Preparation And Identification Of CRISPR/Cas9 Mediated FGF5 Knockout And Noggin Overexpression In Transgenic Mice By Microinjection Technique

The growth and development of hair follicle is regulated by a series of growth factors and signaling pathways,the fibroblast growth factor families and the bone morphogenetic proteins are two groups of regulatory factors of them.The effect of FGF5 on the length of hair shaft has been widely proved,Noggin is involved in the regulation of hair follicles' growth by inhibiting the function of BMPs.CRISPR/Cas,the new third generation genome editing techniqueo,which is simple,time-saving and efficient,its applications in livestock breeding can greatly improve the breeding efficiency,shorten the breeding time,reduce breeding cost,and accomplish many works,which are difficult to complete before.The objective of this study was to establish a mouse model of FGF5 knockout and Noggin overexpression by CRISPR/Cas system and pronuclear microinjection technique.By studying the effect of these two genes on hair follicle development,we provided references to speed up the development of new breed of Cashmere goat.Depilation experiments were used to compare the speed of hair regrowth in four groups;the morphology and quantity of murine hair follicles were observed in paraffin section;the expression of genes related with hair follicles development in the skin tissues were performed by real-time PCR.1.The preparation of transgenic miceInjected FGF5-gRNA,pKI-Noggin vector and Cas9 plasmid into mouse zygotes by pronuclear microinjection technique,these vectors were constructed on the basis of the action mechanism of CRISPR/Cas9 system.268 injected embryos were transplanted into pseudopregnant mice,36 newborn mice were obtained.After identification,we obtained a total of six different types of positive mice.They were three FGF5 knockout mice,two overexpression of Noggin mice and a FGF5 gene knockout and Noggin overexpression mice,the total positive rate was 16.7%.2.The detection of transgenic miceGene knockout mice and positive mice were identified by depilation,paraffin section and real-time PCR.The results showed that,compared with wild-type mice,the speed of hair regrowth of FGF5 knockout mice was slow,their hair follicle growth independently but the distribution was messy,their diameter of hair follicle unchanged and the number of hair follicles increased;the speed of hair regrowth of Noggin overexpression mice was slower than FGF5 knockout mice,their hair follicle clustered growth,and the distribution was regular,their diameter of hair follicle increased and the number of hair follicles decreased;the speed of hair regrowth of FGF5 gene knockout and Noggin overexpression mice was slower than Noggin overexpression mice,their hair follicle clustered growth and the distribution was regularly,their diameter of hair follicle increased and the number of hair follicles decreased.The results of real-time PCR showed that,compared with wild-type mice,the mRNA levels of Noggin in mouse skin tissue in No.92,No.601 Noggin overexpression mice and No.633 FGF5 gene knockout and Noggin overexpression mice were 2.67,8.36,35.35 times.The three transgenic mice can express the mRNA of Noggin.In addition,the relative expression levels of ?-catenin,VEGF,T?4 in these three different types of mice also increased in different degrees.