In genetics, “floxed” describes a segment of DNA intentionally bracketed by specific, engineered sequences. This process is like placing bookmarks around a chapter in a book that you intend to modify or remove later. The term is a blend of “flanked by LoxP,” which alludes to the components used. This genetic modification prepares a gene to be altered in a controlled manner, allowing scientists to study its function with precision. A floxed gene remains fully functional until a specific trigger is introduced.
The Molecular Components of Floxing
The technology behind floxing relies on two molecular tools from a bacteriophage. The first component is the loxP site, a short sequence of DNA not naturally found in organisms like mice or humans. Each loxP site is 34 base pairs long and acts as a recognition signal. These sites are inserted into the DNA to flank the gene of interest.
The second component is an enzyme called Cre recombinase, which functions like molecular scissors. Its purpose is to find and recognize the loxP sites within a genome. When Cre recombinase finds two loxP sites, it binds to them and cuts the DNA at these precise locations.
The interaction between Cre recombinase and loxP sites is highly specific. The enzyme does not act on random DNA sequences, ensuring only the segment of DNA between the two loxP sites is affected. This allows for targeted manipulation of a single gene without causing unintended changes elsewhere in the organism’s genetic code.
How a Gene is Floxed
Creating a floxed gene involves a genetic engineering process within embryonic stem cells. Scientists use homologous recombination to insert two loxP sites into an organism’s genome. This method allows for the targeted placement of these synthetic DNA sequences on either side of a specific gene of interest.
The process begins by designing a piece of DNA in the lab that contains the loxP sites and sequences that match the DNA regions around the target gene. When this engineered DNA is introduced into embryonic stem cells, the cell’s DNA repair machinery recognizes the matching sequences and swaps the original DNA with the engineered version. This results in the loxP sites being integrated into the desired locations.
These modified embryonic stem cells are injected into a developing embryo and carried to term by a surrogate mother. The resulting offspring is a floxed animal, typically a mouse, that carries the modified gene. The floxed gene is still functional because the loxP sites are placed in non-coding regions and do not interfere with its activity. The organism is healthy and shows no signs of the modification but carries the potential for a future genetic alteration.
Activating the Conditional Knockout
The utility of a floxed gene is realized when combined with the Cre recombinase enzyme. A floxed organism is bred with another genetically engineered organism that produces Cre recombinase. The gene for Cre recombinase is placed under the control of a tissue-specific promoter, a switch that only turns the gene on in certain types of cells. This makes the subsequent gene removal “conditional.”
When these two organisms are crossed, their offspring inherit both the floxed gene and the Cre recombinase gene. In these new animals, the floxed gene remains functional in every cell except for those where the tissue-specific promoter is active. In those specific cells, the Cre enzyme finds the loxP sites and snips the DNA, permanently deleting that gene from the cell’s genome.
For example, if scientists want to study a gene’s function in the liver, they use a mouse line where Cre recombinase is only expressed in liver cells. In the resulting offspring, the floxed gene will be deleted exclusively in the liver but remain functional in all other tissues. This creates a conditional knockout, allowing researchers to study the effects of losing a gene in a specific location.
Applications in Scientific Research
Creating conditional knockouts is valuable in studying complex diseases. Many genes are required for embryonic development, and a traditional knockout, where the gene is absent from the start, can be lethal to the embryo. This makes it impossible to study the gene’s function in an adult. With floxing, researchers can allow the gene to function normally throughout development and then trigger its deletion in specific adult tissues.
This technique is used in cancer research to inactivate tumor suppressor genes in specific cell types, mimicking how tumors develop. In neuroscience, scientists can study conditions like Alzheimer’s disease by deleting a specific gene only in brain cells. This allows them to observe how the gene’s absence contributes to disease progression without effects in other parts of the body.
The system can be made inducible, meaning Cre recombinase is only activated when an animal is exposed to a chemical like tamoxifen. This gives researchers control over not only where the gene is deleted but also when. This temporal control helps dissect the functions of genes in health and disease.