Conditional gene knockout (CKO) refers to the process of knocking out a specific gene in particular tissues or at specific stages of cell development. When a gene knockout is lethal in a homozygous state, conditional knockout is required. The conditional knockout generally uses the Cre-loxP system, where the method involves inserting loxP sequences on either side of the critical region of the gene to be knocked out. After preparing the loxP mice, they are crossed with Cre mice to obtain loxP homozygous mice that are also Cre-positive. In these mice, under the action of Cre, loxP recombination occurs, and the sequence in between is deleted, achieving the goal of gene knockout.

There are already hundreds of Cre tool mice available, with fewer rats, but it is also possible to purchase or customize Cre tool mice according to research needs to achieve the knockout of the target gene in different tissues and organs. When Cre is fused with ER/ERT2, tamoxifen can be used to control the nuclear entry of Cre, achieving temporal control of gene knockout.

Figure 1. Through CRISPR assistance, two loxP are inserted on either side of a critical exon through homologous recombination. Under the action of Cre, the exon between the loxP is deleted, achieving conditional gene knockout.

1. Technical Process:

Targeting vector design → Vector construction → Microinjection/EPS targeting → Mouse identification

2. Technical Advantages:

（1）Used to prepare genetically modified animals for genes that are lethal when knocked out homozygously.

（2）For non-lethal genes, conditional knockout can also knock out genes in different tissues at different times to obtain more precise experimental data.

3. Successful Case: Using CRISPR/Cas9 technology, loxP sequences were added to the fourth and fifth exons of the SPOP gene in mice in the same direction, and then mated with Fstl1-CreERT2 knock-in mice. After obtaining double-positive mice, tamoxifen induction can be used to obtain SPOP gene knockout mice (Li Q, et al. 2020). 

Figure 2. Schematic diagram of the preparation of conditional knockout mice for the Spop gene. 

Figure 3. Western blot analysis of SPOP protein expression in mice.

References:

1. Li Q, Wang F, Wang Q, et al. SPOP promotes ubiquitination and degradation of MyD88 to suppress the innate immune response. PLoS Pathog. 2020, 16(5):e1008188.