The conditional alpha-4 integrin knockout

This work attempts to generate a conditional alpha-4 integrin knockout model in order to investigate the role of alpha-4 integrin in adhesion and possibly other functions and also to generate an animal model for the screening of potential alpha-4 integrin antagonists. The homozygous knockout of the alpha-4 integrin is embryonic lethal, the heterozygous knockout however is viable and is also commercially available. The conditional system used in this study was the tet-OFF system. Two different approaches were taken in order to generate the conditional knockout mice. On one hand it was attempted to introduce the elements of the tet-system as a knock-in element into ES cells by homologous recombination. It was postulated that with this approach the expression of the endogenous alpha-4 integrin was to become tetracycline dependent and would only occur in the absence of tetracycline. However none of the selected ES-cell clones transfected with the targeting vector showed a proper targeting event, which is probably due to the very hard to target location of the alpha-4 integrin on the chromosome. However it was shown that the knock-in cassette itself can function in ES-cells by reporter gene expression experiments. On the other hand it was attempted to replace the endogenous alpha-4 integrin with the alpha-4 cDNA under the control of the tet-system. The expression of the tettransactivator was driven by the specific alpha-4 promoter and the function of this construct was shown in tissue culture experiments. The majority of the experiments were done with mice, where both transgenes, the transactivator and the cDNA target gene transgene, were co-injected. Three lines were generated that produced mice with a homozygous null alpha-4 knockout background, all mice had both transgenes integrated, most likely in the same locus. The analysis of the homozygous lines revealed that the expression of the cDNA is not doxycycline dependent in at least one of the three lines and that survival of the homozygous null mice is most likely due to a low level of leakiness of the cDNA. This was postulated after evaluating the outcome of FACS, RPA and Affymetrix experiments. This line has a truncated cDNA transgene in the 5’ part of the gene. A variety of genes are significantly up- or downregulated in these knockout mice in comparison to regular, wild type mice. The findings need to be investigated further but could give insight into other pathways regulating alpha-4. The other two lines showed 3-6 fold lower transgene RNA levels of correct size in comparison to the endogenous alpha-4 RNA level, but further studies have to be conducted for a final conclusion about regulatablility of the transgenes with doxycycline. In conclusion it was not possible to generate a conditional knockout with this approach, but the homozygous knockout mice generated are still of great importance to elucidate alpha-4 integrin functions, since expression of the alpha-4 transgene is either much lower than the endogenous alpha-4 or the protein might not function properly in the line with the truncated transgene. The single injected lines were all tested for leakiness and expression of the transactivator protein before the lines were intercrossed. With this approach it is more likely to generate a conditional alpha-4 knockout. This work is still in progress and no homozygous null mice have been generated with this approach to date.