The KO first (Conditional Ready) mouse model represents a groundbreaking advancement in genetic research. It offers a versatile approach to studying gene functions, enabling scientists to dissect complex genetic pathways with precision. This
model serves as a key tool in understanding gene expression and regulation in mammalian systems.
The KO first (Conditional Ready) model employs a strategic arrangement of LoxP sites, flanking a target gene fragment, and an SA-IRES-reporter fragment, bordered by FRT sites. This unique configuration offers two distinct paths for genetic research:
Cre-expressing Mice Mating:
Results in the removal of the Neo gene and Flox region.
Activates the reporter gene, allowing scientists to trace the expression patterns of the target gene.
Flp-expressing Mice Mating:
Leads to the excision of the SA-IRES-reporter fragment.
Creates conventional Flox mice, which can be crossed with tissue-specific Cre-expressing mice for diverse conditional knockout models.
A schematic diagram representing the process of genetic modification using Cre-Lox and Flp-FRT recombination systems.
KO First Allele: This represents the targeted gene with various elements inserted:
A '1' blue box symbolizes an exonic region that remains unmodified.
SA (splice acceptor) followed by IRES-LacZ-polyA is a reporter cassette allowing for the expression of the LacZ gene, which will produce a blue color in the presence of X-gal, indicating successful recombination.
The 'Neo' cassette is a selectable marker, providing resistance to the antibiotic neomycin.
LoxP sites (green triangles) flank the Neo cassette and part of the gene.
An FRT site (purple diamond) is located after the SA-IRES-LacZ-polyA sequence.
Cre Recombination: The Cre enzyme recognizes the LoxP sites and excises the DNA between them, resulting in the 'Reporter (KO) allele'. This allele retains the SA-IRES-LacZ-polyA sequence but loses the Neo cassette.
Flp Recombination: The Flp enzyme recognizes the FRT sites and excises the DNA between them, resulting in a 'Floxed allele'. This allele has LoxP sites around the second exon (as marked by a '2' blue box), which can be later removed by Cre recombinase.
KO Allele: Finally, Cre recombinase acts on the Floxed allele to remove the second exon, resulting in a KO allele, where the '2' box is missing, indicating that the exon has been successfully knocked out.
The colors and shapes (blue boxes, green triangles, purple diamonds) are used to represent coding and non-coding regions of the gene, and the recombination sites, respectively.
Applications
Tracing Gene Expression:
Understand where and when specific genes are active in living organisms.
Essential in developmental biology and disease modeling.
Conditional Knockout Models:
Study gene function in specific tissues or at particular development stages.
Crucial for researching complex diseases like cancer or neurological disorders.