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 genotype analysis is performed. 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 of this type of gene alteration. We developed and used a sensitive technique of PCR-based restriction fragment length polymorphism (RFLP) to detect point mutation introduced into genomic DNA. Several experiments yielded promising results. Pre-incubation of ssDNA with RecA slightly increased the frequency of genomic DNA modification. A larger increase was observed when a mixture of hRad51 (or mutated hRad51) and hRad54 was co-injected with ssDNA into pronuclei. A similar increase in the frequency of genomic DNA targeted point mutation was observed when the activity of Ku protein was suppressed by co-injection of ssDNA with a mixture of antibodies against the Ku 70 and Ku 86 proteins. Rad51 and Rad 54 are key proteins in the homologous recombination pathway for the repair of double-strand breaks in DNA, and Ku is a key component of the alternative pathway of non-homologous end-joining repair for double-strand DNA breaks. Therefore, our data suggests a crucial role for the DNA homologous recombination pathway in this type of gene alteration, and an inhibitory role for the non-homologous end-joining pathway, which is generally the preferential mode of DNA repair in mammalian cells. However, 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 working toward generating transgenic mouse lines, which will produce a bicistronic mRNA, with coding sequences for two different fluorescent reporter proteins, separated by an IRES element. The gene for one of the fluorescent proteins contains a single point mutation, which eliminates expression. A mouse embryo fluorescing at both wavelengths would then indicate a successful correction of the point mutation by the mutagenic protocol. Such a transgenic mouse model, expressing multiple fluorescent reporter proteins, should be an invaluable tool for in depth study 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.
