Effects Of FGF5 Gene Knockout Mediated By CRISPR-Cas9 System And Noggin Overexpression On Hair Growth Related Factors

Gene editing technology, emerged in the 1980 s, open the upsurge of gene function research. CRISPR-Cas9 is a new gene editing techniques emerging in recent years, which have many advantages, for example, it is easy to build vector and the rate of gene knock out is very high. Cashmere goat as the unique biological resources in our country, has become the best varieties in the world. It is famous for the highest capacity and quality of the cashmere producing. The cashmere even more has the reputation of "biological gold". So screening the high cashmere producing varieties has a very important significance and the huge market potential. Hair growth is a complex mechanism of gene regulation. Previous studies have shown that the overexpression of Noggin gene and knock out of FGF5 gene will promote hair growth. This experiment mainly for hair growth positive regulation gene-Noggin and negative regulation gene-FGF5, using the CRISPR-Cas9 and gene random integration technology, to research the efficiency of CRISPR-Cas9 and detecting after theFGF5geneknockout the overexpression of Noggin gene, the mRNA expression quantity of the gene which related to hair growth at the transcription level, to predict how the FGF5 and Noggin double gene editing affect hair growth.1、The transfection conditions optimized of Mice fetal fibroblastsThis experiment using pCMV-DsRed electrophoretic transfer method, by plasmid quality gradient, electric transfection voltage and pulse combination time gradient, research the best transfection conditions of mice fetal fibroblasts. The results show that the best combination of the total p lasmid quality^ the voltage and pulse time is 10μg/160v/5mm.2、Build the Noggin Random integration vector and the Noggin Fixed -point integrated vectorThe experiment using the CRISPR/Cas9 to knock out the Fixed-point of FGF5 gene, successfully build gRNA-FGF5, the guide RNA, which can be successfully lead Cas9 protein to purpose targeted points to cut the double-stranded DNA. This experiment also successfully constructed two Noggin gene specific expression vector of the skin. The one is Noggin random integration pKI-Noggin, using study the overexpress of the Noggin gene. The other one is Noggin fixed-point integration pKI-Noggin, for building the gRNA-FGF5 induced Noggin Fixed-point integrated animal model.3、Testing of gRNA efficiency and the Noggin Random integration vectorWe test efficiency of the above two vectors:gRNA-FGF5 and Noggin random integration pKI-Noggin of the skin. First, transfect Cas9 and gRNA-FGF5 plasmid together to the mice fetal fibroblasts through the electric transfection method, extracted total cells genome as template with FGF5 point detection primer for PCR, the target fragment purified by PCR product purification kits and be a source of hybrid bands after annealing, using Surveyor mutation detection kit to detect, the results shows that:compared the electrophoresis fragment cut by Surveyor with the kit, the gRNA-FGF5 has high cutting efficiency. The experiment also applies the method of PCR sequencing make the T-A targeted fragment to 19 T vector, pick 20 positive recombinant to Beijing Huada gene company for sequencing. In the 20 positive recombinants, there are eleven recombinants have different degrees of base deletion and insertion, the conclusion obtained that the mutation rate is about 55%.Second, we transfect pKI-Noggin plasmid to the mice fetal fibroblasts, after 48 h, extract total protein, using western blot method, the results show that the Noggin protein relative expression is 1.41 times that of the control cells of the wild type.4、Effects of FGF5 gene knockout and Noggin overexpression on hair growth factors transcriptionsThe experiment transfect Cas9, gRNAFGF5 to the MEF cells get EG1;Transfect pKI-Noggin to the MEF mice cells get EG2;Transfect Cas9, gRNA FGF5, pKI-Noggin to MEF mice cells get EG3, and use nontransgene mice CG MEF cells as control. After 48h transfection, use the total cDNA of 4 kinds of cells as template, by the method of QPCR detection the mRNA relative expression of β-catenin、Vegf、Tβ4. The results showed that the relative expression of β-catenin、Vegf、Tβ4 in the cells of FGF5 knockout was2.82/1.37/1.47 times of the blank control, the relative expression of β-catenin、Vegf、Tβ4 in the cells of Noggin randomly integrated cells was2.44/1.32/1.37 times of the blank control, the relative expression ofβ-catenin、Vegf、Tβ4 in the cells of FGF5 knockout and Noggin randomly integrated cells was3.22/1.69/1.71 times of the blank control.