Department of Animal Science, North Carolina State University, Raleigh, NC 27695, USA.
Mol Biotechnol. 2006 Jun;33(2):115-22. doi: 10.1385/MB:33:2:115.
We have devised a system for the study of in vivo gene correction based on the detection of color variants of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The intensity and spectra of the fluorescence emitted by the blue (BFP) and red-shifted (EGFP) variants of GFP differ from each other. We modified one nucleotide from an EGFP expression vector that we predicted would yield a blue variant (TAC-CAC, Tyr(66)-His(66)). Cells that were either transiently or stably transfected with the reporter system were used to test the functionality and feasibility of the detection of in vivo gene correction. A thio-protected single-stranded oligonucleotide designed to convert the genotype of the blue variant to that of the EGFP variant by the correction of a single base pair was delivered to the reporter cells using a variety of methodologies and strategies.Conversion events were easily observed using fluorescent microscopy because of the enhanced emission intensity and different spectra of the EGFP variant.
我们设计了一种基于水母 Aequorea victoria 的绿色荧光蛋白（GFP）颜色变体检测的体内基因校正研究系统。GFP 的蓝色（BFP）和红移（EGFP）变体发出的荧光强度和光谱彼此不同。我们从我们预测会产生蓝色变体的 EGFP 表达载体中修饰了一个核苷酸（TAC-CAC，Tyr（66）-His（66））。使用报告系统瞬时或稳定转染细胞的方法来测试体内基因校正检测的功能和可行性。设计了一种硫代保护的单链寡核苷酸，通过单个碱基对的校正将蓝色变体的基因型转换为 EGFP 变体的基因型，使用各种方法和策略将其递送到报告细胞中。由于 EGFP 变体的增强发射强度和不同光谱，转换事件很容易通过荧光显微镜观察到。
