[Study of in vitro expression of human platelet ITGB3 gene nonsense mutation c.1476G>A].

OBJECTIVE: To explore the function of a novel nonsense mutation c.1476G>A of ITGB3 gene using an in vitro expression system. METHODS: An eukaryotic expression vector containing ITGB3 c.1476G>A cDNA was generated by site-directed mutagenesis and transformed into E.coli. Plasmid DNA was extracted and sequenced to confirm the target mutations. Wild-type and mutant recombination plasmids were transfected into Chinese hamster ovarian cancer (CHO) cells by nonliposome method, and the stable expression cells were harvested by G418 screening. The ITGB3 gene mRNA transcription and GPIIIa expression level in CHO cells were detected with real-time quantitative PCR, Western blotting and flow cytometry, respectively. RESULTS: The eukaryotic expression vectors of wild ITGB3 cDNA and c.1476G>A mutant were successfully constructed. CHO cells with stable expression were obtained after transfection and screening. Compared with the wild-type transfected cells, the amount of CD61 antigen expression was 37% and mRNA transcription level was only 6% in the mutant-transfected cells. Full length GPIIIa protein was found only in the stably wild-type-transfected cells, but not in mutant-transfected cells by Western blotting analysis. CONCLUSION: The ITGB3 c.1476G>A mutation can decrease the transcription level and further affect GPIIIa synthesis and CD61 antigen expression.

[Analysis of erythroid-specific blood group genes using un-mobilized peripheral stem cells cultured in vitro].

OBJECTIVE: To analyze specific expression of blood group genes using nucleated erythroid cells cultured from un-mobilized peripheral stem cells in vitro. METHODS: Hematopoietic stem cells(HSC) bearing the CD34 antigen were isolated from peripheral blood by centrifugation and magnetic beads sorting, followed by suspension culture in vitro. Cells were collected from medium on various stages and analyzed by immunofluorescence. The RNA transcription of RH and ABO blood group genes was analyzed using culture cells on day 12. RESULTS: A total of(3.19±0.13) ×10 (4) CD34+cells were isolated from about 50 mL peripheral blood with a recovery rate of 67.3%±2.7%. The cells amount in erythroid-lineage culture system on day 9 reached a plateau of a 237.1±15.5-fold amplification of the initial cell input. The stem cell-specific CD34 antigen was dropped off, while the erythroid-specific CD235a and CD240D antigens were increased in culture period. RHD/CE and ABO genes can be amplified using RNA extracted from culture cells on day 12, and genotypes of Rh and ABO systems by DNA sequencing were consistent with their serologic phenotypes. CONCLUSION: A method was established to analyze the gene expression of erythroid blood group derived from un-mobilized peripheral stem cells cultured in vitro. It can be used to study the expression of various erythroid-specific genes.