Chenuet Sebastien, Derouazi Madiha, Hacker David, Wurm Florian
Ecole Polytechnique Féderale de Lausanne, EPFL-SV-IBI-LBTC, Lausanne, Switzerland.
Methods Mol Biol. 2009;518:99-112. doi: 10.1007/978-1-59745-202-1_8.
Gene transfer methods for producing recombinant cell lines are often not very efficient. One reason is that the recombinant DNA is delivered into the cell cytoplasm and only a small fraction reaches the nucleus. This chapter describes a method for microinjecting DNA directly into the nucleus. Direct injection has several advantages including the ability to deliver a defined copy number into the nucleus, the avoidance of DNAses that are present in the cell cytoplasm, and the lack of a need for extensive subcloning to find the recombinant cells. The procedure is described for two cell lines, CHO DG44 and BHK-21, using green fluorescent protein as a reporter gene. However, this method could easily be adapted to other cells lines and using other recombinant genes.
用于产生重组细胞系的基因转移方法通常效率不是很高。一个原因是重组DNA被递送到细胞质中，只有一小部分能到达细胞核。本章描述了一种将DNA直接显微注射到细胞核中的方法。直接注射有几个优点，包括能够将确定的拷贝数递送到细胞核中，避免细胞质中存在的DNA酶，以及无需进行广泛的亚克隆来寻找重组细胞。文中描述了使用绿色荧光蛋白作为报告基因，对两种细胞系CHO DG44和BHK - 21进行该操作的过程。然而，这种方法可以很容易地适用于其他细胞系并使用其他重组基因。
