Characterization of plasmid DNA transfer into mouse skeletal muscle: evaluation of uptake mechanism, expression and secretion of gene products into blood.
Characterization of plasmid DNA transfer into mouse skeletal muscle: evaluation of uptake mechanism, expression and secretion of gene products into blood.
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发表时间：
1996-03
期刊：
影响因子：
5.1
通讯作者：
Levy My;L. G. Barron;Meyer Kb;Francis C. Szoka
中科院分区：
文献类型：
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作者：
Levy My;L. G. Barron;Meyer Kb;Francis C. Szoka
用一种改进的肌肉注射技术研究了萤火虫荧光素酶(PRSVluc)或分泌蛋白人α1抗胰蛋白酶(PRcCMVhAAT)的裸质粒DNA在小鼠骨骼肌中的表达。在沿小鼠股四头肌纵轴、平行于肌纤维的强光引导下，注射产生的荧光素酶表达水平是垂直注射的200倍。荧光素酶的表达被过量的非编码脱氧核糖核酸或葡聚糖硫酸盐抑制，这表明肌肉脱氧核糖核酸摄取机制(S)可以饱和。放射性标记的HAAT质粒的组织分布研究和Southern分析证明，注射的质粒DNA可以迅速从肌肉中清除。然而，聚合酶链式反应分析表明，HAAT基因在肌肉中持续存在至少1个月。免疫组织化学技术表明，HAAT基因在肌纤维中表达。对肌肉注射小鼠血清样本的ELISA分析表明，分泌的Haat蛋白浓度在注射后第7天达到峰值，第14天开始下降，第21天几乎检测不到。RT-PCR分析显示，HAAT转录本在注射部位持续存在至少1个月，提示注射后21d血清HAAT浓度下降不是由于HAAT转录本缺失所致。然而，血清中HAAT蛋白浓度的下降与循环中小鼠抗HAAT抗体的积累呈负相关。
The expression of naked plasmid DNA coding for firefly luciferase (pRSVluc) or a secreted protein, human-alpha-1-antitrypsin (pRcCMVhAAT) in mouse skeletal muscle was characterized following administration by an improved intramuscular injection technique. Injection guided by intense illumination along the longitudinal axis of the mouse quadriceps muscle and parallel to the myofibers yielded 200-fold higher levels of luciferase expression than perpendicular injection. Luciferase expression was inhibited by an excess of non-coding DNA or dextran sulfate suggesting that muscle DNA uptake mechanism(s) can be saturated. Injected plasmid DNA was rapidly eliminated from the muscle as evidenced by tissue distribution studies of radiolabeled hAAT plasmid and Southern analysis. However, PCR analysis demonstrated that hAAT cDNA persisted in the muscle for at least 1 month after injection. Immunohistochemistry techniques indicated that the hAAT gene was expressed by the muscle fibers. ELISA analysis of serum samples collected from intramuscularly injected mice demonstrated that secreted hAAT protein concentration peaked in serum by day 7, started to decline by day 14 and was barely detectable 21 days post-injection. RT-PCR analysis demonstrated that hAAT transcript persisted at the site of injection for at least 1 month indicating that the decline of serum hAAT concentration 21 days post-injection was not due to the absence of hAAT transcript. However, the decline of hAAT protein concentration in the serum was inversely correlated with accumulation of murine anti-hAAT antibodies in circulation.