Maruyama H, Higuchi N, Nishikawa Y, Kameda S, Iino N, Kazama J J, Takahashi N, Sugawa M, Hanawa H, Tada N, Miyazaki J, Gejyo F
Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Niigata 951-8120, Japan.
J Gene Med. 2002 May-Jun;4(3):333-41. doi: 10.1002/jgm.281.
High levels of foreign gene expression in mouse hepatocytes can be achieved by rapid tail vein injection of a large volume of a naked DNA solution, the 'hydrodynamics-based procedure'. Rats are more tolerant of the frequent phlebotomies required for monitoring blood parameters than mice, and thus are better for some biomedical research.
We tested this technique for the delivery of a therapeutic protein in normal rats, using a rat erythropoietin (Epo) expression plasmid vector, pCAGGS-Epo.
We obtained maximal Epo expression when the DNA solution was injected in a volume of 25 ml (approximately 100 ml/kg body weight) within 15 s. We observed a dose-response relationship between serum Epo levels and the amount of injected DNA up to 800 microg. Using quantitative real-time PCR, the vector-derived Epo mRNA expression was mainly detected in the liver. When a lacZ expression plasmid was injected similarly, beta-galactosidase was exclusively detected in the liver, mainly in hepatocytes. Toxicity attributable to the technique was mild and transient, as assessed by histochemical analysis. Epo gene expression and erythropoiesis occurred with Epo gene transfer in a dose-dependent manner, and persisted for at least 12 weeks, the last time point examined. Repeated administration of the plasmid DNA also effectively led to erythropoiesis.
These results demonstrate that gene transfer into the liver via rapid tail vein injection can easily be achieved in the rat, which is more than 10 times larger than the mouse, and has significant value for gene function analysis in rats.
通过快速尾静脉注射大量裸DNA溶液（即“基于流体动力学的方法”），可在小鼠肝细胞中实现高水平的外源基因表达。与小鼠相比，大鼠对监测血液参数所需的频繁采血更耐受，因此更适合某些生物医学研究。
我们使用大鼠促红细胞生成素（Epo）表达质粒载体pCAGGS-Epo，在正常大鼠中测试了这种用于递送治疗性蛋白质的技术。
当在15秒内注射25毫升（约100毫升/千克体重）的DNA溶液时，我们获得了最大的Epo表达。我们观察到血清Epo水平与注射的DNA量之间存在剂量反应关系，直至800微克。使用定量实时PCR，载体衍生的Epo mRNA表达主要在肝脏中检测到。当以类似方式注射lacZ表达质粒时，β-半乳糖苷酶仅在肝脏中检测到，主要在肝细胞中。通过组织化学分析评估，该技术引起的毒性轻微且短暂。Epo基因表达和红细胞生成以剂量依赖的方式随Epo基因转移而发生，并持续至少12周，即检查的最后一个时间点。重复施用质粒DNA也有效地导致了红细胞生成。
这些结果表明，通过快速尾静脉注射将基因导入肝脏在大鼠中很容易实现，大鼠比小鼠大10倍以上，这对于大鼠的基因功能分析具有重要价值。
