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 in order 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. We began this work using chimeric RNA-DNA oligonucleotides, however, our preliminary results and publications from other laboratories prompted us to expand the research to include single stranded DNA (ssDNA) oligonucleotides. Our general protocol involves microinjecting mutagenic oligonucleotides, alone or in combination with other biomolecules, into pronuclei of one celled embryos, or nuclei of two celled embryos. Manipulated embryos are incubated, then harvested at the blastula stage (or earlier if embryonic development ceases) for genotype analysis. In this way, we are searching for biomolecules which enhance such DNA modification, and developing an understanding of factors that control the frequency of this type of gene alteration. We originally developed and used a sensitive technique of PCR-based restriction fragment length polymorphism (RFLP). Several experiments yielded promising results, however, the RFLP-based technique is extremely time inefficient, and, for low DNA copy number, could result in questionable fidelity. To improve the detection method, our recent efforts have been directed toward generating transgenic mouse lines expressing different autofluorescent reporter proteins, which can be detected by fluorescence microscopy during early embryonic development. The gene for one of the fluorescent proteins contains a single point mutation, which eliminates production of the functional protein. An embryo giving a fluorescent signal at the appropriate wavelength would then indicate a successful correction of the point mutation by the mutagenic protocol. This work is currently in the progress. Such a transgenic mouse model, expressing multiple autofluorescent reporter proteins, should be an invaluable tool for systematically studying 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.
