A novel screening system for construction and selection of active ribozymes using DHFR (dihydrofolate reductase) gene...

Ribozyme is thought to be a powerful tool for suppression of specific gene expression, but there are many failure cases in their applications in vivo. One reason may be that there are many cellular proteins in vivo which may inhibit the catalytic activity of ribozymes. Such being the case, many researchers have made efforts to increase the activity of ribozymes. Here we have developed a new screening system for active ribozymes using DHFR (dihydrofolate reductase) gene as a selection marker. In our system, we set the ribozyme target site not to the DHFR gene itself but to the interspace between two ATG codons. One of which is the original initiation codon, ATG, for DHFR gene itself, and the second one locates upstream of the former. The upstream ATG has an ability to cause frame shift for DHFR gene. Since the upstream ATG is preferentially used during translation, unless it is removed, production of DHFR is negligible. By setting the ribozyme target site between the two ATGs, we can make this screening system as positive selection. To our best knowledge, this is the first successful screening system in vivo.