Brooks A I, Halterman M W, Chadwick C A, Davidson B L, Haak-Frendscho M, Radel C, Porter C, Federoff H J
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, NY 14642, USA.
J Neurosci Methods. 1998 Apr 30;80(2):137-47. doi: 10.1016/s0165-0270(97)00207-0.
To develop a reproducible gene transfer method for the murine CNS we evaluated delivery of various gene vehicles using mechanical or manual stereotaxic intracranial inoculation. A microprocessor controlled microsyringe pump (The World Precision Instruments/UltraMicroPump) programmable for volume, rate and syringe size and designed to dispense nanoliter and picoliter volumes was compared to a standard manual deliver method. Gene transfer efficiency of two viral vectors, two synthetic cationic lipid molecules, and naked DNA were evaluated in mice injected unilaterally in two brain regions. Animals received 1 microl over 10 min. of either HSVlac (1 x 10(5) b.f.u), AdLac (1 x 10(5) p.f.u), Tfx-10 or Tfx-20 (2.6 microg DNA in 2.0 microl Tfx; 1:1 charge ratio of DNA to liposome), or naked DNA (HSVlac plasmid, 10 microg/microl). After 4 days, animals from each group were perfused and tissue prepared for X-gal histochemical detection of beta-galactosidase expression. Blue cells were observed in the HSV, Adenovirus, and Tfx-20 groups only at the injection site in animals injected using the UMP. Animals injected manually exhibited fewer blue cells and positive cells were not restricted to the injection site. To quantify expression, tissue punches harvested from the injection sites as well as other brain regions were analyzed using a chemiluminescent reporter assay to detect beta-galactosidase (Galacto-Light). These data indicated increased activity in all animals injected with a lacZ containing vector via the UMP as compared to manual delivery: A 41% increase in the expression levels of beta-gal in HSVlac infected animals (p = 0.0029); a 29% increase in Adlac infected animals (p = 0.01); a 56% increase in Tfx-10 transduced animals (p = 0.04); a 24% increase in Tfx-20 transduced animals (p = 0.01); and a 69% increase in naked DNA gene transfer (p = 0.05). Total beta-galactosidase activity was greatest in HSVlac infected mice followed by Adlac > Tfx-20 > Tfx-10 = naked DNA.
为开发一种可重复的小鼠中枢神经系统基因转移方法，我们使用机械或手动立体定向颅内接种评估了各种基因载体的递送情况。将一种可通过微处理器控制的、可编程设置体积、速率和注射器规格、设计用于分配纳升和皮升体积的微型注射器泵（世界精密仪器公司/超微量泵）与标准手动递送方法进行了比较。在单侧注射到两个脑区的小鼠中评估了两种病毒载体、两种合成阳离子脂质分子和裸DNA的基因转移效率。动物在10分钟内接受1微升的以下物质注射：单纯疱疹病毒 - 乳糖酶基因（HSVlac，1×10⁵ 噬斑形成单位）、腺病毒 - 乳糖酶基因（AdLac，1×10⁵ 空斑形成单位）、Tfx - 10或Tfx - 20（2.6微克DNA溶于2.0微升Tfx中；DNA与脂质体电荷比为1:1），或裸DNA（HSVlac质粒，10微克/微升）。4天后，对每组动物进行灌注，并制备组织用于X - 半乳糖苷酶表达的X - gal组织化学检测。仅在使用超微量泵注射的动物中，于注射部位在HSV、腺病毒和Tfx - 20组中观察到蓝色细胞。手动注射的动物中蓝色细胞较少，且阳性细胞不限于注射部位。为了量化表达，使用化学发光报告基因检测法（Galacto - Light）分析从注射部位以及其他脑区采集的组织小块，以检测β - 半乳糖苷酶。这些数据表明，与手动递送相比，通过超微量泵注射含lacZ载体的所有动物的活性均有所增加：HSVlac感染动物中β - gal表达水平增加41%（p = 0.0029）；Adlac感染动物中增加29%（p = 0.01）；Tfx - 10转导动物中增加56%（p = 0.04）；Tfx - 20转导动物中增加24%（p = 0.01）；裸DNA基因转移增加69%（p = 0.05）。总β - 半乳糖苷酶活性在HSVlac感染的小鼠中最高，其次是Adlac＞Tfx - 20＞Tfx - 10 = 裸DNA。
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