【摘要】 以绵羊 β 乳球蛋白基因 (β Lactoglobulin ,β LG)为转基因表达框架 ,将人G CSF(HumanGranulocyteColonyStimulatingFactor,hG CSF)基因与报告基因 增强型绿色荧光蛋白 (EnhancedGreenFluorescenctProtein ,EGFP)基因作为双表达单元拼接到 β LG基因的第一外显子处 ,并在G CSF基因两侧引入同源重组位点loxP、lox2 2 72 ,将打靶基因表达构件 β LG hG CSF IRES EGFP(总长 9 3kb)分为两段进行构建 ,片段Ⅰ (长 5 9kb)与片段Ⅱ (长 5 6kb)两段重叠部分为 2 2kb。向小鼠受精卵细胞质共注射构件片段Ⅰ、Ⅱ和NLS(核定位信号 ) 3个基因片段。对仔鼠进行整合与表达的检测。PCR Southern杂交检测结果表明 ,片段Ⅰ的整合率为 6 2 3% (86 /138) ,片段Ⅱ的整合率为 5 4 3% (75 /138) ,片段Ⅰ、Ⅱ共整合 (包括两片段分别整合和染色体外同源重组两种情况 )的小鼠为 6 2只 ,整合率为 4 4 9% (6 2 /138) ,其中在双阳性转基因小鼠中发生染色体外同源重组的几率为 80 6 % (5 0 /6 2 )。RT PCR Southern检测了 10只发生染色体外同源重组的转基因雌性小鼠 ,hG CSF基因的表达率为 90 % (9/10 ) ,EGFP基因的表达率为 10 0 % (10 /10 ) ,通过对其乳汁紫外吸收光谱的检测 ,EGFP基因的表达率为 5 0 % (5 /10 )。上述结果更多还原

【Abstract】 An expression construct specified in mammary gland with double cistrons of human G-CSF gene and IRES-EGFP gene under control of ovine beta-lactoglobulin gene flanking sequence,has been constructed in two parts (named fragment Ⅰand fragment Ⅱ) that share an overlapping region of 2.2 kb sequence.Two sites of loxp and lox2272 for homologous recombination were inserted into both flanking regions of G-CSF.The lengths of fragment Ⅰ and fragment Ⅱ are 5.9 kb and 5.6 kb,respectively.The whole length of the expression vector (β-LG-hG-CSF-IRES-EGFP) is 9.3 kb.The two DNA fragments mixed with nuclear locating sequence DNA(NLS) fragment were coinjected into murine zygote cytoplasm.The resulting mice were analyzed for the transgene integration and expression.A total of 138 founders were born.62.3%(86/138) of them was integrated with fragment Ⅰ sequence,and 54.3%(75/138) of them contained fragment Ⅱ,whereas 62 of them contained both fragment Ⅰ and Ⅱ,of whom 80.6% (50/62) were integrated by the whole reconstituted gene construct (result of ex-chromosomal homologous recombination,ECR).By RT-PCR analysis,it was shown that 90% of the ECR mice (9/10) expressed human G-CSF gene and 100% expressed EGFP gene.EGFP expression was also detected by absorption spectrum scanning from 400 nm to 700 nm,and 50%(5/10) of the ECR mice expressed EGFP protein.The high frequency and accuracy of homologous recombination in murine zygotes reported here suggests that some large transgenes could be constructed by ECR pathway.This method does not need to construct a long complex DNA vector directly and to do nucleus-injection,and is easier than traditional method.更多还原