Kuzbanian基因非放射性原位杂交探针制备

Preparation of non-radiolabeled in situ hybridization probes of Kuzbanian gene

目的研究制备地高辛(DIG)标记的Kuzbanian(KUZ)基因非放射性原位杂交探针,用于检测转基因动物、正常组织KUZ基因的表达。方法用体外转录法依自构建质粒pcDNA-KUZFL(质粒:6.0kb;探针:623bp)和pcDNA-KUZDN(质粒:5.8kb;探针:360bp)制备反义链与正义链DIG标记cRNA探针。结果用转基因模型阳性与阴性对照品系动物小鼠胰岛组织非放射性原位杂交实验,证实制备探针具有较高的特异性和敏感性。结论DIG标记KUZ基因杂交探针的成功制备,为进一步研究有关转基因动物的KUZ基因的分布和表达以及为正常组织KUZ基因的有关研究提供了实验基础。

Objective To prepare digoxin （DIG） non-radiolabeled in situ hybridization cRNA probes of Kuzbanian （KUZ） gene in order to detect KUZ gene expression in the trans-gene animal and normal tissue. Methods The plasmid pcDNA-KUZFL（plasmid, 6.0 kb; probe, 623 bp）and pcDNA-KUZDN（plasmid, 5.8 kb; probe, 360 bp） were prepared in Sherwin＇s lab by amplification and purification of plasmid＂ DNA. The four cRNA probes of sense and antisense KUZ were successfully prepared by in vitro transcription of DNA with DIG-labeling methods. Results Nonradioactivity in situ hybridization studies on the expression of KUZ gene in trans-gene mice pancreas islet tissue showed that the sensitivity and specificity of the probes was relatively high. Con- clusion The successful preparation of KUZ gene DIG labeled probes provides an experimental foundation for detecting the distribution, expression of KUZ gene in the trans-gene animal and other related subjects studied.