNA-C1117-3*flag-GFP-PURO were the same as the target gene.Lamin A/C distributed uniformly at the inner nuclear membrane in HEK293 and C2C12 cells transfected by pEGFP-N1-LMNA,but distributed abnormally,forming distinct aggregates in the cells with transfection of the mutant construct.C2C12 exhibited lower transfection efficiency of plasmid than HEK293.However,transfection efficiency of lentivirus in C2C12 was improved significantly.Lamin A/C forming distinct aggregates in nucleus and abnormal nuclear morphology were also showed in C2C12 after transfection of mutant lentivirus.Conclusions Two kinds of eukaryotic expression vectors of LMNA mutant,and HEK293 and C2C12 cell models transfected by LMNA mutant are successfully constructed,which provides scientific basis for the pathogenesis of diseases caused by LMNA mutant.