Animal models are essential for studying basic biological processes, elucidating the root causes of disease and developing treatment regimens for disease. The mouse has proven to be particularly amenable to genetic manipulation, allowing researchers to design mouse models specific for their work. A great number of useful mouse models have been generated by inserting various genes into mice (gain of function), or disrupting/deleting various genes from the mouse genome (loss of function). However, there exist no rapid, simple techniques to introduce a precise mutation at a particular spot in the mouse genome (correction of function or change of function). The current standard technology is extremely cumbersome and always involves concomitant insertions or alterations in addition to the desired point mutation. We are therefore attempting to adapt technologies being developed in the gene therapy field for effecting somatic cell gene repair to inducing specific point mutations into the mouse genome. The most promising and widely applicable approach employs chimeric RNA-DNA oligonucleotides (RDO). These molecules have been shown to mediate gene modifications in vitro (cell-free extracts), ex vivo (cell tissue culture), and in vivo (in some somatic animal cells), but reported conversion frequency has been erratic, and apparently quite tissue-dependent. We have begun a systematic study to determine the conditions under which RDOs can be used to induce point mutations in mouse embryos at a frequency sufficient to make this technology practical for generating mouse models. We have begun with the single celled mouse embryo, microinjecting into pronuclei RDOs, alone or in combination with other molecules. The RDO chosen for initial experiments has been previously shown to correct a point mutation in the tyrosinase gene, effectively reverting an albino phenotype to a pigmented phenotype in cells successfully converted. Initial studies suggest that microinjecting high concentrations of RDO can induce correction of the tyrosinase gene in single celled embryos, but may be toxic to the embryo. Studies are continuing on optimizing conditions.
