[Construction of nonsense-mutated eukaryotic expression vector of factor IX gene and its expression in COS-7 cells].
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.
Nie
Xin
X
Department of Hematology, The Second Hospital, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China.
Yang
Lin-Hua
LH
Chai
Bao-Feng
BF
Shen
Quan
Q
Zhang
Yuan
Y
Zhang
Yao-Fang
YF
Chen
Jian-Fang
JF
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018389:Codon, Nonsense / Q000235:genetics*; D005164:Factor IX / Q000235:genetics*; D005822:Genetic Vectors; D014162:Transfection
D018389:Codon, Nonsense; D005164:Factor IX