用Cre重组酶敲除成熟小鼠脑中N-甲基-D-天门冬氨酸受体基因

Gene of the N-methyl-D-aspartate Receptor in Adult Mouse Brain Was Knocked Out Using Cre Recombinase

目的观察Cre重组酶能否敲除N甲基D天门冬氨酸受体(N methyl D aspartatereceptor,NMDAR)转基因成熟小鼠海马CA1区域的局部基因。方法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NMDAR转基因成熟小鼠海马CA1区域,利用地高辛精UTP作标记,以线性NMDAR质粒作模板,产生带有标记的核糖探针并用其对小鼠脑冷冻切片进行荧光原位杂交,之后立即作PicoGreen对照染色,在Confocal显微镜下观察NMDARmRNA表达。结果所获得与NMDARmRNA特异结合的RNA探针大小为446bp,在注射Cre重组酶病毒载体海马CA1区域无NMDARmRNA表达,而且PicoGreen对照染色提示在无NMDARmRNA表达的区域几乎无细胞损害。结论Cre重组酶成功地敲除了NMDAR转基因成熟小鼠海马CA1区域的NMDAR基因。

Objective To observe if Cre recombinase can knock out the local gene of the CA1 region of hippocampus in N-methyl-D-aspartate receptor (NMDAR) transgenic adult mice. Methods Adeno-associated virus containing a Cre recombinase (AAV-Cre) was injected into the CA1 area of hippocampus in NMDAR transgenic adult mice using stereotaxic microinjection technique. Fluorescent in situ hybridization (FISH) for NMDAR (mRNA) was carried out on cryostat sections of mice brain using riboprobes that were labeled by Digoxigenin-UTP and created from linearized NMDAR plasmid as templates. The sections were immediately counterstained with (PicoGreen) after FISH. The expression of NMDAR mRNA was observed under confocal microscopy. Results The size of riboprobe that can particularly combine NMDAR mRNA was 446 bp. There was no NMDAR mRNA (expression) on the CA1 area of hippocampus where AAV-Cre was injected and no cell damage at the site of no NMDAR mRNA expression as indicated by PicoGreen conunterstaining. Conclusions Cre recombinase can successfully knock out the NMDAR gene of CA1 area of hippocampus in NMDAR transgenic adult mice.