可分解草酸小鼠肠干细胞群的构建

目的 通过提取草酸分解菌的分解草酸功能基因frc和oxc,转染小鼠肠干细胞群使之获得草酸分解功能.方法 (1)构建能同时表达oxc和frc基因的双表达载体质粒pIRES-oxc-frc.(2)分离培养小鼠肠干细胞群,并了解其生长分化功能.(3)将pIRES-oxc-frc通过脂质体转染至小鼠肠干细胞群中,经过G418筛选后,了解验证转基因肠干细胞群生长、分化及基因表达状况.(4)用离子色谱法测定转基因后细胞培养液中草酸浓度,鉴定其草酸分解功能.结果 oxc和frc基因片段与GenBank提供的序列比较有极高的同源性.带有oxc和fre基因片段的重组质粒能顺利转染小鼠肠干细胞群,并能在后者中表达.转基因小鼠肠十细胞群培养液中草酸浓度(2.48±0.03)g/L]低于普通小鼠肠干细胞群和空白对照组(2.69±0.01)、(2.69±0.01)g/L,P<0.01].结论 产甲酸草酸杆菌分解草酸的重要功能基因oxc和frc基因能在体外转入小鼠肠十细胞群,并使后者具有草酸分解功能;原核细胞的多个基因能在一定条件下通过基因工程技术转入真核细胞.

Construction of oxalate-degrading intestinal stem cell population in mice

Objective The oxalate-degradation genes of Oxalobacter formigenes(Ox.F)-frc and oxc genes were cloned and transfected into murine intestinal stem eell population to make the latter acquire oxalate-degradation function.Methotis(1)The dicistronic eukaryotic expression vector,co-expressing OXC and frc genes,pIRES-oxc-frc was constructed.(2)The murine intestinal stem cell population wag isolared and cultured,and the function of cellular growth and differentiation identified.(3)The cells were transfected with pIRES-oxc-frc.After selection by G418,the function of cellular growth and differentiatiell and the expression of objective genes were identified.(4)The concentration of oxalate in the culture medium growing the transgenic cells wag determined by ion chromatography to explore the oxalate-degradation function of cells.Results Ox.F could be isolated and cultured from the feces of Chinese people.Compared with foreign reports,a certain morphologic variation of Ox.F existed.But OXC and frc genes showed a high homology with the sequence reported in GenBank.The recombinant plagmid containing OXC and frc genes could be successfully transfected into the murine intestinal stem coll population.The expression of objeetive genes wag normal.The concentration of oxalate in the culture fluid of ransgenic intestinal stem cell population(2.48±0.03)g/L]was lower than those of the normal group(2.69±0.01)g/L]and the control group(2.69±0.01)g/L,P<0.01].Conclusions Both oxc and frc genes could be transfected into the murine intestinal stem cell population,and the cells could be given oxalate-degrading function.The gene of prokaryecyte could be introduced into the eukaryocyte for u successful expression.