[Application of CRISPR/Cas9 Gene Editing Technique to Establish S1PR5 Gene Knockout Mice]

To establish the S1PR5 gene knockout mouse model by using CRISPR/Cas9 gene editing technique so as to provide the tool for studying the regulating role of sphingosine-1-phosphate receptor 5 (S1PR5) in allogeneic hematopoietic stem cell transplantation.

Single guide RNA (sgRNA) plasmids against the exon 3 of S1PR5 were designed and constructed. Then the sgRNA and hCas9 were transcribed by T7 RNA polymerase in vitro. Cas9 mRNA and sgRNA were mixed and microinjected into fertilized eggs of C57BL/6 mice. T7E1 digestion and gene sequencing were used to detect the mutations of S1PR5. Quantitative PCR (qPCR) and Western blot were used to detect the expression of S1PR5.

Finally 2 kinds of F2 generation of homozygous S1PR5 deficent mice (S1PR5-170/-170 mice and S1PR5-215/-215 mice) were gained, and in these 2 model mice the S1PR5 did not express at mRNA and protein levels.

A mouse model with S1PR5 dificiency has been successfully established, which shall lay a foundation for future investigation of S1PR5.