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. We began this work using chimeric RNA-DNA oligonucleotides, however, our preliminary results and publications from other laboratories [Igoucheva, O. et al. Gene Therapy 8, 391-399, (2001); Liu, L. et al. Nucleic Acids Res. 29, 4238-4250, (2001)] 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 have developed sensitive techniques to detect targeted SNPs using very few cells, (i.e. those present in preimplantation stage embryos), and are currently using them to screen for induced polymorphisms. Several experiments have yielded promising results. We are continuing a systematic study to determine 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.
