Important Facts about the Producing a Floxed Allele to Create Complete Knockouts
An allele is essentially a version of a given gene. While some genes are only present once in the body, depending on the species and the particular gene in question, others have two or more alleles of the same gene that result in various phenotypic traits, such as different colored eyes, skin or hair. In humans, as in the case of most multicellular organisms, each gene has two alleles of the same gene, which are associated with the two sets of chromosomes they have. In many cases, subtle mutations occur in germ cells, which then pass on the mutated allele to the following generation, leading to physiological changes that drive the species’ evolution. These mutations can also bring about genetic conditions that may cause severe harm.
The reason why genetic researchers study the production of floxed alleles in animal models such as mice is to learn more about these genetic conditions. Since the mouse genome is significantly close in comparison to human DNA, it makes for an excellent subject for studying these conditions. The means by which that is done is the Cre/lox recombination system together with the formation of a floxed allele that leads to the creation of complete knockout mice.
The production of knockout mouse models that specifically target a mutated allele is very important for the further study of genetically transmitted diseases. Since some diseases impair a certain gene by generating mutated alleles that nullify it, creating knockout mouse models of that particular gene might be necessary to observe the effects that eliminating the gene may have in the development of the mouse. At the same time, certain mutations can be more likely to cause genetic disorders than others, and the careful and intelligent use of floxed alleles and complete knockouts can help classify them more easily.
Lesser Known Facts about Cre/lox Recombination and Floxed Allele
Anyone who seeks to use the Cre/lox recombination system to produce a floxed allele and knockout mice should be aware of a few important and lesser-known facts that experts tend to point out. The first of these is that the system is actually more efficient in converting floxed alleles to complete knockouts if the alleles are passed down from the mother. While this isn’t always the case, and the parental inheritance pattern doesn’t matter in the case of all Cre strains, it is always best to compare maternal and paternal transmission results when possible.
Another essential fact to remember is that it’s actually possible to generate a knockout out of a floxed allele even if the mouse doesn’t necessarily carry a Cre transgene. This option is prevalent in the case of Vasa-Cre strains, where the Cre mRNA protein is expressed even when the transgene is not present within the oocyte. Strains that feature maternal Cre expression in the oocyte can help save a significant amount of time during the process of converting floxed alleles to knockouts, since no additional breeding will be needed in order to eliminate the Cre strain after the recombination process is confirmed.
It is also important to remember that Cre recombinase can be toxic in ES cells, causing difficulties when linking expression to genes that are active during embryonic time points. Random integration transgenic mice produced via pronuclear injection have been used as an alternative to avoid embryonic stem cell lethality; however, these models do not faithfully reflect endogenous gene expression patterns. To solve this problem, an approach is needed that enables native gene expression patterns while avoiding ES cell toxicity. To eliminate concerns about ES cell toxicity, it’s possible to generate your mouse line by splitting Cre recombinase in half using a FRT-flanked neomycin selection cassette. This split design can be used for gene replacement or co-expression knockin, generating Cre recombinase expressing mouse lines that follow the native expression patterns of any target gene, regardless of expression location or timepoints.