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Generation of an Msx2-GFP conditional null allele
Vardina Bensoussan1, Yvan Lallemand, Julie Moreau
1Institut Pasteur, Unité de Génétique Moléculaire de la Morphogenèse, CNRS URA 2578, Paris, F-75015, France.
Abstract:
Msx1 and Msx2, two members of the Msx gene family, encode homeoprotein transcription factors and play critical roles during mouse development. Because of the redundancy between the two genes, many of these roles can only be studied in double Msx1; Msx2 mutants. However, these animals die around 14.5 dpc, which precludes analysis of Msx gene function beyond this stage. Moreover, the pleiotropic defects displayed by these embryos make phenotypic analysis difficult. To overcome these restrictions and study the double Msx mutant phenotype at later stages, we generated an Msx2 conditional null allele using Cre/loxP technology. The strategy consisted of flanking the Msx2 gene coding sequence with two loxP sites. In addition, a green fluorescent protein (GFP) reporter gene was placed under Msx2 regulatory sequences in the modified locus. Our results demonstrate that the Msx2-GFP conditional allele behaves as a normal one, whereas Cre-mediated recombination creates an Msx2 null allele. With either allele, expression patterns of the GFP reporter gene and the Msx2 endogenous gene are identical.
Insights
Researchers developed a new Msx2 conditional null allele using Cre/loxP technology to study gene function in mouse development. This tool allows for analysis beyond embryonic lethality, overcoming previous limitations in Msx gene research.
Area of Science:
- Developmental Biology
- Genetics
- Molecular Biology
Background:
- Msx1 and Msx2 are homeoprotein transcription factors crucial for mouse development.
- Redundancy between Msx1 and Msx2 necessitates double mutants for functional studies.
- Existing Msx1; Msx2 double mutants exhibit embryonic lethality around 14.5 days post coitum (dpc), limiting later-stage analysis.
Purpose of the Study:
- To overcome the limitations of embryonic lethality in Msx1; Msx2 double mutants.
- To enable the study of Msx gene function at later developmental stages.
- To generate a tool for conditional gene knockout of Msx2.
Main Methods:
- Generation of an Msx2 conditional null allele utilizing Cre/loxP technology.
- Flanking the Msx2 gene coding sequence with loxP sites.
- Incorporation of a green fluorescent protein (GFP) reporter gene under Msx2 regulatory control.
Main Results:
- The generated Msx2-GFP conditional allele functions normally.
- Cre-mediated recombination successfully creates an Msx2 null allele.
- Expression patterns of the GFP reporter precisely mirror those of the endogenous Msx2 gene.
Conclusions:
- The Msx2 conditional null allele is a viable tool for studying Msx gene function.
- This technology allows for the investigation of Msx gene roles beyond the previously observed embryonic lethality.
- The GFP reporter provides a reliable indicator of Msx2 gene expression and recombination events.
