Cyagen has generated a number of conditional knockout mouse models with the using of CRISPR technology and the Cre-Loxp system for Cre conditional knockout (cKO). In this case, gene Slc30a7 conditional knockout mouse line was created in the C57BL/6N background and subsequently crossed with the proper Cre animals. The strategy of gene Slc30a7 cKO mice was to select exon 4 and exon 5 as conditional knockout regions. CRISPR technology was used for insertion of two LoxP sites flanking the cKO region for conditional removal using Cre conditional knockout technology.

Diagram 1. The schematic describes the first stage in developing a conditional knockout mouse model using CRISPR to generate a floxed mouse.

Figure 1. PCR genotype screening of the floxed mice. Amplify 5'LoxP and 3'LoxP fragments by using two different PCR primers, candidate positive transformant genomic DNA as template, wild-type and water as control. PCR results showed four mice were identified as potential founders.

Figure 2. Chromatograms of 5'LoxP and 3'LoxP PCR fragments from the floxed mouse. Genomic DNA extracted from individual mice born from microinjection of the embryos were subjected to genotyping PCR. Sequencing results showed candidate positive mouse No. 51 has LoxP insertions.