Caelers Antje, Maclean Norman, Hwang Gyulin, Eppler Elisabeth, Reinecke Manfred
Division of Neuroendocrinology, Institute of Anatomy, University of Zürich, Zürich, Switzerland.
Transgenic Res. 2005 Feb;14(1):95-104. doi: 10.1007/s11248-004-5791-y.
We have previously produced transgenic fish from crosses between a wild-type female tilapia (Oreochromis niloticus) and a G transgenic male. This line of growth-enhanced tilapia carries a single copy of a chinook salmon (s) growth hormone (GH) gene spliced to an ocean pout antifreeze promoter (OPA-FPcsGH) co-ligated to a carp beta-actin/lacZ reporter gene construct, integrated into the tilapia genome. Because little is known about the expression sites of transgenes, we have characterised the gene expression patterns of sGH and tilapia (t)GH in transgenic tilapia using a newly established real-time PCR to measure the absolute mRNA amounts of both hormones. The sGH gene, which was expected to be expressed mainly in liver, was also found to be expressed in other organs, such as gills, heart, brain, skeletal muscle, kidney, spleen, intestine and testes. However, in pituitary no sGH mRNA but only tGH mRNA was found. Tilapia GH mRNA in wild-type pituitary amounted to 226 +/- 30 pg/microg total RNA but in transgenics only to 187 +/- 43 pg/microg total RNA. Liver exhibited the highest level of sGH mRNA (8.3 +/- 2.5 pg/microg total RNA) but the extrahepatic sites expressed considerable amounts of sGH mRNA ranging from 4.1 +/- 2.0 pg/microg total RNA in gills to 0.2 +/- 0.08 pg/microg total RNA in kidney. The widespread expression of the sGH gene is assumed to be due to the tissue specificity of the type III AFP gene promoter. It is assumed that our transgenic experiments, which in contrast to some other approaches caused no obvious organ abnormalities, mimick the GH expression during ontogeny. Because sGH mRNA is expressed both in liver and in extrahepatic sites it may not only promote secretion and release of liver-derived (endocrine) IGF-I leading to an overall growth enhancement but also stimulate IGF-I expression within the different organs in a paracrine/autocrine manner and, thus, further promote organ growth.
我们之前通过野生型雌性罗非鱼(尼罗罗非鱼)与转基因雄性罗非鱼杂交培育出了转基因鱼。这种生长增强型罗非鱼品系携带一个拼接至海洋鲽抗冻蛋白启动子(OPA - FPcsGH)的奇努克鲑(s)生长激素(GH)基因单拷贝,该启动子与鲤鱼β - 肌动蛋白/乳糖酶报告基因构建体共连接,并整合到罗非鱼基因组中。由于对转基因的表达位点了解甚少,我们使用新建立的实时PCR来测量两种激素的绝对mRNA量,从而对转基因罗非鱼中sGH和罗非鱼(t)GH的基因表达模式进行了表征。预期主要在肝脏中表达的sGH基因,也在其他器官中表达,如鳃、心脏、脑、骨骼肌、肾脏、脾脏、肠道和睾丸。然而,在垂体中未发现sGH mRNA,仅发现了tGH mRNA。野生型垂体中的罗非鱼GH mRNA总量为226±30 pg/μg总RNA,但转基因鱼中仅为187±43 pg/μg总RNA。肝脏中sGH mRNA水平最高(8.3±2.5 pg/μg总RNA),但肝外位点也表达了相当数量的sGH mRNA,范围从鳃中的4.1±2.0 pg/μg总RNA到肾脏中的0.2±0.08 pg/μg总RNA。sGH基因的广泛表达被认为是由于III型抗冻蛋白基因启动子的组织特异性。据推测,我们的转基因实验与其他一些方法不同,未引起明显的器官异常,模拟了个体发育过程中的GH表达。由于sGH mRNA在肝脏和肝外位点均有表达,它可能不仅促进肝脏来源的(内分泌)IGF - I的分泌和释放,导致整体生长增强,还可能以旁分泌/自分泌方式刺激不同器官内的IGF - I表达,从而进一步促进器官生长。