Derouazi Madiha, Flaction Rachel, Girard Philippe, de Jesus Maria, Jordan Martin, Wurm Florian M
Laboratory of Cellular Biotechnology, Institute of Biological Engineering and Biotechnology, Ecole Polytechnique Fédérale de Lausanne, Lausanne, CH-1015, Switzerland.
Biotechnol Lett. 2006 Mar;28(6):373-82. doi: 10.1007/s10529-005-6062-6.
Microinjection is a gene transfer technique enabling partial control of plasmid delivery into the nucleus or cytoplasm of cultured animal cells. Here this method was used to establish various recombinant mammalian cell lines. The injection volume was estimated by fluorescence quantification of injected fluorescein isothyocynate (FITC)-dextran. The DNA concentration and injection pressure were then optimized for microinjection into the nucleus or cytoplasm using a reporter plasmid encoding the green fluorescent protein (GFP). Nuclear microinjection was more sensitive to changes in these two parameters than was cytoplasmic microinjection. Under optimal conditions, 80-90% of the cells were GFP-positive 1 day after microinjection into the nucleus or the cytoplasm. Recombinant cell lines were recovered following microinjection or calcium phosphate transfection and analyzed for the level and stability of recombinant protein production. In general, the efficiency of recovery of recombinant cell lines and the stability of reporter protein expression over time were higher following microinjection as compared to CaPi transfection. The results demonstrate the feasibility of using microinjection as a method to generate recombinant cell lines.
显微注射是一种基因转移技术，能够部分控制将质粒递送至培养的动物细胞核或细胞质中。在此，该方法被用于建立各种重组哺乳动物细胞系。通过对注射的异硫氰酸荧光素（FITC）-葡聚糖进行荧光定量来估计注射体积。然后使用编码绿色荧光蛋白（GFP）的报告质粒，针对向细胞核或细胞质的显微注射优化DNA浓度和注射压力。细胞核显微注射比细胞质显微注射对这两个参数的变化更敏感。在最佳条件下，向细胞核或细胞质显微注射1天后，80-90%的细胞为GFP阳性。显微注射或磷酸钙转染后回收重组细胞系，并分析重组蛋白产生的水平和稳定性。一般来说，与磷酸钙转染相比，显微注射后重组细胞系的回收效率和报告蛋白表达随时间的稳定性更高。结果证明了使用显微注射作为产生重组细胞系的方法的可行性。
