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Published on: April 26, 2017
[Construction of nonsense-mutated eukaryotic expression vector of factor IX gene and its expression in COS-7 cells]
Xin Nie1, Lin-Hua Yang, Bao-Feng Chai
1Department of Hematology, The Second Hospital, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China.
Abstract:
The purpose of this study was to construct 4 types of nonsense-mutated eukaryotic expression plasmids of fIX gene, using pcDNA3.1 plasmid containing fIX cDNA as template, and to identify, then to perform their expression in COS-7 cells. These stop mutants constructed by site-directed mutagenesis based on PCR, and further confirmed by DNA sequencing. COS-7 cells were transfected with either the wild-type or mutated fIX expression constructs, then the relative expression levels of fIX mRNA were detected by real time fluorescent quantitative PCR. The result showed that except the designed sites, there were no other nucleotide mutation in the sequences of four nonsense mutants. The results of real time PCR proved that the nonsense-mutated vectors can be effectively expressed in COS-7 cells. It is concluded that the nonsense-mutated eukaryotic expression vectors of fIX gene have been successfully constructed and can express in COS-7 cells, which provides the material basis for further researches on mechanism and treatment of FIX deficiency and the function defects caused by nonsense mutation.
Insights
Researchers successfully created four nonsense-mutated eukaryotic expression plasmids for the fIX gene. These novel plasmids effectively express Factor IX (fIX) in COS-7 cells, paving the way for studying Factor IX deficiency.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Nonsense mutations in the Factor IX (fIX) gene can lead to Factor IX deficiency, a bleeding disorder.
- Understanding the functional consequences of these mutations is crucial for developing effective treatments.
Purpose of the Study:
- To construct four distinct nonsense-mutated eukaryotic expression plasmids of the fIX gene.
- To confirm the successful construction and expression of these mutated plasmids in COS-7 cells.
Main Methods:
- Site-directed mutagenesis using PCR to create nonsense mutations in the fIX gene.
- DNA sequencing to verify the integrity of the constructed plasmids.
- Transfection of COS-7 cells with wild-type and mutated fIX constructs.
- Real-time fluorescent quantitative PCR to assess fIX mRNA expression levels.
Main Results:
- Four nonsense-mutated fIX expression plasmids were successfully constructed without unintended nucleotide alterations.
- The constructed nonsense-mutated vectors demonstrated effective expression in COS-7 cells, as confirmed by real-time PCR.
- fIX mRNA expression was detected from the mutated vectors in transfected cells.
Conclusions:
- Nonsense-mutated eukaryotic expression vectors for the fIX gene have been successfully engineered.
- These vectors are capable of expressing functional fIX in COS-7 cells.
- This provides a valuable tool for further research into the mechanisms and potential treatments for Factor IX deficiency caused by nonsense mutations.
