嗜热四膜虫的电穿孔转化及影响细胞存活的参数

Transformation of Tetrahymena thermophila by electroporation and parameters effecting cell survival.

作者信息

Brunk C F, Navas P

机构信息

Biology Department, University of California, Los Angeles 90024.

出版信息

Exp Cell Res. 1988 Feb;174(2):525-32. doi: 10.1016/0014-4827(88)90322-9.

DOI:10.1016/0014-4827(88)90322-9

PMID:2892691

Abstract

We have successfully transformed Tetrahymena thermophila by electroporation, a process of electrically introducing DNA. The DNA used for transformation contains a mutant ribosomal RNA gene (rDNA) that confers resistance to paromomycin on the transformed cells. This mutant rDNA replicates more rapidly than the endogenous rDNA of the transformed cells so that the mutant rDNA becomes predominant within several generations. This mutant rDNA also carries a restriction polymorphism that readily distinguishes it from the endogenous rDNA of the transformed cells. Substantial nuclease activity is released from the cells during electroporation and must be neutralized in order for transformation to be effective. Cell survival is inversely proportional to the electrical energy dissipated (joules) in the medium. Electroporation is a convenient and effective means of introducing transforming DNA into T. thermophila.

摘要

我们已通过电穿孔成功转化嗜热四膜虫，电穿孔是一种电导入DNA的过程。用于转化的DNA含有一个突变核糖体RNA基因（rDNA），该基因赋予转化细胞对巴龙霉素的抗性。这种突变rDNA比转化细胞的内源性rDNA复制得更快，因此突变rDNA在几代内就会占主导地位。这种突变rDNA还具有一种限制性多态性，很容易将其与转化细胞的内源性rDNA区分开来。在电穿孔过程中，细胞会释放大量核酸酶活性，为使转化有效，必须将其中和。细胞存活率与培养基中耗散的电能（焦耳）成反比。电穿孔是将转化DNA导入嗜热四膜虫的一种方便有效的方法。