Development of an efficient Agrobacterium-mediated transformation method and its application in tryptophan pathway modification in Catharanthus roseus.

The biosynthetic pathway of Catharanthus roseus vinca alkaloids has a long research history, including not only identification of metabolic intermediates but also the mechanisms of inter-cellular transport and accumulation of biosynthesized components. Vinca alkaloids pathway begins with strictosidine, which is biosynthesized by condensing tryptamine from the tryptophan pathway and secologanin from the isoprenoid pathway. Therefore, increasing the supply of precursor tryptophan may enhance vinca alkaloid content or their metabolic intermediates. Many reports on the genetic modification of C. roseus use cultured cells or hairy roots, but few reports cover the production of transgenic plants. In this study, we first investigated a method for stably producing transgenic plants of C. roseus, then, using this technique, we modified the tryptophan metabolism system to produce transgenic plants with increased tryptophan content. Transformed plants were obtained by infecting cotyledons two weeks after sowing with Agrobacterium strain A13 containing a plant expression vector, then selecting with 1/2 B5 medium supplemented with 50 mg l-1 kanamycin and 20 mg l-1 meropenem. Sixty-eight regenerated plants were obtained from 4,200 cotyledons infected with Agrobacterium, after which genomic PCR analysis using NPTII-specific primers confirmed gene presence in 24 plants with a transformation rate of 0.6%. Furthermore, we performed transformation into C. roseus using an expression vector to join trpE8 and aroG4 genes, which are feedback-resistant mutant genes derived from Escherichia coli. The resulting transformed plants showed exactly the same morphology as the wild-type, albeit with a marked increase in tryptophan and alkaloids content, especially catharanthine in leaves.

Maxizyme technology.

Ribozymes are small and versatile nucleic acids that can cleave RNAs at specific sites. These molecules have great potential to be used as effective gene-therapeutic agents. However, because of the limitation for cleavable sequences within the target mRNA, in some cases conventional ribozymes have failed to exhibit precise cleavage specificity. A maxizyme is the dimer of minimized ribozymes (minizymes), which can specifically cleave two distinct target sites. The maxizyme also has an allosteric function in that it can form an active conformation and cleave the two target sites only when it recognizes two distinct target sites. We demonstrated previously that an allosterically controllable maxizyme was a powerful tool in the disruption of an abnormal chimeric RNA (bcr-abl) in cells and in mice. Furthermore, more than five custom-designed maxizymes have clearly demonstrated these allosteric functions in vitro and in vivo. Thus, maxizyme technology is not limited to one specific case, but may have broad general applicability in molecular biology and in molecular gene therapy.

siRNAs generated by recombinant human Dicer induce specific and significant but target site-independent gene silencing in human cells.

RNA interference has emerged as a powerful tool for the silencing of gene expression in animals and plants. It was reported recently that 21 nt synthetic small interfering RNAs (siRNAs) specifically suppressed the expression of endogenous genes in several lines of mammalian cells. However, the efficacy of siRNAs is dependent on the presence of a specific target site within the target mRNA and it remains very difficult to predict the best or most effective target site. In this study, we demonstrate that siRNAs that have been generated in vitro by recombinant human Dicer (re-hDicer) significantly suppress not only the exogenous expression of a puromycin-resistance gene but also the endogenous expression of H-ras, c-jun and c-fos. In our system, selection of a target site is not necessary in the design of siRNAs. However, it is important to avoid homologous sequences within a target mRNA in a given protein family. Our diced siRNA system should be a powerful tool for the inactivation of genes in mammalian cells.

Allosterically controllable maxizymes for molecular gene therapy.

Ribozymes are small and versatile nucleic acids that can cleave RNAs at specific sites. These molecules have great potential to be used as effective gene therapeutic agents. However, conventional ribozymes have, in some cases, failed to exhibit precise cleavage specificity because they require cleavable sequences in the target mRNA. Recently, we demonstrated that an allosterically controllable novel ribozyme, designated the maxizyme, is a powerful tool for disruption of an abnormal chimeric RNA target (BCR-ABL (b2a2) mRNA) in cells and in mice. Furthermore, more than five custom-designed maxizymes have demonstrated these allosteric functions in vitro and in vivo. Thus, maxizyme technology is not limited to a specific case but may have broad general applicability in molecular biology and in molecular gene therapy.

Construction of an allosteric trans-maxizyme targeting for two distinct oncogenes.

A maxizyme is dimmer of minimized ribozymes (minizymes) and can specifically cleave two target sites. The maxizyme also can allosterically cleave the target RNA only when it recognizes two target sites. In this study, for a cancer gene therapy, we focused two distinct oncogenes, cyclinD1 and hst-1, which are overexpressed in breast cancer cells. If we use conventional ribozymes for suppression of expression of those genes, these ribozymes affect not only these mRNAs in cancer cells but also those in normal cells because those genes are necessary for a growth factor-dependent signal transduction and a cell cycle in normal cells. To overcome this problem, we tried to design the trans-maxizyme that can cleave these mRNAs only in the breast cancer cells.

A functional gene discovery in cell differentiation by hybrid ribozyme and siRNA libraries.

Recently, we developed a gene discovery system that can identify functional genes using a randomized hybrid ribozyme library. In this system, inhibition of the expression of a particular gene by active ribozymes was reflected by a change in a particular phenotype, the method allowed the identification of functional genes. In the case of identification of functional genes for apoptosis pathways, we identified many pro-apoptotic genes in TNF-alpha and Fas-mediated apoptosis pathways. In this study, we tried to identify the functional genes that are necessary for the retinoic acid (RA)-induced cell differentiation using randomized ribozyme and siRNA libraries. We succeeded to identify the several differentiation factors. Therefore, our gene discovery system based on randomized ribozyme and siRNA libraries are high potential to identify the differentiation and undifferentiation factors in the post genome era.