Expression and function of ribozymes in transgenic mice

Abstract

Hammerhead and hairpin ribozymes are short RNAs that act as sequence-specific endoribonucleases by cleaving a target RNA after hybridization to complementary sequences. By changing the specific target hybridization sequence of the ribozyme, virtually any RNA can be targeted, making ribozymes a versatile tool for gene down-regulation (knockdown). This thesis addresses questions regarding expression and function of ribozymes in transgenic mice. A series of hammerhead and hairpin ribozyme transgenic mouse models were generated. The first transgenic mouse model, expressing a hammerhead ribozyme targeting beta-2-microglobulin (ß2-m), proved the principle that ribozymes can efficiently down-regulate the expression specific mRNAs in vivo in a mammalian organism. A second transgenic mouse model expressing hammerhead ribozymes targeting Wilms tumor gene 1 (WT1) generated interesting preliminary data on the involvement of WT1 in male sexual development. However, ribozyme transgene expression was gradually shut-off during breeding, correlating with a disappearance of the observed abnormalities. A third transgenic model showed the feasibility of using ribozyme transgenic mice as a model for ribozyme gene therapy against HIV-1 infection. Transgenic spleen cells expressing an anti-HIV-1 hairpin ribozyme, showed a dose-dependent resistance to infection compared with control spleen cells, when infected with HIV-1/MuLV pseudotype virus.

The different trans-cleaving ribozymes targeting ß2-m, WT1 or HIV-1 in transgenic mice were expressed in an expression cassette, in which a 3' cis-cleaving ribozyme, downstream of the trans-targeting ribozyme, was used to terminate the transcript. This cis-cleavage was found to be essential for the activity of the transcleaving ribozyme. Direct proof of hammerhead ribozyme cleavage in mammalian cells in vivo was given by detection of cis-cleavage products in transgenic mouse organs. Steady state levels of cleavage products, relative to the level of the non-cleaved transcript, showed a consistent ranking order: kidney > liver > lung > spleen, which suggests a difference in hammerhead ribozyme cleavage activity in these organs. In vitro analyses of ribozyme - RNA substrate kinetics have primarily relied on indirect detection methods. We have developed a real-time analysis of interactions (i.e. association, dissociation and cleavage) between hammerhead ribozymes and substrates using the surface plasmon resonance (SPR) technology. The SPR technology permits an inclusion of proteins in in vitro analyses, which may improve the capabilities to predict ribozyme function in living cells. In summary, this work has shown that ribozymes, expressed in transgenic mice, can down-regulate the mRNA of specific target genes in a dose-dependent manner, thereby satisfying the general aim of the thesis. It has also shed light on some of the basic aspects of ribozyme expression and function in transgenic mice.