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 induce specific point mutations into the mouse genome. Our general protocol involves microinjecting of modified single stranded DNA (ssDNA), alone or in combination with other factors, into pronuclei of one celled mouse embryos. Manipulated embryos are incubated up to the blastula stage, at which point they are examined for presence of the desired mutation. In this way, we are searching for factors that could increase the frequency of targeted DNA point mutations, and developing an understanding of the mechanisms controlling this type of gene alteration. While working to produce an efficient high throughput assay, we have been using PCR-based restriction fragment length polymorphism (RFLP) assay to detect point mutation introduced into genomic DNA. Several experiments, reported previously, have yielded promising results. Our results are consistent with mounting evidence in the literature that gene editing by this mechanism involves components of the homologous recombination pathway, which have been shown to be present in mouse ova and preimplantation stage embryos, and suggest an inhibitory role for the non-homologous end-joining pathway, which is generally the preferential mode of DNA repair in mammalian cells. The RFLP-based technique is extremely time consuming, and, for low DNA copy number, could result in questionable fidelity. To improve the detection method, we are developing transgenic mouse lines in which correction of a mutation in a fluorescent protein will give an instant read-out, and many embryos can be screened simultaneously. We previously engineered a cassette encoding a bicistronic mRNA (mutated red fluorescent protein [DsRed] / an IRES element / green fluorescent protein). All mouse embryos expressing the transgene should fluoresce green (endogenous control for transgene expression), but only embryos in which the point mutation in DsRed has been corrected should also fluoresce red. We have used several enhancer/promoter elements, which have been used successfully in preimplantation stage mouse embryos or mouse ES cells, to drive expression of the cassette. In testing the system, a cassette containing the non-mutated DsRed was used. Previoulsy we tested constructs containing the mPGKpromoter / adenovirus tripartite leader and the hCMV IE promoter / enhancer, neither of which produced transgenic mice suitable for mutagenesis experiments. We are currently working on two new constructs to express the cassette in transgenic mouse embryos. The first uses the transcriptional regulatory elements from the widely used pCAGGS plasmid containing the hCMV IE enhancer / chicken betta-actin promoter and partial intron / rabbit betta-globin partial intron and polyadenylation site. The second uses the EF1alpha promoter. Both have been constructed and show intense expression of EGFP and DsRed in transient assays in mouse embryos. Transgenic mouse production for these constructs is ongoing. This mouse model should be an invaluable tool to study in depth the conditions under which mutagenic oligonucleotides can be used to induce point mutations in mouse embryos at a frequency sufficient to make this technology practical for generating mouse models of human genetic diseases.
