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

摘要在日本发现了一株自发性矮鼠（SDR, dr），并对其进行了充分的鉴定。它们小而短，是由大鼠生长激素基因的第3内含子和第4外显子之间的剪接点突变引起的，这是我们和其他人发现的。它们为我们提供了一个机会来研究igf -1独立的生长激素对各种组织的作用，如脂肪和软骨。为了阐明生长激素对脂肪组织或软骨的不依赖于igf -1的直接作用，我们首先生成了人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kimeric基因。通过将Lep-hGH基因和冷冻精子注射到dr的卵母细胞中，我们成功地获得了一株靶向表达到脂肪组织的人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hGH。少量的生长激素基因在胃、肾上腺和肾脏中表达。Lep-hGH-dr显示体重（比对照组增加1.3-1.5倍）和体脂（比对照组增加140-150%）。Lep-hGH-dr的血清hGH水平通过超灵敏的EIA检测，为8.1-949.1pg/ml。常规细菌学检查发现转基因大鼠群体中存在肺支原体污染。包括Lep-hGH-dr在内的所有转基因大鼠均被处死。随后对Lep-hGH-dr进行生理和组织学分析，并终止α 1C-hGH-dr的产生。基于这些结果，我们认为人类瘦素基因上游-2.4kbp的启动子片段足以将hGH基因的表达靶向到脂肪组织。问题仍然没有解决，即脂肪中hGH基因表达量不足还是循环hGH水平低导致了意想不到的肥胖和Lep-hGH-dr的适度加速生长。

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.