In genetic research, many scientists will mention terms like “gene recombination” and “flox sequence,” which might sound simple, but are actually extremely significant and carry a lot of weight in the field. Flox sequences, for instance, are DNA sequences that are flanked by LoxP sites for the purpose of targeting the genetic sequence in question with the Cre-lox recombination system. This system is one of the most well-designed and useful methods for genetic manipulation, which uses the Cre enzyme to target floxed sequences and either delete, translocate or invert the gene.
A flox sequence is obtained by inserting loxP sites on both sides of a gene to target it most typically for deletion, as part of the process of creating a knockout gene. Depending on the loxP sites’ orientation, the use of two loxP sites with the same orientation will lead to the excision of the gene, while opposite orientations will cause inversion. Flox sequences allow for the complete removal of the gene, but partial deletion is also possible. The incomplete deletion of Cre-mediated excisions has been found to lead to phenotype differences that can be used to study a variety of unusual genetic changes that can occur in the case of some diseases.
A flox sequence is obtained and targeted through mouse ES cells. Laboratory mice are used because their genome presents genetic researchers with a unique opportunity to study a mammal whose genetic code is similar to humans, but is also small and easy to work with to obtain data. The Cre-lox process allows for the targeting and floxing of a specific gene within the ES cells of a mouse. The gene is then subsequently deleted or inverted, depending on what the experiment might require. The gene is targeted through floxing, and the process of flanking the gene by specific LoxP sites is usually completed using homologous recombination of a targeting vector in the mouse’s ES cells, often deleting the entire gene to obtain a null mutation.
Once a flox sequence is obtained, the Cre-lox recombination method is used to target that sequence and either completely remove it, invert it, or make a unique change that precisely manipulates the genome of the mouse. Some methods can include the gradual inactivation of the gene over time, triggering its deletion by a certain event that would occur in the mouse’s development, or the specific targeting of ES cells that would only be used in some of the mouse’s tissues or organs, such as the skin or the heart, in order to study tissue-specific gene inactivation.
Floxed genes and the Cre-lox system can be very efficient when it comes to facilitating the study of various genetic changes, disorders and developmental impairments. However, the Cre-lox process is by no means perfect, and further research and progress are needed to obtain better results. Because there’s still not enough knowledge about usable insertion sites and the use of hemizygous mice to produce a certain flox sequence, the results can still be uncertain, leading many researchers to conclude that homozygous mice should be used to a greater extent for the time being.
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