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Dissecting the role of N-myc in development using a single targeting vector to generate a series of alleles
1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada. nagy@mshri.on.ca
Abstract:
The N-myc proto-oncogene is expressed in many organs of the mouse embryo, suggesting that it has multiple functions. A null mutation leads to mid-gestation lethality [1-4], obscuring the later roles of the gene in organogenesis. We have generated a multi-purpose gene alteration by combining the potential for homologous and site-specific recombination in a single targeting vector, and using the selectable marker for neomycin-resistance, neo, to downregulate gene activity. This allowed us to create a series of alleles that led to different levels of N-myc expression. The phenotypes revealed a spectrum of developmental problems. The hypomorphic allele produced can be repaired in situ by Cre-recombinase-mediated DNA excision. We show here for the first time the use of a single targeting vector to generate an allelic series. This, and the possibility of subsequent lineage-specific or conditional allele repair in situ, represent new genome modification strategies that can be used to investigate multiple functions of a single gene.
Insights
Researchers developed a novel gene targeting method to study the N-myc proto-oncogene's multiple roles in embryonic development. This approach allows for precise control over gene expression, revealing new insights into developmental processes.
Area of Science:
- Developmental Biology
- Molecular Genetics
- Genomics
Background:
- The N-myc proto-oncogene is crucial for embryonic development, but its diverse functions are not fully understood due to early lethality from null mutations.
- Investigating N-myc's roles in later organogenesis requires methods to modulate its activity without causing complete loss-of-function.
Purpose of the Study:
- To develop a versatile gene targeting strategy to generate a series of N-myc alleles with varying expression levels.
- To investigate the spectrum of developmental phenotypes associated with different N-myc activities.
- To establish a platform for conditional N-myc allele repair for lineage-specific functional studies.
Main Methods:
- Construction of a single targeting vector enabling both homologous and site-specific recombination.
- Utilizing the neomycin-resistance (neo) gene as a selectable marker to modulate N-myc expression.
- Generating and analyzing a series of hypomorphic N-myc alleles in mouse models.
- Employing Cre-recombinase-mediated DNA excision for in situ allele repair.
Main Results:
- Successfully generated multiple N-myc alleles with distinct expression levels using a single targeting vector.
- Observed a range of developmental abnormalities correlating with altered N-myc expression.
- Demonstrated the feasibility of in situ repair of hypomorphic alleles via Cre-recombinase.
Conclusions:
- The developed genome modification strategy allows for the generation of an allelic series from a single vector, offering a powerful tool for gene function analysis.
- This approach facilitates the investigation of multiple gene functions, including those essential for later developmental stages.
- The capacity for conditional in situ allele repair opens new avenues for studying gene roles in specific cell lineages and developmental contexts.