Objective: To investigate the molecular mechanism in stable cell strains expressing Mini-hF9 gene with nonsense mutation.
Methods: Mini-hF9 gene and its nonsense mutants were transfected into HeLa cells independently, and stable cell strains were obtained after G418 resistance screening and monoclonal transformation. The altered splicing and protein expression of mRNA in Mini-hF9 gene in stable cell strains were detected by using RT-PCR and Western blot.
Results: The wild type and nonsense mutated human coagulation factor IX stable cell strains were constructed successfully, which were named HeLa-F9-WT, HeLa-F9-M1 and HeLa-F9-M2. Only normal splicing Norm was detected in the wild-type cell strain HeLa-F9-WT; Norm and Alt-S1 splicing were detected in HeLa-F9-M1; while Norm, Alt-S1 and Alt-S2 splicing were detected in HeLa-F9-M2.
Conclusion: The nonsense associated altered splicing (NAS) pathway, which generated alternately spliced transcripts, might be triggered in coagulation factor IX gene with nonsense mutation.
题目: 无义突变的人凝血因子IX小基因稳定细胞株选择性剪接的研究.
目的: 探讨无义突变的人凝血因子IX小基因稳定细胞株发生选择性剪接的分子机制.
方法: 将人凝血因子IX小基因（Mini-hF9）及其无义突变体分别转染哺乳动物细胞HeLa，经G418抗性筛选、单克隆化得到稳定细胞株；通过RT-PCR及Western blot分别检测稳定细胞株中Mini-hF9基因mRNA的选择性剪接及蛋白表达情况.
结果: 成功构建了野生型和无义突变的人凝血因子IX小基因稳定细胞株HeLa-F9-WT、HeLa-F9-M1和HeLa-F9-M2。野生型稳定细胞株HeLa-F9-WT中扩增到正常剪接产物Norm；无义突变稳定细胞株HeLa-F9-M1中扩增到Norm和Alt-S1这两种剪接产物，HeLa-F9-M2中扩增到Norm、Alt-S1和Alt-S2这三种剪接产物.
结论: 无义突变的凝血因子IX基因可能触发无义相关的选择性剪接（NAS）途径，导致选择性剪接的产生.