Tonheim Tom Christian, Dalmo Roy Ambli, Bøgwald Jarl, Seternes Tore
Department of Marine Biotechnology, The Norwegian College of Fishery Science, University of Tromsø, N-9037 Tromsø, Norway.
Fish Shellfish Immunol. 2008 Jan;24(1):90-101. doi: 10.1016/j.fsi.2007.09.006. Epub 2007 Oct 5.
In this study we investigated tissue distribution of pDNA after intramuscular and intravenous administration, cellular localisation, receptor-specific uptake, integrity of pDNA and transgene expression in Atlantic salmon (Salmo salar L). Anatomical distribution of plasmid DNA was determined using both radiotracing and fluorescence microscopy. Cellular uptake was studied in cultures of adherent anterior kidney leucocytes. The integrity of the pDNA in vivo was investigated by Southern blot analysis. Transcription of plasmid DNA encoded luciferase gene and protein synthesis were investigated in salmon tissues by means of real-time reverse transcription-polymerase chain reaction and enzyme activity measurements, respectively. Approximately 50% of the total recovered radioactivity was redistributed from the carcass 168h after intramuscular administration and accumulated mainly in the kidneys (37% of total). The majority of radiolabelled plasmid DNA administered intravenously was taken up within the first 15min mainly by the kidney. Intravenous co-administration of trace amounts of radiolabelled plasmid DNA with excess amounts of unlabelled plasmid DNA or formaldehyde treated albumin (a ligand for the scavenger receptors) significantly inhibited accumulation of the radiotracer in the kidney. Fluorescence microscopy demonstrated that fluorescence was localised intracellularly in cells lining the sinusoids of the kidney after intravenous administration of rhodamine-labelled plasmid DNA. Southern blot analysis demonstrated presence of supercoiled plasmid DNA in all organs and tissue samples 168h after intramuscular administration, but degradation products were only revealed at the administration site. Luciferase transcript and activity were only detectable at the administration site 24-168h after intramuscular administration of plasmid DNA. After incubation with trace amounts of radiolabelled plasmid DNA, only minor amounts of radiolabelled plasmid DNA were cell associated in cultures of adherent anterior kidney leucocytes. These results suggested that a substantial portion of radiolabelled plasmid DNA was redistributed from the carcass and was mainly cleared by a receptor-specific uptake in the kidney. Although intact plasmid DNA was detected in the kidney and other tissues, no luciferase transcripts or activity were detected in these samples at any time points investigated (24-168h), except for the administration site following intramuscular administration.
在本研究中，我们调查了肌内和静脉注射后质粒DNA（pDNA）在大西洋鲑（Salmo salar L）体内的组织分布、细胞定位、受体特异性摄取、pDNA完整性及转基因表达情况。使用放射性示踪和荧光显微镜确定质粒DNA的解剖分布。在贴壁前肾白细胞培养物中研究细胞摄取。通过Southern印迹分析研究体内pDNA的完整性。分别通过实时逆转录-聚合酶链反应和酶活性测量，在鲑鱼组织中研究质粒DNA编码的荧光素酶基因的转录和蛋白质合成。肌内注射后168小时，约50%回收的总放射性从鱼体重新分布，主要积聚在肾脏（占总量的37%）。静脉注射的大多数放射性标记质粒DNA在最初15分钟内主要被肾脏摄取。静脉共同注射微量放射性标记质粒DNA与过量未标记质粒DNA或甲醛处理的白蛋白（清道夫受体的配体）可显著抑制放射性示踪剂在肾脏中的积聚。荧光显微镜显示，静脉注射罗丹明标记的质粒DNA后，荧光定位于肾脏血窦内衬细胞的细胞内。Southern印迹分析表明，肌内注射后168小时，所有器官和组织样本中均存在超螺旋质粒DNA，但仅在给药部位发现降解产物。肌内注射质粒DNA后24 - 168小时，仅在给药部位可检测到荧光素酶转录本和活性。用微量放射性标记质粒DNA孵育后，贴壁前肾白细胞培养物中仅少量放射性标记质粒DNA与细胞相关。这些结果表明，相当一部分放射性标记质粒DNA从鱼体重新分布，主要通过肾脏中的受体特异性摄取清除。尽管在肾脏和其他组织中检测到完整的质粒DNA，但在任何研究时间点（24 - 168小时），除肌内注射后的给药部位外，这些样本中均未检测到荧光素酶转录本或活性。
