Objective

Mice and humans with reduced growth hormone (GH) action before birth are conferred positive health- and life-span advantages. However, little work has been performed to study the effect of conditional disruption of GH action in adult life. With this as our objective, we sought to elucidate a reproducible protocol that allows generation of adult mice with a global disruption of the GH receptor (Ghr) gene, using the tamoxifen (TAM)-inducible Cre-lox system, driven by the ROSA26 enhancer/promoter. Here we report the optimum conditions for the gene disruption.

Design

Six month old mice, homozygous for the ROSA26-Cre and the Ghr-floxed gene, were injected, once daily for five days with four distinct TAM doses (from 0.08 to 0.32 mg of TAM/g of body weight). To evaluate the most effective TAM dose that leads to global disruption of the GHR, mRNA expression of the Ghr and insulin growth factor-1 (Igf1) genes were assessed in liver, adipose tissue, kidney, and skeletal and cardiac muscles of experimental and control mice. Additionally, serum GH and IGF-1 levels were evaluated one month after TAM injections in both, TAM-treated and TAM-untreated control mice.

Results

A dose of 0.25 mg of TAM/g of body weight was sufficient to significantly reduce the Ghr and Igf1 expression levels in the liver, fat, kidney, and skeletal and cardiac muscle of six-month old mice that are homozygous for the Ghr floxed gene and Cre recombinase. The reduction of the Ghr mRNA levels of the TAM-treated mice was variable between tissues, with liver and adipose tissue showing the lowest and skeletal and cardiac muscle the highest levels of Ghr gene expression when compared to control mice. Moreover, liver tissue showed the ‘best’ Ghr gene disruption, resulting in decreased total circulating IGF-1 levels while GH levels were increased versus control mice.

Conclusion

The results show that in mice at six months of age, a total TAM dose of at least 0.25 mg of TAM/g of body weight is needed for a global downregulation of Ghr gene expression with a regimen of 100 μL intraperitoneal (ip) TAM injections, once daily for five consecutive days. Furthermore, we found that even though this system does not achieve an equivalent disruption of the Ghr between tissues, the circulating IGF-1 is >95% decreased. This work helped to create adult mice with a global GHR knockdown.

中文翻译：

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使用 Cre-lox 系统处理小鼠生长激素受体基因破坏的标准化方案。

客观的

出生前生长激素 (GH) 作用降低的小鼠和人类具有积极的健康和寿命优势。然而，很少有人研究 GH 作用条件性中断对成人生活的影响。以此为我们的目标，我们试图阐明一种可重现的方案，该方案允许使用由 ROSA26 增强子驱动的他莫昔芬 (TAM) 诱导型 Cre-lox 系统生成 GH 受体 ( Ghr ) 基因全局破坏的成年小鼠/发起人。在这里，我们报告了基因破坏的最佳条件。

设计

对 ROSA26-Cre 和Ghr -floxed 基因纯合的六个月大小鼠进行注射，每天一次，持续五天，注射四种不同的 TAM 剂量（从 0.08 到 0.32 mg TAM/g 体重）。为了评估导致 GHR 全面破坏的最有效 TAM 剂量，在实验组和对照组的肝脏、脂肪组织、肾脏、骨骼肌和心肌中评估了Ghr和胰岛素生长因子 1 ( Igf1 ) 基因的 mRNA 表达老鼠。此外，在 TAM 治疗和未治疗的对照小鼠中注射 TAM 一个月后评估血清 GH 和 IGF-1 水平。

结果

剂量为 0.25 mg TAM/g 体重足以显着降低6 个月大的Ghr floxed 基因纯合小鼠的肝脏、脂肪、肾脏、骨骼肌和心肌中的Ghr和Igf1表达水平和 Cre 重组酶。经 TAM 处理的小鼠Ghr mRNA 水平的降低在不同组织之间存在差异，与对照小鼠相比，肝脏和脂肪组织的 Ghr 基因表达水平最低，而骨骼肌和心肌的Ghr基因表达水平最高。此外，肝组织显示出“最佳” Ghr基因破坏，导致总循环 IGF-1 水平降低，而 GH 水平与对照小鼠相比有所增加。

结论

结果表明，在 6 个月大的小鼠中，需要至少 0.25 mg TAM/g 体重的 TAM 总剂量才能通过 100 μL 腹膜内 (ip) TAM 注射方案全面下调Ghr基因表达，连续五天每天一次。此外，我们发现即使该系统没有实现组织间Ghr的等效破坏，循环中的 IGF-1 也减少了 >95%。这项工作有助于创造具有全球 GHR 击倒的成年小鼠。