title: [Construction and significance of recombinant hF9 minigene and its stable nonsense mutant cell lines].
abstract: This study was purposed to construct the recombinant hF9 minigene and its stable nonsense mutant cell lines, and to investigate its significance. Minigene hF9 was cloned into the mammalian expression vector pCMV-Tag3B; a nonsense mutant containing a premature termination codon (PTC) in the 121(st) amino acid residue was obtained by PCR site-directed mutagenesis; minigene hF9 and nonsense mutant were respectively transfected into HepG2 cells with G418 treatment to get stable HepG2-WT and HepG2-N cell lines. The results confirmed that the minigene hF9 and nonsense mutant were constructed successfully. The gene of interest was amplified by RT-PCR from the stable cell lines, and the minigene hF9 was expressed in the stable cell lines. It is concluded that the recombinant hF9 minigene and its stable nonsense mutant cell lines are constructed successfully. The cell lines can be used to screen the drugs treating the nonsense mutation-caused hemophilia according to PTC read-through approaches.
authors.affiliation: Shanxi University, Taiyuan, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.forename: Bo-Wen
authors.initials: BW
authors.lastname: Jiang
authors.forename: Lin-Hua
authors.initials: LH
authors.lastname: Yang
authors.forename: Xin
authors.initials: X
authors.lastname: Nie
authors.forename: Chen-Liang
authors.initials: CL
authors.lastname: Jia
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Quan
authors.initials: Q
authors.lastname: Shen
authors.forename: Bao-Feng
authors.initials: BF
authors.lastname: Chai
mesh_terms: D045744:Cell Line, Tumor; D018389:Codon, Nonsense; D005164:Factor IX / Q000235:genetics*; D005822:Genetic Vectors; D002836:Hemophilia B / Q000235:genetics*; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics
chemical_list: D018389:Codon, Nonsense; D011994:Recombinant Proteins; D005164:Factor IX