摘要
建立一种能方便检测utrophin表达的方法,制备筛选Duchenne肌营养不良症治疗药物的细胞模型。将Utrophin基因上18号和19号外显子及其周围约6.7kb的DNA,分三段克隆下来,并将TETC报告基因插入插入片段Ⅰ(其中的18号外显子)中,构建打靶基因。经过电转化的方法,将测序正确的打靶基因转染到C2C12细胞株中。经G418筛选,FlAsH荧光染色,PCR扩增鉴定得到正确同源重组的细胞株。为筛选utro-phin表达促进剂建立了细胞模型,为筛选DMD治疗药物打下基础。
To establish a convenient method for observing the expression of utrophin and a cellular model for drug screening of duchenne muscular dystrophy. Three fragments of utrophin gene at 18 exon and 19 exon and the sequence surround them by PCR. TETC gene was inserted in 3＇ of fragmentⅠ. The target gene was constructed with＂ pGPKneobpA-LoxP-A and fragment Ⅰ, Ⅱ, Ⅲ, then transformed into C2C12 cells by electroporation. To identify homologous recombinants, transfected C2C12 cells were screened by G418, FlAsH staining and PCR detection. The target gene sequence was correct by sequencing, and two homologous recombinated C2C12 cells were got. A cellular model for detecting utrophin expression and a platform for drug screening of duchenne muscular dystrophy were constructed.
出处
《药物生物技术》
CAS
CSCD
2006年第4期250-254,共5页
Pharmaceutical Biotechnology
基金
教育部留学回国人员科研启动基金资助项目(编号:21001BA710B)
