[Construction of a trans-splicing ribozyme for restoring EGFP truncation mutation].
Special designed group I intron ribozymes can specifically splice objective RNA, repair the mutant gene in RNA level. The specificity of ribozyme is determined by nucleotides specific internal guide sequence (IGS) introduced to the enzyme. In this study, fragment sequence containing Tetrahymena thermophilia intron I of 26S rRNA gene was cloned and cis-splicing activity of this ribozyme was confirmed by in vitro transcription. For evaluating the trans-splicing activity of this ribozyme, a truncated mutant Green Fluorescence Protein (GFP) vector, XYQ5/XYQ10- pEGFP-C2, was constructed. This vector deleted the 3' end 564bp fragment of EGFP coding sequence, led to the lost the activity of emitting green fluorescence. Trans-splicing ribozyme plasmids ptrans-rib-CMV2 for remedy of the truncated mutant EGFP was constructed by PCR and molecular cloning techniques. This vector utilizing cloned 26S rRNA intron 1 as core enzyme; selecting T-G site at 194bp of EGFP coding sequence as splicing receptor, designed an IGS which is inversely complement to the 188-193nt of EGFP mRNA; the 195-890bp fragment of EGFP coding sequence was ligated to the 3'-end of ribozyme core. The fragment containing these components was inserted to a eukayotic expression vector pRC-CMV2. Using linearized XYQ5/XYQ10- pEGFP-C2 and ptrans-rib-CMV2 as templates, truncated EGFP mRNA and the constructed ribozyme vector were transcribed and mixed to evaluate the trans-splicing activity. Analysis of in vitro transcription products mix by RT-PCR verified the existence of wild type EGFP mRNA molecule. Co-transfection of XYQ5/XYQ10- pEGFP-C2 with ptrans-rib-CMV2 to Hela cells proved this ribozyme restored green fluorescence within cell, but the efficiency was low.
Li
Bing
B
Experimental Medical Research Center, Guangzhou Medical College, Guangzhou 510182, China. lbfree@21cn.com
Xiong
Yu-Quan
YQ
Tu
Hong-Bin
HB
Liu
Qi-Cai
QC
Zou
Dong-Ting
DT
Zhou
Wen-Qu
WQ
Chen
Yao-Yong
YY
D000818:Animals; D001483:Base Sequence; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics*; D009154:Mutation; D016337:RNA, Catalytic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D013768:Tetrahymena / Q000201:enzymology*; D020040:Trans-Splicing; D014158:Transcription, Genetic
C116167:GIR1 ribozyme; D050505:Mutant Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins