Patrício Maria I, MacLaren Robert E
Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.
Oxford Eye Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, UK.
Methods Mol Biol. 2018;1715:89-97. doi: 10.1007/978-1-4939-7522-8_7.
As gene therapy of choroideremia is becoming a clinical reality, there is a need for reliable and sensitive assays to determine the expression of exogenously delivered Rab Escort Protein-1 (REP1), in particular to test new gene therapy vectors and as a quality control screen for clinical vector stocks. Here we describe an in vitro protocol to test transgene expression following AAV2/2-REP1 transduction of a human cell line. Gene augmentation can be confirmed by western blot and quantification of the fold-increase of human REP1 levels over untransduced controls.
随着脉络膜视网膜炎的基因治疗逐渐成为临床现实，需要可靠且灵敏的检测方法来测定外源性递送的Rab护送蛋白1（REP1）的表达，特别是用于测试新的基因治疗载体以及作为临床载体储备的质量控制筛选。在此，我们描述了一种体外实验方案，用于检测人细胞系经AAV2/2-REP1转导后的转基因表达。基因增强可通过蛋白质免疫印迹法以及对人REP1水平相对于未转导对照的增加倍数进行定量来确认。
