Construction Of FGF21 Knockout Mice Using The CRISPR-Cas9 System And The Study Of Its Effect Of Hair Growth Action

Fibroblast growth factor21(FGF21) is a member of FGF gene family, which can promote the transition to catagen in secondary hair follicles, presumably the gene deletion or mutation can promote hair follicle development. CRISPR-Cas9 system is a new gene targ-ting modification technology developed from bacterial acquired immunity. In this study, FGF21 global knockout mice were generated bymicroinjection with Cas9 and gRNA plasmid. Depilation experiment was made to compare the speed of hair regrowth in two groups; The morphology and quantity of murine hair follicles were observed in paraffin section; The expression of genes related with hair follicle development in the skin tissues was performed by real time-PCR and Western Blot.1. The design and construction of gRNA expression vectorIn this study, according to the coding sequence(CDs) of FGF21, our pairs of gRNA with 20nt long were designed through the CRI-SPR Design software of the Massachusetts Institute of technology(h-ttp://crispr.Mit.edu/). gRNA-T2 served as a template for PCR to ob- tain complete gRNA.A suitable overlapping target site PCR primers for PCR amplification was designed. Four pairs hCas9 vector and gRNA were transfected respectively together into mouse embryonic fibroblasts to choose two gRNA with higher efficiency to produce knockout mice by pronucleus injection.2. The generation of knockout mice by microinjectionIn this study,340 injected embryos were transplanted into receptors and 63 mice were born in which 15 heterozygous mice and 3 homozygous mice, with a total knockout efficiency of 28.6%.3.The detection of FGF21 knockout miceGene knockout mice and wild-type mice were identified by depilation, paraffin section, real time quantitative PCR and Western Blot. Results of depilation indicated that compared with the wild-type mice, the speed of hair regrowth was slower and there was no hair on the depilated skin in knockout mice. Both the hair follicle diameter and quantity of the knockout mice reduced from paraffin section observation. Compared with wild-type mice, the mRNA levels of FGFR2, AKT and β-catenin were decreased dramatically (P< 0.05), while the level of GSK-3β was stable (P>0.05). Compared to wild-type mice, the FGF21 knockout mice had decreased the protein levels and phosphorylation of ERK1/2 and AKT. The protein expression of p-LEF gene wasn’t detected in wild-type mice and knockout mice. Further, in knockout mice the protein level of p-P38 was lower than that of wild-type mice.These results provided experimental evidence to understand the biological effects and growth cycle regulation mechanism of FGF21 on hair follicle development.