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Bypass of lethality with mosaic mice generated by Cre-loxP-mediated recombination
U A Betz1, C A Vosshenrich, K Rajewsky
1Institute for Genetics, University of Cologne, Germany. ubetz@mac.genetik.uni-koeln.de
Background:
The analysis of gene function based on the generation of mutant mice by homologous recombination in embryonic stem cells is limited if gene disruption results in embryonic lethality. Mosaic mice, which contain a certain proportion of mutant cells in all organs, allow lethality to be circumvented and the potential of mutant cells to contribute to different cell lineages to be analyzed. To generate mosaic animals, we used the bacteriophage P1-derived Cre-loxP recombination system, which allows gene alteration by Cre-mediated deletion of loxP-flanked gene segments.
Results:
We generated nestin-cre transgenic mouse lines, which expressed the Cre recombinase under the control of the rat nestin promoter and its second intron enhancer. In crosses to animals carrying a loxP-flanked target gene, partial deletion of the loxP-flanked allele occurred before day 10.5 post coitum and was detectable in all adult organs examined, including germ-line cells. Using this approach, we generated mosaic mice containing cells deficient in the gamma-chain of the interleukin-2 receptor (IL-2R gamma); in these animals, the IL-2R gamma-deficient cells were underrepresented in the thymus and spleen. Because mice deficient in DNA polymerase beta die perinatally, we studied the effects of DNA polymerase beta deficiency in mosaic animals. We found that some of the mosaic polymerase beta-deficient animals were viable, but were often reduced in size and weight. The fraction of DNA polymerase beta-deficient cells in mosaic embryos decreased during embryonic development, presumably because wild-type cells had a competitive advantage.
Conclusions:
The nestin-cre transgenic mice can be used to generate mosaic animals in which target genes are mutated by Cre-mediated recombination of loxP-flanked target genes. By using mosaic animals, embryonic lethality can be bypassed and cell lineages for whose development a given target gene is critical can be identified. In the case of DNA polymerase beta, deficient cells are already selected against during embryonic development, demonstrating the general importance of this protein in multiple cell types.
Insights
Mosaic mice enable gene function analysis when mutations cause embryonic lethality. This study used nestin-cre mice to generate mosaic animals, bypassing lethality and identifying critical gene roles during development.
Area of Science:
- Developmental Biology
- Genetics
- Molecular Biology
Background:
- Gene function studies are limited by embryonic lethality in knockout mice.
- Mosaic mice, containing a mix of mutant and wild-type cells, overcome this limitation.
- The Cre-loxP system facilitates targeted gene alteration in embryonic stem cells.
Purpose of the Study:
- To develop a method for generating mosaic mice using the nestin-cre system.
- To analyze gene function in cases of embryonic lethality.
- To identify cell lineages dependent on specific target genes.
Main Methods:
- Generation of nestin-cre transgenic mouse lines.
- Cre-mediated deletion of loxP-flanked genes before embryonic day 10.5.
- Analysis of gene-deficient cells in various adult organs and germ lines.
- Study of mosaic mice with deficiencies in IL-2R gamma and DNA polymerase beta.
Main Results:
- Nestin-cre mediated recombination occurred early in development, affecting all examined organs.
- Mosaic mice with IL-2R gamma deficiency showed underrepresentation in thymus and spleen.
- Mosaic mice with DNA polymerase beta deficiency were viable but smaller; deficient cells decreased during development due to competitive disadvantage.
Conclusions:
- Nestin-cre transgenic mice are effective for generating mosaic animals to study gene function.
- Mosaic analysis bypasses embryonic lethality, allowing identification of critical genes for specific cell lineages.
- DNA polymerase beta deficiency highlights the protein's importance across multiple cell types, with deficient cells selected against during embryogenesis.