Cre重组酶敲除小鼠大脑脚背盖背侧核lacZ基因研究

The LacZ Gene of the Laterodorsal Tegmentum in Mouse was Knocked Out Using Cre Recombinase

目的观察Cre重组酶能否敲除双报告Z/EG转基因成熟小鼠大脑脚背盖背侧核局部lacZ基因。方法用PCR技术对双报告Z/EG转基因鼠系进行基因鉴定,利用立体定位微量注射技术将含有Cre重组酶的病毒载体注射到双报告Z/EG转基因成熟小鼠左侧大脑脚背盖背侧核,将鼠脑冷冻切片行抗绿色荧光蛋白免疫组织荧光染色及PicoGreen对照染色,于Confocal显微镜下观察绿色荧光蛋白的表达。结果双报告Z/EG转基因鼠所获PCR产物为631bp;未作任何染色切片在Confocal显微镜下可观察到注射Cre重组酶病毒载体左侧大脑脚背盖背侧核区域有弱绿色荧光,经抗绿色荧光蛋白免疫组织荧光染色后发现此区域有红色荧光。结论Cre重组酶成功地敲除了双报告Z/EG转基因成熟小鼠大脑脚背盖背侧核局部lacZ基因。

Objective The aim is to observe if Cre recombinase can knock out the local LacZ gene of the laterodorsal tegmentum nucleus (LDT) in double reporter Z/EG transgenic adult mouse. Methods Double reporter Z/EG transgenic adult mice were genotyped by PCR. Adeno-associated virus vector containing a Cre recombinase (AAV-Cre) was injected into the left laterodorsal tegmentum nucleus in double reporter Z/EG transgenic adult mice using stereotaxic microinjection technique. Anti-green fluorescent protein immunohistofluorescence stain and PicoGreen counterstain were carried out on cryostat sections of mice brain. The expression of green fluorescent protein was observed under confocal microscopy. Results The products of PCR in double reporter Z/EG transgenic adult mice were 631 bp band. Weak green fluorescence signal on the left laterodorsal tegmentum nucleus where AAV-Cre was injected on cryostat section without any stain was observed under confocal microscopy. The red fluorescence signal on the same area was found after anti-green fluorescent protein immunohistofluorescence stain. Conclusions Cre recombinase can successfully knock out the local LacZ gene of the laterodorsal tegmentum nucleus in double reporter Z/EG transgenic adult mice.