应用荧光原位杂交技术检测人βE珠蛋白基因在转基因小鼠染色体上的整合状态

为将荧光原位杂交技术应用于基因定位研究中,探讨一种能有效地检测转基因动物染色体上外源基因整合状态的实验方法,对小鼠腹腔注射秋水仙素后,取转基因小鼠骨髓制备中期染色体,将传统的FISH方法加以改进,检测外源基因在转基因小鼠染色体上的整合状态.检测结果表明,外源人βE珠蛋白基因已稳定地整合于小鼠染色体上.FISH能直观地反映外源基因在转基因动物染色体上的整合状态,该方法可对转基因动物及基因转移研究中的外源基因整合后进行染色体定位检测。 Abstract:To determine the integration site of human βE globin gene in the chromosomes of transgenic mice, transgenic mice carrying human βE globin gene were injected intraperitoneally with colchicines, then, bone marrow cells wereisolated and metaphase chromosomes were prepared, the traditional FISH method was improved to detect the integration site of humanβE globin gene in transgenic mice when combined with G-banding. Human t3E globin gene can bedetected in different position of different chromosomes in transgenic mice and FISH signals showed that two mice were heterozygous of human 13E globin gene and one was homozygous. Human t3E globin gene was integrated into thechromosomes of transgenic mice in a random pattern and the results demonstrated that FISH can be used to investigate the integration site of foreign genes in transgenic mice.     

应用荧光原位杂交技术检测人βE珠蛋白基因在转基因小鼠染色体上的整合状态

为将荧光原位杂交技术应用于基因定位研究中,探讨一种能有效地检测转基因动物染色体上外源基因整合状态的实验方法,对小鼠腹腔注射秋水仙素后,取转基因小鼠骨髓制备中期染色体,将传统的FISH方法加以改进,检测外源基因在转基因小鼠染色体上的整合状态.检测结果表明,外源人βE珠蛋白基因已稳定地整合于小鼠染色体上.FISH能直观地反映外源基因在转基因动物染色体上的整合状态,该方法可对转基因动物及基因转移研究中的外源基因整合后进行染色体定位检测。 Abstract:To determine the integration site of human βE globin gene in the chromosomes of transgenic mice, transgenic mice carrying human βE globin gene were injected intraperitoneally with colchicines, then, bone marrow cells wereisolated and metaphase chromosomes were prepared, the traditional FISH method was improved to detect the integration site of humanβE globin gene in transgenic mice when combined with G-banding. Human t3E globin gene can bedetected in different position of different chromosomes in transgenic mice and FISH signals showed that two mice were heterozygous of human 13E globin gene and one was homozygous. Human t3E globin gene was integrated into thechromosomes of transgenic mice in a random pattern and the results demonstrated that FISH can be used to investigate the integration site of foreign genes in transgenic mice.     

反转录病毒载体介导的人β—珠蛋白基因低水平表达的机制初探

以人 β 珠蛋白基因簇基因座控制区DNaseI高敏位点 2和高敏位点 3组合的微小基因座控制区作为调控元件 ,以 374bp第二内含子部分缺失的 2 .0kbβ 珠蛋白基因作为结构基因的反转录病毒重组体 ,通过建立 ψ 2产病毒细胞系和瞬时转染双向性包装细胞系 ψ A两种方法获得重组病毒。感染鼠红白血病细胞后 ,Southern杂交鉴定前病毒整合完整性 ,用RNase保护实验检测人 β 珠蛋白基因的表达 ,通过PCR产物测序确定前病毒结构。结果表明 ,在瞬时转染 ψ A来源的重组病毒转导和质粒转染的鼠红白血病细胞中 ,人 β 珠蛋白基因的表达水平分别达到内源性鼠α 基因表达的 (5 2 .4± 11.2 ) % (n =12 )和 (73.8± 14 .3) % (n =12 ,未确定整合拷贝数 ) ;而在 ψ 2产病毒细胞系来源的病毒转导的细胞中 ,表达水平低于 3% ,前病毒序列分析发现其高敏位点 2中出现一个点突变 ,这可能是人β 珠蛋白基因呈低水平表达的一个原因。     

腺相关病毒介导基因组来源的基因转移与整合

构建一个带β-珠蛋白基因组序列的腺相关病毒载体AV53HS2Δβ2Neo.经包装成重组腺相关病毒后,转导红系细胞.DNA印迹证实包含红系增强子、β-珠蛋白基因和筛选标志基因的前病毒基因组完整整合于红系细胞基因组中.结果说明腺相关病毒载体能介导基因组序列来源的目的基因稳定整合于受体细胞基因组中.