转hDAF基因小鼠的构建(英文)

本工作构建了含有ｈＤＡＦ基因的转基因小鼠，以便研究ｈＤＡＦ基因能否消除异种器官移植中的排斥反应。 采用ＤＮＡ重组的方法构建ｈＤＡＦ基因的表达载体ｐＳＰ６４ＨＰ（Ｆｉｇ．１）。通过受精卵显微注射，将其中的目的基因片段，转移到小鼠体内，建立转基因小鼠。再通过Ｄｏｔ ｂｌｏｔｔｉｎｇ和Ｓｏｕｔｈｅｒｎ ｂｌｏｔｔｉｎｇ杂交方法对出生小鼠的基因组特征进行查证。 连续两次对直接裂解菌液做ＰＣＲ扩增，筛选出重组质粒（Ｆｉｇ．２＆３），酶切图谱（Ｆｉｇ．４）和Ｓｏｕｔｈｅｒｎ杂交（Ｆｉｇ．５）分析结果与预期吻合，出现预期条带；小鼠受精卵注射后存活比率为７７．９％，受精卵的发育率为３．４％，出生小鼠中，１０．５％出现清晰杂交信号。 表明：ｈＤＡＦ基因表达载体构建成功；并整合入小鼠基因组中。

The Construction of Transgenic Mice with hDAF Gene for Xenotransplantation

Production of transgenic mice with human decay accelerating factor (hDAF) gene will provide an effective way to study how to overcome xenogenic hyperacute organ. The expression vector of hDAF gene, PSP 64HD, has been constructed. The PSP 64 HD DNA were lineared and microinjected into the fertilized eggs of mice by using male pronucleus microinjection technique. The transgenic eggs were transplanted into the uterus mice. When newborn mice were 4 weeks old, their tail DNAs were isolated to do Dot and Southern blot analysis for their-self identification. The results show that the constructed foreign gene has been integrated into the mouse genome. It means that the expression vector of hDAF gene and its transgenic mice have been constructed successfully in our experiments. For the transgenic mice, 77.9% of injected eggs survived after microinjection, and 3.4% in pups after development. The integration rate for hDAF gene is 10.5%.