Zhang W, Feng J Q, Harris S E, Contag P R, Stevenson D K, Contag C H
Department of Pediatrics, Stanford University Medical Center, Stanford University, CA 94305-5208, USA.
Transgenic Res. 2001 Oct;10(5):423-34. doi: 10.1023/a:1012042506002.
The use of transgenic animals in biomedical research is increasing rapidly and may be the best means of determining gene function. Generating transgenic animals typically requires time-consuming screening processes, and gene function is assessed by an array of difficult phenotypic and biochemical assays performed ex vivo. To address the unmet need in transgenic research for functional assays performed with ease in living animals, we demonstrate here that in vivo detection of luciferase enzyme as a transcriptional reporter facilitates rapid screening for both the presence and function of transgenes in intact living mice. Using this approach we identified three bioluminescent transgenic founders where the transgene consisted of the heme oxygenase promoter fused to the modified coding sequence of the luciferase gene. These founders were identified from 183 pups and confirmed by PCR analysis. Identification of HO-1-luc homozygotes from back- crossed F2 littermates was then accelerated by in vivo imaging. In another transgenic mouse line, where the transgene was comprised of the bone morphogenic-4 (BMP4) promoter fused to the modified luciferase gene, we were able to identify transgenic animals and in each line we were able to visualize patterns of expression in living animals over time. The light production from these transgenic mice indicated that the desired DNA fragment was functional and different expression profiles apparent at different ages and after gene induction.
转基因动物在生物医学研究中的应用正在迅速增加，可能是确定基因功能的最佳手段。生成转基因动物通常需要耗时的筛选过程，并且基因功能是通过一系列在体外进行的困难的表型和生化分析来评估的。为了解决转基因研究中对在活体动物中轻松进行功能分析的未满足需求，我们在此证明，作为转录报告基因的荧光素酶的体内检测有助于在完整的活体小鼠中快速筛选转基因的存在和功能。使用这种方法，我们鉴定出三只生物发光转基因奠基动物，其转基因由与荧光素酶基因的修饰编码序列融合的血红素加氧酶启动子组成。这些奠基动物是从183只幼崽中鉴定出来的，并通过PCR分析得到证实。然后通过体内成像加速了从回交F2同窝仔中鉴定HO-1-luc纯合子的过程。在另一个转基因小鼠品系中，其转基因由与修饰的荧光素酶基因融合的骨形态发生蛋白4（BMP4）启动子组成，我们能够鉴定转基因动物，并且在每个品系中我们都能够随着时间推移观察活体动物中的表达模式。这些转基因小鼠发出的光表明所需的DNA片段具有功能，并且在不同年龄和基因诱导后有明显不同的表达谱。
