In order to improve efficiency, 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 (Morozov V, Wawrousek EF, Gene Ther 15:468,2008). This novel methodology of supplying protein factors to stimulate gene modification in the nucleus has not been reported previously. Our first attempt to produce mice with an alphaB-crystallin targeted gene resulted in an extremely low number of born pups (born to transferred 1.5% - 5% for different factors) compared to the average 20 % (Auerbach A.B. et al. Transgenic Research 12:59,2003), suggesting potential toxicity of the injected mixtures. In order to optimize gene alteration conditions by determining a reasonable efficiency to toxicity balance, and to improve the detection method, we have developed transgenic mouse lines in which correction of a mutation in a fluorescent protein gives an instant read-out for many embryos that can be screened simultaneously. We engineered a cassette encoding a bicistronic mRNA under the EF1alpha promoter (mutated red fluorescent protein DsRed/an IRES element/green fluorescent protein). 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 copy) and one has a high copy number (26 copies of the construct). In addition to our work with ssDNA as a targeting vector, we are also working on another type of gene targeting which we have named Forced Homologous Recombination (FHR). In this case, we use a significantly larger targeting vector, similar to those currently used in ES cell targeting procedures, with regions of homology to a selected gene or chromosomal locus at both 5'and 3'ends. With the introduction of this type of DNA construct as an intermediate product of the homologous recombination pathway into pro-nucleus of one-celled embryo, we expect an increase in gene targeting efficiency. The presence of long homology arms at both ends should increase the specificity of gene modification and also dramatically decrease any random integration of the targeting vector, comparable to our ssDNA strategies. The ROSA 26 region of FVB/N mouse genome was selected for in vivo testing of this FHR strategy, and those experiments are currently underway.
| Morozov, V; Wawrousek, E F (2008) Single-strand DNA-mediated targeted mutagenesis of genomic DNA in early mouse embryos is stimulated by Rad51/54 and by Ku70/86 inhibition. Gene Ther 15:468-72 |
