In Drosophila, loss-of-function Ultrabithorax mutations result in the posterior thoracic segments differentiating into body parts with an identity normally found in the anterior thoracic segments. When the Ultrabithorax gene was cloned, it was shown to encode a transcription factor and to be expressed only in the posterior region of the thorax. Thus, Ultrabithorax acts to specify the identity of the posterior thoracic segments. Similar genes were soon discovered in other animals, including mice and humans. You have found that mice possess two closely related genes, Hoxa7 and Hoxb4, which are orthologs of Ultrabithorax. You wish to know whether the two mouse genes act to specify the identity of body segments in mice.
How will you create loss-of-function alleles of the mouse genes?

Verified step by step guidance

1. Step 1: Understand the concept of loss-of-function alleles. These are mutations that result in the reduction or complete loss of the normal function of a gene. In this case, you aim to create loss-of-function alleles for the mouse genes Hoxa7 and Hoxb4.
2. Step 2: Use gene-targeting techniques to create loss-of-function alleles. One common method is homologous recombination in embryonic stem (ES) cells. Design a targeting vector containing a selectable marker (e.g., neomycin resistance gene) flanked by sequences homologous to the regions surrounding the Hoxa7 or Hoxb4 gene.
3. Step 3: Introduce the targeting vector into mouse ES cells. The homologous recombination process will replace the functional gene with the disrupted version containing the selectable marker, effectively creating a loss-of-function allele.
4. Step 4: Select and screen ES cells for successful recombination events. Use PCR or Southern blotting to confirm that the targeted gene has been disrupted in the ES cells.
5. Step 5: Inject the modified ES cells into mouse blastocysts to create chimeric mice. Breed these chimeric mice to produce offspring that are homozygous for the loss-of-function alleles of Hoxa7 and Hoxb4. Analyze the phenotypes of these mice to determine the role of these genes in specifying body segment identity.

Key Concepts

Loss-of-Function Mutations

Loss-of-function mutations are genetic alterations that result in the reduced or abolished activity of a gene product, typically a protein. In the context of the Ultrabithorax gene in Drosophila, such mutations lead to the transformation of posterior thoracic segments into anterior-like structures. Understanding how to create these mutations in mouse genes, like Hoxa7 and Hoxb4, is crucial for studying their roles in segment identity.

Transcription Factors

Transcription factors are proteins that bind to specific DNA sequences to regulate the transcription of genes. The Ultrabithorax gene encodes a transcription factor that is essential for specifying the identity of thoracic segments in Drosophila. By understanding the function of transcription factors, researchers can infer how similar genes in mice might influence body segment identity and development.

Orthologs

Orthologs are genes in different species that evolved from a common ancestral gene and typically retain similar functions. The Hoxa7 and Hoxb4 genes in mice are orthologs of the Ultrabithorax gene in Drosophila, suggesting that they may have conserved roles in segment identity. Recognizing the significance of orthologs helps in predicting the function of genes across species and designing experiments to test their roles.