Hansen T R, Imakawa K, Polites H G, Marotti K R, Anthony R V, Roberts R M
Department of Animal Science, University of Missouri, Columbia 65211.
J Biol Chem. 1988 Sep 15;263(26):12801-4.
Ovine trophoblast protein-1 (oTP-1), an interferon of embryonic origin, is produced during the peri-implantation period of early pregnancy. Secretion of oTP-1 is detectable between days 13 and 21, but not beyond. In this study, the levels of oTP-1 mRNA in embryos were analyzed to determine if they reflected the transient nature of oTP-1 production. Total cellular RNA (tcRNA) was isolated from embryos representing day 12 (n = 5), 14 (n = 7), 16 (n = 5), 18 (n = 6), 20 (n = 4), and 22 (n = 5) of pregnancy and spotted on nylon membranes. Complementary RNA was transcribed from a specific oTP-1 cDNA (550 base pairs) template and applied (16-1000 pg) to nylon membranes to develop a standard curve. The fixed RNA samples were then allowed to hybridize with the 32P-labeled oTP-1 cDNA. oTP-1 mRNA was not detectable on day 12, increased to high levels (3.6 +/- 1.6 ng/ug of embryo DNA) on day 14, decreased about 5-fold by day 16, 15-fold by day 18, 170-fold by day 20, and 200-fold by day 22 of pregnancy. At day 14 oTP-1 mRNA comprised 0.060 +/- 0.019% of the tcRNA and was more abundant than actin mRNA. Northern analyses of pooled tcRNA representing each day of pregnancy showed that the oTP-1 probe hybridized to a single class of mRNA (approximately 1.1 kilobases) and confirmed the results obtained with dot blots.
绵羊滋养层蛋白-1(oTP-1)是一种胚胎来源的干扰素,在妊娠早期着床前后产生。在第13至21天之间可检测到oTP-1的分泌,但之后则无法检测到。在本研究中,分析了胚胎中oTP-1 mRNA的水平,以确定它们是否反映了oTP-1产生的短暂性。从代表妊娠第12天(n = 5)、14天(n = 7)、16天(n = 5)、18天(n = 6)、20天(n = 4)和22天(n = 5)的胚胎中分离出总细胞RNA(tcRNA),并点样于尼龙膜上。从特定的oTP-1 cDNA(550个碱基对)模板转录互补RNA,并将其(16 - 1000 pg)应用于尼龙膜以绘制标准曲线。然后让固定的RNA样品与32P标记的oTP-1 cDNA杂交。在第12天未检测到oTP-1 mRNA,在第14天增加到高水平(3.6 +/- 1.6 ng/μg胚胎DNA),到第16天下降约5倍,第18天下降15倍,第20天下降170倍,第22天下降200倍。在第14天,oTP-1 mRNA占tcRNA的0.060 +/- 0.019%,且比肌动蛋白mRNA更丰富。对代表妊娠每一天的合并tcRNA进行的Northern分析表明,oTP-1探针与一类单一的mRNA(约1.1千碱基)杂交,并证实了点杂交获得的结果。