[Altered Splicing in Stable Cell Strains Expressing Mini-hF9 Gene with Nonsense Mutation]

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.

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