大鼠脑组织中CNTF基因克隆及其探针制备

目的　克隆大鼠CNTF基因并制备地高辛标记的CNTF探针。方法　采用RT PCR法 ,从大鼠脑组织mRNA中扩增CNTF基因ORF区片段 ,克隆入T载体 ,并经序列测定。以地高辛素标记CNTF基因片段为探针 ,对成年大鼠脊髓组织切片进行原位杂交。结果　RT PCR法扩增出一特异产物与预期长度 6 16bp相符 ,序列测定与CNTF基因 10 0 %同源。原位杂交显示阳性信号出现在成年大鼠脊髓白质的前索及侧索的周边部分 ,为杵棒形和三角形带有纤细突起的胶质细胞。结论　采用RT PCR和T载体技术获得了大鼠脑组织中的CNTF基因克隆 ,地高辛标记的CNTF探针原位杂交显示正常大鼠脊髓白质的部分胶质细胞中表达CNTFmRNA。

Gene cloning of CNTF from rat brain and its probe labelled

Objective To develop the gene cloning of CNTF from rat brain and label its probe using digoxin.Methods The open reading frame of CNTF was amplified by RT PCR from rat brain.RT PCR product was ligated into pGEM T vector and the DNA sequence was obtained.The expression of CNTF mRNA in spinal cord of adult rat was examined by in situ hydridization with Dig CNTF probe.Results The product of RT PCR was the 616bp which matched the size of purpose.The sequence of CNTF was completely homogeneous with the CNIF sequence reported.Hybridized signals of CNTF mRNA were found in part of glial cells on the ventral white matter and surrounding of lateral white matter of spinal cord of adult rat.Positive glial cells showed oblong and triangular bodies with short thin projections.Conclusions CNTF gene has been cloned from rat brain by using RT CNTF probe.The CNTF mRNA expresses in part of glial cells in the white matter of spinal cord in adult rats.