Generation Of Csnk1 Epsilon Overexpression Transgenic Mice By Pronuclear Microinjection And Its Detection

Casein Kinase 1 (Csnkl) belongs to the serine-threonine protein kinase. Mammals have seven family members, they are alpha, beta 1, gamma 1, gamma 2, gamma 3, delta, and epsilon. It has been suggested that the deletion or the mutation of Csnkl epsilon may have an effect on the hair follicles development and growth CRISPR/Cas system is derived from a natural adaptive immune system in bacteria that the system can modify the target gene by RNA guidance Cas protein. For further investigation in Csnkl epsilon on the role of the hair follicles’growth, in this study we designed a CRISPR/Cas9 system makes Csnkl epsilon knock in rosa26 site in the murine genome. Then a Csnkl epsilon overexpression transgenic mice were produced by pronuclear microinjection techniques. A positive mouse was identified by PCR and Southern blot, and the expression levels of the positive mouse through real-time PCR and western blot method were performed respectively.1. The construction of sgRNA vector and targeting vectorIn this study, five 20nt sgRNA fragments were designed by CRISPR software from MIT. Taking the sgRNA-T2 as a template, U6 promoter fragments and sgRNA backbones were amplified by PCR, then the complete sgRNA vectors were constructed finally. The targeting vector consists of a human K14 promoter fragment, which can direct a skin specific gene expression, and the homology between human and mice is known as 98% by sequencing. In order to testify the activity of the promoter, we constructed a pK14-DsRed vector which contains the red fluorescent protein gene to detect the expression in cellular level next step. According to the principle of the homologous recombination, two 1kilo-basepairs size upstream and downstream homologous arms at the site within the first intron in murine Rosa 26 gene were constructed respectively. The complete targeting vector was constructed.2. The cellular level relevant detectionThe red fluorescent protein expression vector pCMV-DsRed was introduced into mouse embryonic fibroblasts (MEF) by electroporation, the transfection condition was optimized as 160V/5. Then Cas9 and sgRNA vectors were co-transfected into MEF under the same condition. The genome DNA was extracted after 48 hours cell culture. The polymerase chain reaction was performed by primers overlapping the target sites. After that the hybridized PCR productions were identified by Surveyor mutation detection kit. Then the sgRNA targeting site was determined at 10960bp in the first intron of Rosa26 gene, the knockout efficiency is 36.52%. In the same way, the pK14-DsRed vector was also transfected into MEF. Through observation of the red fluorescence expressing in cells, we could know the promoter is working. That could be used for the next step. Finally, Cas9 and sgRNA as well as targeting vectors were co-transfected into MEF, PCR results show that the vector confirms with the conditions of homologous recombination, it can be used for the next step study on establishment of transgenic mice. At the same time, the detecting primers can be used for the identification of the positive mice.3. Production and identification of Csnk1 epsilon transgenic mice18 transgenic mice were obtained using the Csnkl targeting vector by pronuclear microinjection, according to the homologous recombination principle. One reandom integration positive mouse was identified by PCR and Southern blot. While there was no targeted integration offspring identified. One positive F1 generation from random integration mouse was obtained. That indicates the founder could stably inherited exogenous DNA to offspring. The results showed we successfully produced Csnkl epsilon transgenic mouse model, and it provided an experimental basis for investigation of the function of the gene in future.4. Detection of csnkl epsilon transgenic mice in expressing levelFirstly, the transgenic mouse was detected in mRNA level. Setting GAPDH as a reference gene, the real time PCR reactions were performed. The results showed that mRNA expression quantity of the positive was 2.65 fold than littermates. Secondly, a protein level detection was performed. Setting alpha tubulin as a reference gene, the results showed that protein expression quantity was 1.234 fold than littermates. All these results indicated that the target gene has been successfully integrated into murine genome and could be stably expressed.