摘要
目的 应用逆转录病毒载体四环素调控系统 ,定量调节促血小板生成素 (TPO)基因在NIH3T3细胞中的表达。方法 pRevTet On质粒转染逆转录病毒包装细胞PT6 7细胞 ,应用包装成Tet On重组逆转录病毒 ,并将此病毒感染NIH3T3细胞 ,建立整合入Tet On逆转录病毒的阳性细胞株(RevTet On3T3) ,并通过强力霉素调控荧光素酶基因的表达证明此细胞株具有调控作用。构建pRevTRETPO重组质粒 ,转染PT6 7细胞 ,包装成含四环素反应元件和血小板生成素基因的重组逆转录病毒。再将此病毒感染上述细胞株 ,建立整和双病毒的细胞株 (RevTet On3T3TPO) ,通过强力霉素调节TPO基因在此细胞株中表达。培养基中加入 5mg·L-1强力霉素 (Dox)或不加Dox ,培养 72h后检测促血小板生成素基因表达情况。结果 RevTet On3T3在培养基中加入Dox时荧光素酶活性高 ,平均为5 .9× 10 4 RLU·S ,当培养基中不加Dox时荧光素酶活性低 ,平均为 2 .6× 10 3RLU·S。RevTet On3T3TPO在培养基中加入Dox时TPO表达量高 ,平均为 12 .8ng/ml。在培养基中不加入Dox时TPO表达量低 ,平均为 0 .5 6ng/ml,加Dox组为不加Dox组的 2 2 8倍。结论 建立逆转录病毒整合的NIH3T3细胞株 ,可利用四环素及其衍生物定时、定量调节多种外源基因的表达 ,增加基因治疗的精确性和?
Objective To use the retrovirus vector Tet On regulation system to control the expression of TPO and luciferase gene by doxycycline (Dox). Methods Packing the recombinant Tet On retrovirus, and infecting it to NIH3T3 cells to establish recombined Tet On retrovirus cell lines, RevTet On3T3. To construct the pRevTRETPO, the new cell line was transfered to PT67 cell to package the recombinant retrovirus which contained both the TRE and TPO. This virus was RevTet On3T3 used to establish the two inserting recombinant retrovirus cell strain RevTet On3T3TPO. Doxycycline was used to control the expression of TPO in RevTet On3T3. Results When Dox (5 mg/L) was added to the RevTet On3T3 cells, a higher activity of luciferase was seen (mean, 5.9×10 4 RLU·S) compared with the mean activity (2.6×10 3 RLU·S) in the absence of Dox. When Dox was added to the RevTet On3T3TPO cells, cell populations had high TPO expression (mean, 12.8 ng/ml) in 5 mg/L of Dox (On state), and a low TPO expression (mean, 0.56 ng/ml)in the absence of Dox (Off state). Conclusion The RevTet On gene expression system could modulate the expression of multiple genes by tetracyline and its derivatives, providing a useful tool for gene therapy.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2000年第10期773-775,共3页
National Medical Journal of China
基金
国家九五科技攻关项目!资助 (96 90 6 0 1 19)
