A floxed gene is a genetic engineering tool used to precisely manipulate the genome of a living organism, typically a laboratory mouse. The term “floxed” is a portmanteau, meaning the gene sequence has been flanked by loxP sites, which are specific DNA markers. This technique is fundamental to modern genetic research because it enables researchers to study the function of a single gene with spatial and temporal control. Scientists effectively “tag” a gene for future removal or inactivation, allowing them to understand its role in specific tissues or at particular life stages.
Defining a Floxed Gene
A floxed gene refers to a target gene sandwiched between two short recognition sequences known as loxP sites. These sites delineate the exact segment of DNA intended for later manipulation. The loxP site is a specific, short sequence derived from the P1 bacteriophage, a virus that infects bacteria.
Each loxP site consists of 34 base pairs, including two 13 base pair palindromic repeats separated by an 8 base pair core spacer sequence. This spacer region introduces an asymmetry that gives the loxP site a specific directionality. Since the loxP sequence does not naturally occur in model organisms, inserting it at specific locations allows for highly targeted genetic changes. When a gene is engineered with two of these sites placed in the same orientation on either side, it becomes a “floxed allele.”
The Role of Cre Recombinase
The floxed gene structure becomes active when introduced to the enzyme Cre recombinase. Cre, which stands for “cyclization recombinase,” is derived from the P1 bacteriophage. Its function is to recognize the loxP sequences and initiate a site-specific genetic recombination event.
The Cre enzyme binds to the 13 base pair repeats within the loxP sites, bringing the two distant sites together. If the two loxP sites flanking the target gene are oriented in the same direction, Cre recombinase excises the entire segment of DNA between them. This action physically removes the floxed gene from the chromosome, effectively deleting it and turning its function “off” within the cell. The gene sequence remains intact and functional until the Cre enzyme is introduced into the cell’s nucleus.
Conditional Gene Deletion
The floxed gene system facilitates conditional gene deletion, meaning gene removal can be controlled by location and timing. This precision is necessary because deleting an important gene throughout the body early in development often results in the death of the embryo. Conditional deletion avoids this lethality, allowing researchers to study the gene’s function in specific contexts, such as a particular disease stage in an adult.
Spatial control is achieved by linking the expression of the Cre recombinase gene to a promoter active only in a specific cell type or tissue. For example, a promoter turned on only in liver cells ensures that Cre recombinase is produced exclusively there, leading to the deletion of the floxed gene only in that organ. The gene remains functional in all other tissues, allowing the organism to survive while scientists study the gene’s specific role in the liver.
Temporal control uses a modified enzyme called CreERT2, which is fused to a mutant estrogen receptor. This fusion protein is normally trapped outside the nucleus, preventing it from accessing the DNA and initiating recombination.
The system is activated when a synthetic drug, typically the Tamoxifen metabolite 4-Hydroxytamoxifen, is administered. Upon binding to the drug, CreERT2 changes shape, releases from the trapping proteins, and moves into the nucleus to delete the floxed gene at the exact time chosen by the researcher.
Impact on Biomedical Research
The ability to control gene manipulation has advanced biomedical research, particularly in creating precise animal models. The Cre-Lox system allows for the generation of models for human diseases that require studying gene function in a localized or time-dependent manner. Researchers can model complex conditions like Alzheimer’s disease or specific cancers by deleting a tumor suppressor gene only in a particular brain region or after the animal has reached adulthood.
The technology has also been instrumental in mapping cell lineages, allowing scientists to trace the developmental fate of a single cell type across an organism’s lifetime. By using a floxed reporter gene that permanently changes color upon Cre-mediated deletion, researchers can visualize the family tree of specific cells. This helps scientists understand how these cells contribute to tissue formation, repair, and disease progression.