fmrl基因敲除小鼠中间神经元发育研究的新方法

A viable model for the study of GABAergic neurons in fmrl knockout mice

目的:通过杂交技术获得能表达GAD67-GFP的fmrl基因敲除小鼠模型。方法:fmrl基因敲除(fmrl-ko)雌性小鼠(X^-X^-)和GAD67-GFP敲入基因杂合雄性小鼠(2^+2^-)杂交。鼠尾巴提取基因组DNA,用PCR方法鉴定所有子代基因型。对杂交后代小鼠进行行为学观察,并行脑组织切片观察绿色荧光蛋白在γ-氨基丁酸能神经元的表达。结果:PCR方法证实通过杂交繁殖出四种基因型小鼠:同时带有杂合fmrlko及GAD67-GFP位点的雌性小鼠(X^+X^-/2^+2^-)、带有纯合fmrl-ko及GAD67-GFP位点的雄性小鼠(X^-Y/2^+2^-)、不带有GAD67-GFP的雌性和雄性小鼠(X^+X^-/2^-2^-,X^-Y/2^-2^-)。观察发现X^-Y/2^+2^-小鼠具有fmrl-ko小鼠类似的行为学表现,同时在显微镜下观察到X^-Y/2^+2^-小鼠脑组织切片有发绿色荧光的GABA神经元。结论:该杂交小鼠成功表达了GAD67-GFP,为研究γ-氨基丁酸能神经元在脆性X综合征发病机制中的作用提供一种可靠的模型。

Objective： To obtain Fragile X syndrome rat models with GAD67-GFP expression by hybridization technique. Methods： One fmrl-knockout female （X-X-） rat was mated with GAD67-GFP heterozygous male （2 ＋ 2-）. All hybrid offspring were genotyped by polymerase chain reaction （PCR） according to genomic DNA extracted from the tail. The behaviors and the expression of GFP on GABAergic neurons of brain slice were also observed. Results： Four genotypes of mice have been generated ： the heterozygous female mice with both the f mrl-gfp-ko and GAD67-GFP locus （X ＋ X-/2 ＋2-） , fmrl-ko male mice heterozygous with GAD67-GFP locus （ X-Y/2＋2-）, female and male mice without GAD67-GFP locus （ X ＋ X/-2-2-, X-Y/2-2- ）. Mice X-Y2 ＋ 2- were noted to have similar behaviors with fmrl ko mice. Morphological observation of GABAergic neuron had a good match between the mice X-Y/2 ＋ 2-and fmrl ko mice. Conclusions：The hybrid mice X-Y＋2 ＋ 2- successfully expressed the GAD67-GFP protein, which provides a viable model for the study on the pathogenetic effect of GABAergic neurons in the fragile X syndrome.