一种发育期皮质脊髓投射神经细胞基因表达谱的研究方法

A Method for Examining the Gene Expression Profile of Developing Cortico-Spinal Projection Neuron

皮质脊髓投射神经细胞(cortico-spinal projection neurons,CSPN)对于大脑的功能非常重要,但目前还缺乏研究小鼠发育期CSPN基因表达谱的有效方法。将出生后2 d(P2)的小鼠在脊髓交叉处注射逆行标记的神经示踪剂,并在P8时进行激光显微切割。提取捕获细胞的RNA并线性放大,而后利用定量PCR进行皮层标记基因的验证,线性放大后的RNA与大脑皮层组织的RNA相比,第五皮层标记基因Fezf2(Fez family zinc fi nger 2)表达明显升高,第四、第六皮层标记基因Rorβ(RAR related orphan receptorβ)、Foxp2(forkhead box P2)的表达相对较低。之后,进行小鼠基因芯片表达谱检测,通过基因功能富集度分析发现,在CSPN中大多数富表达的基因都与G蛋白偶联受体信号通路以及运动行为有关。而且,染色质的修饰和编排相关基因在CSPN里也有富集表达。通过定量PCR进一步验证了芯片中CSPN的富集基因,结果发现,Meg3(maternally expressed gene 3)等7个基因在大脑第五皮层表达丰富。该研究将在体神经细胞荧光标记、激光捕获显微切割、RNA线性放大、基因芯片及定量PCR等多种技术结合起来,提供了一种研究发育期小鼠CSPN基因表达谱的方法。

The cortico-spinal projection neurons （CSPN） are very important for the brain function However, an efficient method to study the gene expression profile of developmental CSPN is still lacking. In the present study, mice were injected Red Retrobeads at spinal cross at postnatal day 2 （P2） for retrograde labeling of CSPN and sacrificed at P8 for laser capture micro-dissection （LCMD）. Total RNAs of isolated cells were amplified linearly and verified through the marker genes of each layer by qPCR. While layer V marker gene Fezf2 （Fez family zinc finger 2） was increased, layer IV maker gene Ror~ （RAR related orphan receptor ~） and layer VI maker gene Foxp2 （forkhead box P2） were decreased in amplified RNAs from LCMD, compared with those from cortical tissue. The RNA products were determined by Affymetrix Mouse GeneChip. GO category analysis found that the most enriched biological processes in the CSPN were the regulations of G-protein coupled receptor signaling pathway and motor behavior. Interestingly, genes for chromatin modification and organization are also enriched in the CSPN. Additionally, we verified the enriched genes from GeneChip by qPCR and found that 7 genes have high level of expression in the CSPN, such as Meg3 （maternally expressed gene 3） etc. Our study provides a method for examining the gene expression profile of developmental CSPN by composing several techniques such as retrograde labeling, LCMD, RNA amplification, microarray screening and qPCR.