靶基因xylE质粒载体转基因小鼠的建立

目的建立在基因组中整合有ｐＵＣ１１８ＮＸ质粒的转基因小鼠致突变检测模型。方法将含ｘｙｌＥ靶基因的ｐＵＣ１１８ＮＸ质粒ＤＮＡ显微注射至Ｃ５７ＢＬ／６Ｊ小鼠３７６枚受精卵雄性原核中，存活的２２５枚受精卵移入１１只假孕母鼠双侧输卵管内发育成个体后，用ＰＣＲ－Ｓｏｕｔｈｅｒｎｂｌｏｔ、质粒回收转化试验和酶切鉴定方法筛选存活仔鼠。结果假孕母鼠中有７只妊娠，共产仔鼠２９只，存活２５只，其中ＰＣＲ－Ｓｏｕｔｈｅｒｎｂｌｏｔ检测阳性小鼠１８只，阳性率为７２％。最后选择２只在基因组中完整整合ｐＵＣ１１８ＮＸ质粒的健康雄性小鼠作为Ｆｏｕｎｄｅｒ鼠，进行转基因小鼠致突变检测和建系工作。结论成功地建立了在基因组ＤＮＡ中整合有ｐＵＣ１１８ＮＸ质粒的Ｃ５７ＢＬ／６Ｊ转基因小鼠。

Development of Plasmid based Transgenic Mice Carrying with Target Gene xylE

Objective To develop a transgenic mouse model carrying with plasmid pUC118NX integrated into its genomic DNA for detecting mutagenesis. Methods DNA of plasmid UC118NX in target gene xy1E was injected microscopically into male protonucleus of 376 mouse spermatova,and 225 survival spermatova were transferred into the oviducts in both sides of 11 pseudopregnant female mice to develop their offspring.The genomic DNA in survival young mice were analyzed with polymerase chain reaction (PCR) Southern blot, plasmid transformation test and endonuclease digestion. Results Seven pseudopregnant mice got pregnant, and 29 offspring were delivered and 25 survived of which 18 were identified with positive for PCR Southern blot(72%). The two stout male mice with intact integration of plasmid pUC118NX in their genome were finally chosen as founders to detect gene mutation in vivo and establish transgenic mouse lineages. Conclusion Transgenic C57BL/6J mice integrated with plasmid pUC118NX into their genomic DNA have been successfully developed.