Low and variable efficiency is a major problem in targeted gene alteration, which is used as a primary tool in gene therapy and animal model studies. We tested several types of constructs, alone, or in combination with other factors, to introduce a point mutation into the alphaB-crystallin gene in one-celled mouse embryos. We found that co-injection of single stranded DNA (ssDNA) along with antibodies against Ku70/86, or supplementing the system with hRad51/hRad54, increases efficiency of targeted mutagenesis. These findings suggest that proteins in the homologous recombination DNA repair pathway contribute, and that proteins involved in the alternative non-homologous end joining pathway inhibit, ssDNA-mediated targeted mutagenesis. This is the first successful demonstration of targeted mutation in early mouse embryos. This novel methodology of supplying protein factors to stimulate gene modification in the nucleus has not been reported previously. For this initial study we have been using an extremely time consuming PCR-based restriction fragment length polymorphism (RFLP) assay to detect point mutations introduced into genomic DNA.? ? 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.? ? A successful promoter would induce easily detectable levels of fluorescent protein expression at the earliest stages of embryo development. All promoter/fluorescent protein transgenes were constructed and tested for expression of EGFP and DsRed in both transient assays in mouse embryos and in F1 transgenic mouse embryos. Expression of green or red fluorescent proteins from the mPGK promoter was detected neither in transient assays nor in transgenic mouse embryos. The mPGK promoter construct, with an adenovirus tripartite leader enhancer, was able to induce expression of EGFP, but not DsRed, in transient assays, and a transgenic mouse line failed to express any detectable fluorescent protein. The hCMV IE promoter / enhancer, the hCMV IE enhancer / chicken beta-actin promoter and partial intron / rabbit beta-globin partial intron, and the EF1alpha promoter all induce detectable EGFP and DsRed expression in mouse embryos during transient assays. For the last two constructs, expression was extremely high. However, transgenic mice with the hCMV IE promoter / enhancer construct expressed EGFP only after embryo hatching, and did not express DsRed. The construct carrying the chicken beta-actin promoter with the hCMV IE enhancer produces detectable expression of both fluorescent proteins in transgenic mouse embryos. EGEP was visible at the early blastula stage, and DsRed fluorescence appeared at the late blastula stage. These data imply that expression of DsRed protein is not lethal for mouse embryos, which has been suggested in the literature. Surprisingly, the EF1alpha promoter construct was able to generate green fluorescent protein in eight cell stage embryos (48 hr pf) and red fluorescent protein in 72 hr pf. A line of transgenic mice with the EF1alpha promoter / mutated red fluorescent protein / an IRES element / green fluorescent protein construct appears to be extremely promising at this time. Four transgenic mice lines with mutated DsRed protein were established. All four lines show expression of EGFP at 48 hr pf. Sequence analysis of a PCR-amplified region of the DsRed gene, from mouse genomic DNA, confirmed presence of the nonsense mutation in codon 15 of DsRed. RT-PCR analysis shows that three transgenic mouse lines have low copy number of the construct (2-3 copy) and one has a high copy number (26 copies of the construct). Established mouse lines should be an invaluable tool for systematic study of the conditions under which mutagenic constructs can be used to induce point mutations into embryos at a frequency sufficient to make this technology practical.
