地高辛标记Ucp2基因RNA探针的制备和应用

The Preparation and Application of the Ucp2 Gene RNA Probe Labeled by Digoxin

目的：制备用于检测小鼠胚胎早期Ucp2基因表达的地高辛标记的特异性RNA探针。方法：提取小鼠胚胎脑组织总RNA,设计引物,通过RT-PCR方法获取Ucp2基因片段,将其克隆到pGEM-T载体。分别利用Sp6、T7和Ucp2特异性引物,PCR扩增获得转录模板,通过Sp6及T7 RNA聚合酶,获得地高辛标记的正义、反义Ucp2 RNA原位杂交探针。检测标记探针的效价后,通过全胚胎原位杂交分析制备探针的特异性和杂交效果。结果：成功获得Ucp2基因正义、反义探针,反义探针能高效灵敏检测到Ucp2基因在小鼠胚胎Ed9.5、Ed10.5神经系统呈现高表达,而正义探针未能检测到表达信号。结论：成功制备了特异高效的地高辛标记Ucp2 RNA原位杂交探针,为进一步研究Ucp2基因在小鼠胚胎组织中的表达,尤其在神经组织的定位奠定基础。

Objective： To prepare the Ucp2 gene RNA probe labeled by digoxin,and was used to detect the early Ucp2 gene spatiotemporal expression in mouse embryos. Methods： The primers were designed and the total RNA was extracted from fetal mice nervous tissue. The Ucp2 gene fragment was obtained by RT-PCR and cloned into the p GEM-T vector. Then transcription template was obtained using Sp6,T7 and Ucp2 primers by PCR. Using Sp6 and T7 RNA polymerase,the digoxingenin-labeled antisense and sense probes were synthesized and confirmed by whole embryo in situ hybridization. Results： The plasmid p GEMT-UCP2 was constructed. The digoxingenin-labeled antisense Ucp2 probe was effectively detected in mice Ed9. 5 and Ed10. 5,and the expression was not detected by the sense probe. Conclusion： The antisense Ucp2 probe can be used specifically and sensitively for study in situ hybridization and lay the foundation for further research of Ucp2 expression in the tissue of mouse embryo,especially in the nerve tissue.