One design can obtain the whole-gene knockout mouse model first, and then obtain the conditional knockout mouse model after mating with a proper mouse model. Furthermore, a reporter gene can be knocked in during the target gene knockout.
2.Advantages and disadvantages
Advantages: (to be discussed) the target gene's expression status can be tracked according to the reporter gene's expression.
Disadvantages: the gene needs to be extensively analyzed when designing to avoid the down-regulated expression of an endogenous gene or expression of a truncated protein caused by gene knockout. The knockout of the target gene may cause embryonic death or expression dysregulation of other genes, so this risk should be fully analyzed when designing the project.
The KO first, conditional ready designing scheme knockouts the target gene by inserting a gene disruption box (a reporter gene with shear receptor and a resistance gene, as shown in the figure) in the intron. By inserting a loxP locus on the ends of the knocked out exon, a floxed (flanked by loxP) mouse obtained with the target gene knocked out and a reporter gene and resistance gene knocked in. According to the experimental needs, the whole-gene knockout and conditional knockout mouse models can be obtained simultaneously by using the Cre/LoxP recombination system or Flp/FRT recombination system. For example, when the floxed mouse mates with a mouse expressing Cre throughout the body, the mouse with the target gene knocked out and the reporter gene knocked in can be obtained. When the floxed mouse mates with the Flp mouse, the knocked out gene can be repaired, and the mouse with a conditional knockout of target gene can be obtained by mating with a mouse expressing Cre tissue specifically. The targeting strategy is shown in the following figure:
Track the knocked out target gene's expression according to the reporter gene's expression, thereby facilitating gene study
Drug target affirmation
Drug toxicological study