肺缺陷的体细胞核移植牛TTF-1 mRNA的低表达与启动子区CpG的甲基化无关

Low mRNA expression of thyroid-specific transcription factor 1 gene is not associated with promoter CpG methylation in cloned cattle with defective lung symptom

通过体细胞核移植技术获得克隆哺乳动物的种类在不断增加,但成功率仍然较低,常表现为围产期的高死亡率.其中,肺发育异常是最常见的发育缺陷之一.哺乳动物肺器官的发生受一系列蛋白因子的调控,其中隶属于同源结构域转录因子家族的TTF-1参与肺的发生和出生后功能的维持,是一种重要的转录因子,其表达水平的异常很可能与肺的发育缺陷有关.SCNT胚胎异常的甲基化可能影响发育关键基因的表达.本研究克隆了牛TTF-1基因,包括启动子区(921bp)、外显子1(373bp)、内含子1(932bp)和外显子2的一部分(273bp)在内约2.5kb的核苷酸序列,发现3头肺发育有缺陷的SCNT牛TTF-1mRNA的表达量均显著低于肺发育正常的SCNT牛及对照牛(P<0.05).对表达量最低的个体和对照牛肺组织TTF-1基因启动子区63个CpG位点的甲基化状态进行了分析,发现在大多数位点上,二者均处于去甲基化状态,甲基化百分比差异不显著(P>0.05),说明肺发育缺陷的体细胞核移植牛TTF-1mRNA的低表达与启动子区CpG的甲基化无关.

Although more and more mammalian species are cloned by using somatic cell nuclear transfer （SCNT） approach,the success rate is low.Most cloned embryos die during post-implantation development,and those that survive to term are frequently defective.In cloned cattle,various developmental defects were revealed,of which,respiratory problem is one of the commonly found anomalies.Lung morphogenesis in mammals is regulated by a series of protein factors.Thyroid-specific transcription factor 1 （TTF-1） participates the branching of lobar bronchi and alveolarization process,and co-regulates the expression of pulmonary surfactant proteins,therefore,is essential for the lung to perform normal functions.The aberrant expression of TTF-1 likely affects lung development and the abnormal methylation in SCNT embryos likely affects the expression of developmentally important genes.In present study,the bovine TTF-1 genomic sequence of ~2.5 kb was obtained,including 921 bp of TTF-1 promoter region,373 bp of exon 1932 bp of intron 1 and 273 bp of partial exon 2.The relative mRNA levels of TTF-1 expression in 3 cloned cattle with defective lung symptom were much lower than that of cloned cattle with normal lung structure and of the two controls （N1 and N2） （P〈0.05）.Comparison of the methylation level of 63 CpG dinucleotides in TTF-1 promoter region of C2,which has the lowest TTF-1 expression,and N1 revealed that most CpG dinucleotides were unmethylated,the CpG methylation percentage was not significantly different （P〉0.05）.The results indicated that,in the lung tissues of SCNT cattle with defective lung symptom,the decreased expression of TTF-1 mRNA was not associated with promoter CpG methylation.