Conditional knockout mice (also known as Flox mice) are mice whose target genes contain paired loxp sites. After mating them with Cre mice, target genes can be knocked out in specific tissues or cells.
If Flox mice are mated with Cre mice widely expressing Cre or expressing Cre only in germ cells, systemic KO mice can be obtained. The breeding procedure is shown in the figure. In the ES targeting vector, the screening gene neo is located between two loxp sites and it is not necessary to remove neo separately. The structure is 5' arm-loxp-flox-frt-neo-frt-loxp-3' arm and the neo gene can be simultaneously removed by directly mating with mice expressing Cre in the gonad.
Figure 3. Breeding procedure to obtain systemic and homozygous KO mice from conditional knockout mice.
The procedure shown in Figure 4 can be adopted if breeding is carried out by mating with mice showing tissue-specific Cre expression. In general, heterozygous flox mice transfected with Cre recombinase are mated with heterozygous flox mice to obtain homozygous conditional knockout mice carrying integrated Cre enzyme. At this time, homozygous flox mice in the same litter that do not carry integrated Cre enzyme are generally selected as the control group for the experiment.
Figure 4. Breeding procedure 1 to obtain tissue-specific knockout mice.
The protocol in Figure 5 can also be used to increase the proportion of mice in the experimental group.
Figure 5. Breeding procedure 2 to obtain tissue-specific knockout mice.