Construction And Evaluation Of PIN1 Gene Knockout Adult Mouse Neural Stem Cells Lines

BackgroundPeptidyl-prolyl cis/trans isomerases,NIIMA-interacting1(PIN1)is the only cis-trans isomerase that can specifically recognize the phosphorylated serine/threonine-proline(PSER/Thr-pro)in proteins.It plays an important role in cell proliferation,differentiation and survival by catalyzing cis-trans isomerization of substrates.The expression of PIN1 is tightly regulated,and its dysregulation plays an important role in many pathological conditions,especially cancer and neuropathic diseases such as Alzheimer's disease and ischemic stroke.Adult neural stem cells(NSCs)have the ability of self-renewal,differentiation and migration.When the nervous system is injured,such as neurodegenerative diseases and ischemia and hypoxia injury,NSCs can proliferate,migrate and differentiate into new nerve cells to compensate for structural and functional damage.PIN1 is highly expressed in the nervous system,but the role of PIN1 in adult NSCs has not been reported.PIN1 gene knockout can lead to abnormal symptoms such as abnormal organs,premature senility and sterility in PIN1 null mice,it is necessary to establish the mouse strain with conditional gene knockout and the adult neural stem cell line with PIN1 gene knockout in vitro.Conditional gene knockout of PIN1 in adult NSCs in vivo will be obtained by injecting Tam at 2–month old Nestin-Cre ER^T2;PIN1^F/F mice.In order to establish PIN1 gene knockout line in adult NSCs cultured in vitro,high-quality adult NSCs must be obtained first.At present,it is somewhat difficult to obtain a large number of NSCs in vitro,and their proliferation rate is low.With the increase of in vitro culture generations,NSCs are more prone to differentiation.Therefore,it is necessary to optimize the in vitro culture conditions of NSCs,improve the survival rate and life span of NSCs in vitro isolation and culture,and maintain the characteristics of NSCs.In addition,there are few reports on targeted gene editing of adult NSCs in vitro.We optimized and used CRISPR/Cas9 gene editing technology to establish a PIN1 gene knockout NSCs line for the first time,which can not only provide new ideas for precise gene editing of NSCs,but also to study the role of PIN1gene in NSCs.ObjectiveBy optimizing the culture conditions of NSCs in vitro,the life and survival rate of NSCs cultured in vitro were prolonged and the characteristics of NSCs was maintained.The PIN1 gene of NSCs was knocked out by CRISPR/Cas9 gene editing technology,and the PIN1 gene knockout neural stem cells line was established.The self-renewal,proliferation and differentiation abilities of PIN1 gene knockout neural stem cell lines were analyzed.In addition,the proliferation and differentiation levels of adult NSCs in vivo were detected,and Nestin-Cre ER^T2;PIN1^F/+mice were identified,which laid a foundation for the further study of PIN1 gene function in adult NSCs.Methods1.NSCs were extracted from the SVZ region of 8-week-old C57BL/6 mice,and cultured in vitro under optimized in vitro culture conditions.Immunofluorescence(IF)was used to specifically express the protein Nestin,proliferation ability and multidirectional differentiation potential of neural stem cells Perform testing.2.Fluorescence quantitative PCR was used to detect the expression level of PIN1 gene in NSCs and mouse tissues,and IF was used to detect the expression of PIN1 in NSCs cultured in vitro.3.Design and synthesize Single guide RNA(sg RNA)targeting mouse PIN1 gene using online tool(https://zlab.bio/guide-design-resources),using p X330 plasmid as skeleton.The Cas9 target vector of PIN1 gene was constructed,and mouse adult NSCs were transfected.The PIN1 gene knockout monoclonal cells were identified by Puromycin screening and sequencing.The expression level of PIN1 protein was determined by Western blot,and the self-renewal,proliferation and differentiation abilities of PIN1 knockout NSCs were analyzed by IF.4.Use Immunohistochemistry(IHC)and IF to detect the expression and distribution of PIN1 in SVZ of adult mice,and obtain Nestin-Cre ER^T2;PIN1^F/Fmice by breeding and crossing.ResultsThere are NSCs in the SVZ and SGZ area of adult mice.Brd U labeling and Ki67staining show that the NSCs in the SVZ area are in a proliferating state.Using optimized in vitro culture conditions for NSCs,IF results showed that the positive rate of Nestin in multiple generations of NSCs cultured in vitro reached more than90%.The results of Brd U labeling experiments showed that the Brd U positive rate of primary NSCs cultured in vitro for 24 hours was 71.8%.After NSCs induce differentiation,immunofluorescence detection of astrocyte specific marker GFAP,neuron specific marker TUJ-1,and oligodendrocyte specific marker OLIG 2 were all positively expressed.NSCs cultured in vitro still have the characteristics of proliferation and multi-differentiation stem cells after multiple passages.IHC and IF results show that PIN1 is highly expressed in neural stem cells cultured in vivo and in vitro.CRISPR/Cas9 gene editing technology was used to successfully construct PIN1knockout NSCs,and 9 single cell clones were obtained.Western blot and IF detection results showed that there was no PIN1 protein expression in PIN1 knockout NSCs.IF results showed that the proliferation ability of adult NSCs cultured in vitro was improved after PIN1 gene knockout,and the differentiation of neurons was significantly inhibited without affecting the production of astrocytes and oligodendrocytes.At present,Nestin-Cre ER^T2;PIN1^F/+mice have been obtained,which lays the foundation for the follow-up study of the role PIN1 gene in adult NSCs in vivo.ConclusionsIn this study,by optimizing the in vitro culture conditions of NSCs,the lifespan and survival rate of NSCs cultured in vitro were prolonged,and the ability of self-renewal was maintained.The use of CRISPR/Cas9 gene editing technology to successfully obtain a PIN1 gene knockout neural stem cell line proves that this technology provides a fast,flexible and efficient tool for precise gene editing of NSCs.The proliferation ability of adult NSCs cultured in vitro was improved after PIN1gene knockout,and the differentiation of neurons was significantly inhibited without affecting the production of astrocytes and oligodendrocytes.The Nestin-Cre ER^T2;PIN1^F/+mice were obtained through breeding,which laid the foundation for the later study of the role of PIN1 gene in adult NSCs in vivo.