Question

Describe the Cre-Lox system for generating conditional knockout mice. What type of genes can be studied by using an inducible promoter to activate the Cre gene?

Short answer

Answer: The Cre-Lox system helps researchers study essential or embryonic lethal genes by allowing conditional disruption of these genes in adult mice or specific tissues, avoiding the lethality associated with traditional knockout methods. By using an inducible promoter to control the expression of the Cre gene, the knockout of essential or embryonic lethal genes can be controlled both spatially and temporally, which allows for a more detailed understanding of the gene's functions and their role in development and survival.

Step-by-step solution

Understanding the Cre-Lox System

The Cre-Lox system is based on the Cre recombinase enzyme derived from the P1 bacteriophage and its substrate, the LoxP (locus of X-over P1) site. The Cre enzyme recognizes the LoxP sites and can either recombine or excise the DNA sequence between two LoxP sites, depending on their relative orientation. This allows for genomic manipulation, such as gene knockout or activation, in a precise and controlled manner.

Generating Conditional Knockout Mice Using the Cre-Lox System

To generate conditional knockout mice using the Cre-Lox system, three components are required: 1. A "floxed" gene, which is the target gene flanked by two LoxP sites. 2. The Cre recombinase gene introduced into the mouse genome, often under the control of a tissue-specific or inducible promoter, determining when and where the Cre enzyme is expressed. 3. Mating of mice carrying floxed genes and Cre recombinase genes. When the Cre enzyme is expressed, it triggers the recombination between the LoxP sites flanking the target gene, resulting in its excision and creating a knockout for that specific gene in a specific tissue or developmental stage, depending on the Cre promoter used.

Studying Genes with an Inducible Promoter to Activate the Cre Gene

By using an inducible promoter to control the expression of the Cre gene, researchers can study a wide range of genes in a more controlled manner. This approach is particularly useful for studying: 1. Essential or embryonic lethal genes: Genes that are crucial for organism development or survival can be conditionally disrupted in adult mice or specific tissues, avoiding the lethality associated with traditional knockout methods. 2. Genes with redundant functions: If multiple genes can compensate for each other's loss, analyzing a knockout phenotype becomes challenging. Conditional knockouts can help determine the specific role of each gene in a particular tissue or time point. 3. Genes with pleiotropic effects: When a gene has multiple functions in different tissues or developmental stages, an inducible promoter allows the investigation of each function separately by controlling when the gene is disrupted. 4. Gene interactions: Conditional knockouts can be used to study the interactions between genes or the effects of disrupting multiple genes in combination during specific periods of development or adulthood. By using an inducible promoter to activate the Cre gene in the Cre-Lox system, researchers can manipulate genes of interest under specific temporal and spatial conditions, which allows for more accurate and detailed study of gene functions and interactions.