过度表达突变核纤层蛋白A受精卵移植至假孕鼠体内的成活能力

Survival ability of mouse zygotes microinjected with over-expressive mutant lamin A in pseudopregnant mice

目的:制备Hutchinson-Gilford早老症转基因小鼠模型,观察突变的核纤层蛋白A对小鼠受精卵着床和成活情况的影响。方法:实验于2004-09/2007-04在北京大学医学部医学遗传学系和中国农业大学生物技术国家重点实验室完成。①实验方法:将缺失了第11外显子末端150个碱基的LMNA基因cDNA克隆到pcDNA3.1载体,应用原核显微注射方法将线性化的pcDNA3.1-mutant LMNA注射到小鼠受精卵的雄原核,再将注射后的受精卵移植至假孕鼠的输卵管。②实验评估:观察受精卵的着床和成活情况。通过聚合酶链反应检测着床的胚胎,鉴定有无阳性转基因动物。结果:①178只注射pcDNA3.1-mutant LMNA后的存活受精卵移植至假孕鼠输卵管后,出生13只子代鼠,出生率只有7%;58只注射pcDNA3.1-mutant LMNA后的存活受精卵移植到假孕鼠输卵管后,在移植后12￣15d的受体母鼠子宫内收集到1只活胚和5只死胚。②聚合酶链反应方法检测所收集到的胚胎,没有得到pcDNA3.1-mutant LMNA阳性转基因鼠。在着床的胚胎中也没有检测到pcDNA3.1-mutant LMNA。结论:过量表达的突变核纤层蛋白A对小鼠受精卵有致死性,只有无目的基因整合的受精卵才能存活至出生。

AIM： To establish a transgenic mouse model of Hutchinson-Gilford progeria syndrome and observe the effect of mutant lamin A on the implantation and survival ability of injected mouse zygotes. METHODS： The experiment was conducted in the Department of Medical Genetics, Peking University Health Science Center and the National Key Laboratory for Agrobiotechnology, China Agricultural University from September 2004 to April 2007. （1）Lamin A gene （LMNA） cDNA lacking of 150 basyls at the end of the eleventh extron was cloned into pcDNA3.1 carrier, and the recombinant plasmid pcDNA3.1-mutant LMNA cDNA was injected into the male pronucleus of mouse zygotes, then the zytotes was replanted into the oviducts of pseudopregnant mice. （2）The implantation and survival conditions of injected zygotes were observed. The implanted embryo was detected by PCR to identify if there were positive transgenic ones. RESULTS： （1）A total of 178 injected and survived zygotes were replanted. Thirteen mice were born with the birth rate of 7%. Fifty-eight injected zygotes were replanted and 1 living embryo and 5 dead embryos were collected from the oviducts of their foster mothers on the 12-15 ^th days. （2）No positive transgenic mice was found by PCR screening, and no pcDNA3.1-mutan LMNA was found in the implanted embryos. CONCLUSION： Over expressive mutant lamin A is toxic to the development of zygotes. Only zygotes with no target gene integration could survive until to be born.