二氢叶酸还原酶和谷氨酰胺合成酶双基因筛选扩增系统对外源基因表达的影响

Dual gene amplification and selection system with dihydrofolate reductase and glutamine synthetase genes effectively increase the foreign gene expression

目的 研究二氢叶酸还原酶 (DHFR ,D)与谷氨酰胺合成酶 (GS ,G)单基因和二氢叶酸还原酶与谷氨酰胺合成酶 (DHFR +GS ,DG)双基因筛选扩增系统对外源基因表达的影响。方法 选择N端部分TPO基因 (T1 84)作为靶基因 ,以DHFR和GS基因作为筛选及扩增基因构建重组质粒pDCT1 84与pGCT1 84,将两种质粒分别转染CHOdhfr- 细胞形成单基因筛选扩增系统 ,两种质粒共转染CHOdhfr-细胞形成双基因筛选扩增系统。在双基因筛选扩增系统中设计三种药物加压方式。第一种为DG方式 :先用DHFR系统压力 (MTX)筛选 ,再用GS系统压力 (MSX)筛选 ;第二种为GD方式 :先用GS系统压力筛选 (MSX) ,再用DHFR系统压力筛选 (MTX) ;第三种为共加压方式 :同时利用DHFR与GS系统压力筛选 (MTX +MSX)。通过比较T1 84基因拷贝数及表达水平来研究DHFR和GS双基因筛选扩增系统对外源基因表达的影响。结果 T1 84基因拷贝数及表达水平在DG双基因筛选扩增系统不同药物加压方式中 ,均比DHFR或GS单基因筛选扩增系统高 ,但不同药物选择方式没有明显差异。结论 采用DHFR +GS双基因筛选扩增系统共加压方式是提高CHOdhfr-

Objective To study the effect of gene amplification and selection system with DHFR plus GS and DHFR or GS gene on the foreign gene expression. Methods Using the N-terminal truncated hTPO(T 184) gene as target gene,two plasmidsre were constructed :pDC-T 184 and pGC-T 184 where DHFR and GS gene were used respectively as the selective amplification marker. They were cotransfected into CHO dhfr'cells to establish dual gene amplification and selection system of DHFR plus GS gene and respectively transfected to establish single gene amplfication and selection system of DHFR or GS gene.Three selective methods in dual selective system to compare expression efficiency of hTPO were designed:the first method (DG) was to use drug pressure of MTX,then use MSX; the second method (GD) was reversed; the third method was simultanetously to use MTX and MSX as drug pessure. Results DHFR+GS dual system had not only higher gene amplification efficiency but also higher level expression. There was no distinct affect in different method of drug pressure. Conclusion MTX plus MSX dual drug pressure in dual selection system was an efficient and simple method to increase the expression of foreign gene in mammalian cells.