【摘要】 为制备地高辛标记的小鼠雌激素受体α(ERα)RNA探针,用其研究ERα在小鼠胚胎发育过程中的表达。通过RT-PCR,获得ERα基因片段,构建ERα/pGEM-3Z重组质粒,分别用HindⅢ和EcoRⅠ进行酶切得到线性化DNA片段,以T7和Sp6聚合酶合成地高辛标记的正反义RNA探针,然后通过胚胎整体原位杂交技术分析ERα在小鼠胚胎中的表达。结果构建了ERα/pGEM-3Z质粒,获得高效价的正反义dig-ERαRNA探针。运用该探针检测到ERα在10.5dpc胚胎的前脑、脊神经管、生殖嵴、肢芽及颌弓中表达,在13.5dpc胚胎的端脑、中脑、脊髓、肢芽及生殖系统中表达,为进一步研究ERα在小鼠胚胎发育中的功能打下了基础。更多还原

【Abstract】 Objective:In order to study the expression of estrogen receptor-α(ERα)in mouse embryo,the sense and anti-sense RNA probes for hybridization of ERα were prepared.Methods:The ERα fragment which was obtained by RT-PCR was subcloned into plasmids pGEM-3Z,and then the recombinant plasmids ERα/pGEM-3Z were linearized with the restriction enzymes,Hind Ⅲ and EcoRⅠ.Using T7 and Sp6 RNA polymerase,respectively,the digoxigenin(dig)labeled sense and antisense probes were transcribed in vitro.Then the expression of ERα in mouse embryo was examined with the probes by whole mount in situ hybridization.Results:The gene fragment of ERα was cloned and the cRNA probes were prepared with digoxigenin-labeled.ERα gene was expressed in the forebrain,spinal neural tube,genital ridge,limb bud,mandibular arch of 10.5 dpc embryo and was also expressed in the telencephalon,mesencephalon,spinal cord,limb bud,and reproductive system of 13.5 dpc embryo.更多还原