Targeted expression of human growth hormone gene to the fat or to the cartilage in spontaneous dwarf rats.

在日本发现了一种自发性侏儒大鼠（SDR，dr），并对其进行了充分的鉴定。它们是由我们等鉴定的大鼠GH基因第三内含子和第四外显子之间的剪接点突变引起的。他们为我们提供了一个机会，研究IGF-1的非依赖性生长激素的行动，以各种组织，如脂肪和软骨。为了阐明生长激素对脂肪组织或软骨的直接作用，我们首先构建了人GH基因（2.1kbp）与瘦素基因启动子（-3.5kbp，Lep-hGH：5.7kbp）或人α 1-胶原基因启动子（-5.2kbp，α 1C-hGH：7.3kbp）偶联的嵌合基因。通过将Lep-hGH基因和冷冻精子注射到dr的卵母细胞中，成功地获得了脂肪组织靶向表达的人GH转基因dr（Lep-hGH-dr）。Lep-hGH-dr不仅在脂肪组织中表达hGH，而且在睾丸或卵巢中也表达。少量的hGH基因在胃、肾上腺和肾脏中表达。Lep-hGH-dr显示体重（与对照dr相比，1.3-1.5倍）和体脂（与对照dr相比，140- 150%）均增加。用超灵敏的酶免疫法检测Lep-hGH-dr的血清hGH浓度为8.1-949.1pg/ml。在转基因大鼠群体中的肺结核。处死所有转基因大鼠，包括Lep-hGH-dr。随后对Lep-hGH-dr进行了生理学和组织学分析，并终止了α 1C-hGH-dr的产生，由此我们得出结论：人leptin基因上游启动子-2.4kbp片段足以使hGH基因靶向脂肪组织表达。这些问题仍然没有解决，即，脂肪中hGH基因表达量不足或循环hGH水平低是否会导致Lep-hGH-dr的意外肥胖和适度生长加速。

A strain of spontaneous dwarf rats (SDR, dr) is discovered and fully characterized in Japan. They are small and short caused by a point mutation at the splice junction between the third intron and the forth exon of the rat GH gene, which was identified by us and others. They provide us an opportunity to study the IGF-1-independent GH actions to various tissues, such as fat and cartilage. In order to elucidate the IGF-1-independent direct actions of growth hormone to the fat tissue or cartilage, we first generated kimeric genes of the human GH gene (2.1kbp) coupled with leptin gene promoter (-3.5kbp, Lep-hGH : 5.7kbp) or human alpha 1-collagne gene promoter (-5.2kbp, α 1C-hGH : 7.3kbp). By injecting the Lep-hGH gene and frozen sperm into oocytes of dr, we successfully generated a line of human GH-transgenic dr with targeted expression to the fat tissue (Lep-hGH-dr). Lep-hGH-dr expressed hGH not only in fat tissues, but also in testes or ovaries. Small amounts of the hGH gene expressed in the stomach, adrenal and kidney. Lep-hGH-dr showed increased both in weight (1.3-1.5 times, vs. control dr) and body fat (140-150%, vs. control dr). Serum hGH levels in Lep-hGH-dr were detectable by an ultrasensitive EIA, 8.1-949.1pg/ml.Routine bacteriological examination detected contamination with M. pulmonis in the transgenic rat colony. All of the transgenic rats, including Lep-hGH-dr, were sacrificed. Subsequent physiological and histological analyses of Lep-hGH-dr, and generation of α 1C-hGH-dr were both terminated.Based on these results, we conclude that the segment of -2.4kbp upstream promoter of human leptin gene is sufficient to target the hGH gene expression to the fat tissue. The questions have remained unsolved, i.e., whether insufficient amount of expressed hGH gene in the fat or low levels of circulating hGH resulted in unexpected obesity and modest acceleration of growth in Lep-hGH-dr.