McClarrinon M, Gilkey L, Watral V, Fox B, Bullock C, Fradkin L, Liggitt D, Roche L, Bussey L B, Fox E, Gorman C
Valentis, Inc., Burlingame, California 94010, USA.
DNA Cell Biol. 1999 Jul;18(7):533-47. doi: 10.1089/104454999315088.
As the sequencing of the human genome proceeds, the need for a new screen for in vivo function is becoming apparent. Many investigators are turning to various transgenic models as a means of studying function. However, these approaches are very time consuming, with a transgene-expressing mouse model often taking months to establish. We have developed an efficient system for delivering genes in vivo, which allows the gene product to be studied as early as 24 h after introduction into the mouse model. The delivery system employs a novel cationic lipid, 1-[2-(9-(Z)-octadecenoyloxy)ethyl]-2-(8-(Z)-heptadecenyl)-3- (hydroxyethyl)imidazolinium chloride (DOTIM), and a neutral lipid, cholesterol, complexed with an expression vector containing the reporter gene chloramphenicol acetyl transferase (CAT). After a single intravenous injection of these complexes, several tissues were seen to express the transgene. High, persistent expression in the vascular endothelial cells in the mouse lung was obtained. Delivery of DNA in vivo has been evaluated by quantitative polymerase chain reaction and protein expression by CAT activity assays. In vivo studies showed reproducible expression in more than 500 mice injected via the tail vein. An early peak of expression was followed by lower, but sustained, expression for > 50 days. Transgene expression of CAT could also be identified by immunohistochemistry staining in mouse lung and appeared to be located within the capillaries. The pattern of in vivo expression could be modulated and targeted to specific organs by altering the lipid-DNA formulation. New expression vectors with altered introns and polyadenylation sites further improved expression. The expression reported here may be sufficient in magnitude, duration, and flexibility to be an attractive alternative, in some cases, to establishing transgenic animals by stable gene transfer.
随着人类基因组测序工作的推进，对一种用于体内功能新筛选方法的需求日益明显。许多研究人员正在转向各种转基因模型作为研究功能的手段。然而，这些方法非常耗时，构建一个表达转基因的小鼠模型通常需要数月时间。我们开发了一种在体内递送基因的高效系统，该系统能使基因产物在引入小鼠模型后最早24小时就可进行研究。该递送系统采用一种新型阳离子脂质1-[2-(9-(Z)-十八碳烯酰氧基)乙基]-2-(8-(Z)-十七碳烯基)-3-(羟乙基)咪唑啉氯化物（DOTIM）和一种中性脂质胆固醇，它们与含有报告基因氯霉素乙酰转移酶（CAT）的表达载体复合。单次静脉注射这些复合物后，可见多个组织表达转基因。在小鼠肺血管内皮细胞中获得了高水平的持续表达。通过定量聚合酶链反应评估体内DNA递送情况，并通过CAT活性测定评估蛋白质表达。体内研究表明，通过尾静脉注射的500多只小鼠中均出现了可重复的表达。表达先出现一个早期高峰，随后是较低但持续的表达，持续时间超过50天。通过免疫组织化学染色也可在小鼠肺中鉴定出CAT的转基因表达，且其似乎位于毛细血管内。通过改变脂质-DNA配方，可调节体内表达模式并使其靶向特定器官。具有改变的内含子和聚腺苷酸化位点的新表达载体进一步提高了表达水平。本文报道的表达在强度、持续时间和灵活性方面可能足以在某些情况下成为通过稳定基因转移建立转基因动物的一种有吸引力的替代方法。
