关键词: FoxA1, 慢病毒, 载体构建, 过表达

Abstract: 【Abstract】〓Objective〓To construct the restructuring lentiviral expression vector FoxA1 gene and lay a foundation for us to explore the functions and mechanisms of FoxA1. Methods〓The primers were designed for the amplification of FoxA1 by polymerase chain reaction(PCR),and cDNA was constructed to the lentiviral expression vector PCDH-CMV-MCS-EF1a-GFP-puro. After PCR,double digests and DNA sequencing, the positive recombinant FoxA1 expression vector, pCMV-VSV-G and pCMV-dR8.91 were cotransfected into HEK-293T cells to produce lentiviral particles, and then the latter was transfected into 293T cells to calculate the virus titer by observing the GFP expression. Finally the mRNA expression of transfected FoxA1 in primary human skin fibroblasts HFFs was identified by real-time quantitative PCR. Results〓PCR, double digests and DNA sequencing assays demonstrated that the inserted sequences was correct. The recombinant lentiviral expression vector FoxA1 was successfully constructed with a concentration of 1×108 TU/mL of viral titer.The level of FoxA1 expression was significantly increased after infection with PCDH-CMV-FoxA1-EF1a-GFP-puro. Conclusion The lentiviral vector over-expressing FoxA1 gene was successfully constructed and highly overexpresses in HFFs. It lays an experiment foundation to explore the functions and mechanisms of FoxA1 for further study.

Key words: lentivirus, Vector construction, Over-expression, FoxA1