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| Experimental Screening mRNA Targets by Reverse Transcription for Ribozyme to GPA Expression Interference |
| FU Hui1,2, LI Fei-fei2, MA Qiong2, FU Huai-xiu2, CUI Yu-fang2, MAO Jian-ping2 |
1. Medical University of Anhui, Hefei 230032, China;
2. Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China |
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Abstract An oligodeoxynucleotide library which the sequence were defined at 5 prime and randomized at 3 prime was employed to screen mRNA accessible targets, in reverse transcription and PCR after hybridized the library with mRNA. The mRNA of Glycophorin A(GPA), type I transmembrane glycoprotein, was screened and obtained 4 targets sequences. Accordingly 4 antisense nucleic acids designed respectively, the binding efficiency of every target were verified by using RNase H with antisense nucleic acids. Among them 2 targets showed better effects on binding and cutting. Designed 2 ribozymes to these targets, packaged in lentivirus system, then infected K562 cells(human erythroid leukemia line), the down-regulation effect of gene expression was validated by Real Time RT-PCR and by Western Blot. It was found that the screened targets showed the best effective knocking down effects on gene expression. The study provided a reference for mRNA targets screening and Ribozyme design, and was helpful in membrane receptor expression interference, since GPA is a transmembrane protein.
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Received: 21 January 2014
Published: 25 March 2014
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