Chen Lifeng, Li Jianhua, Zhang Xichen, Liu Quan, Yin Jigang, Yao Longquan, Zhao Yueping, Cao Lili
College of Animal Science and Veterinary Medicine, Jilin University, Changchun, China.
Vet Parasitol. 2007 Jan 19;143(1):14-20. doi: 10.1016/j.vetpar.2006.07.029. Epub 2006 Sep 18.
Giardia, a most primitive eukaryote, infects several species including human and it is a major agent of waterborne outbreak of diarrhea. It has been difficult to employ standard genetic methods in the study of Giardia, but the RNA virus-based transfection system has been developed and used for the genetic manipulation. KRR1 protein is responsible for ribosome biosynthesis in Giardia. In this study, cDNA encoding hammerhead ribozyme flanked with various lengths of antisense Krr1 RNA were cloned into a viral vector pGCV634/GFP/GCV2174 derived from the genome of Giardia canis virus (GCV). RNA transcripts of the plasmids showed high cleavage activities on Krr1 mRNA in vitro. They were electroporated into GCV-infected G. canis trophozoites and Krr1 mRNA level was decreased by 72% with the ribozyme KRzS and 86% with the ribozyme KRzL, while the control ribozyme TRzS showed no effect on the level of Krr1 mRNA. The two hammerhead ribozyme transfected cells grew slowly, their internal structures got blurred and the cells were deformed. These results indicated that GCV could be useful tool for gene manipulation of G. canis.
贾第虫是一种最原始的真核生物，可感染包括人类在内的多种物种，是水源性腹泻暴发的主要病原体。在贾第虫研究中，采用标准遗传方法一直存在困难，但基于RNA病毒的转染系统已被开发并用于基因操作。KRR1蛋白负责贾第虫中的核糖体生物合成。在本研究中，将编码锤头状核酶且两侧带有不同长度反义Krr1 RNA的cDNA克隆到源自犬贾第虫病毒（GCV）基因组的病毒载体pGCV634/GFP/GCV2174中。质粒的RNA转录本在体外对Krr1 mRNA显示出高切割活性。将它们电穿孔导入GCV感染的犬贾第虫滋养体中，核酶KRzS使Krr1 mRNA水平降低了72%，核酶KRzL使其降低了86%，而对照核酶TRzS对Krr1 mRNA水平没有影响。两种锤头状核酶转染的细胞生长缓慢，其内部结构变得模糊，细胞发生变形。这些结果表明，GCV可能是犬贾第虫基因操作的有用工具。
