Establishment Of Cell Model Based On C9orf72 Gene And In Vitro Experimental Study

Amyotrophic lateral sclerosis(ALS)and frontotemporal dementia(FTD)are fatal and devastating neurodegenerative diseases.The researches have shown that a hexanucleotide GGGGCC repeat expansion in the first intron of the C9orf72 gene is the most frequent genetic cause of ALS and FTD diseases(C9orf72 associated ALS and FTD,C9ALS/FTD).C9ALS/FTD patients generally carry one normal allele comprised of 3-30 copies of the repeats and an expanded pathogenic allele with repeats numbering in the hundreds to thousands.At present,there are three credible possible mechanisms by which the repeat expansion may cause neurodegenerative diseases.First,the mRNA expression of C9orf72 was decreased because of the GGGGCC repeats expansion,so haploinsufficiency for C9orf72 protein may trigger disease pathogenesis.Second,sense and antisense transcripts containing expanded GGGGCC repeats can form RNA foci which can recruit RNA binding proteins,therefore interfering with the normal functions of these proteins.Third,sense and antisense transcripts containing expanded GGGGCC repeats can also be translated into dipeptide repeat proteins that may have toxicity to neurons by the protein translation termed repeat-associated non-ATG(RAN)translation.However,which mechanism may have a crucial effect on the disease is still unknown.In order that our lab has an advantage of revealing the pathogenic mechanism of the C9orf72 GGGGCC hexanucleotide repeat expansion in C9ALS/FTD disease pathogenesis,we preliminarily conducted our study from the following four aspects:1.Preparation of monoclonal antibodies against human C9orf72 protein:Monoclonal antibodies against human C9orf72 protein were prepared by cross-immunizing BALB/c mice with purified C9orf72 prokaryotic protein and DNA vaccine expressing C9orf72 protein.Finally,it was confirmed by Western blot that the obtained the antibody(46A8)was specific to C9orf72 protein expressed by prokaryoctic or eukaryotic vector.So the antibody can be used as an important experimental tool for the subsequent study of C9orf72 protein function.2.Construction of the HEK293 cell model of C9orf72-T2A-Luciferase KI:First,we constructed a targeting vector for co-expression of sgRNA and Cas9 which can generate double-strand break(DSB)in the region after the last coding exon of the C9orf72 gene and constructed the donor vector mediating a specific integration of T2A-Luciferase into the site right after open reading frame of C9orf72 by up-stream homologous arm and down-stream homologous arm.Then the targeting vector and the donor vector were co-transfected into HEK293 cells.After screening with drug-resistance and cell cloning,it was demonstrated by PCR and sequencing that we had successfully obtained the C9orf72-T2A-Luciferase KI HEK293 cell model with the insertion of T2A-Luciferase gene into C9orf72 single allele.Finally,the Luciferase kit and Real-Time PCR assay indicated that the quantity of the expression of Luciferase in this cell model was positively correlated with C9orf72 gene mRNA.So the expression of the C9orf72 gene can be initially determined by detecting the activity of Luciferase in this cell model.The C9orf72-T2A-Luciferase KI HEK293 cell model has laid an experimental foundation for high-throughput screening for small molecule deficiency drugs targeting C9orf72 gene leading to the haploinsufficiency of C9orf72 protein and has great potential application in the field.3.The C9orf72 gene knockout cell model was utilized to study the role of C9orf72 gene in regulating cellular inflammation:We designed sgRNAs to target the first encoding exon of C9orf72 gene and constructed lentiviral vector co-expressing sgRNA and Cas9.U937 cells were infected with lentivirus carrying sgRNA and Cas9,then screened by drug-resistance and cell cloning,we successfully obtained the C9orf72 knock-out cell models(C9orf72+/-)in which one of allele of C9orf72 gene was mutated,which was confirmed by PCR and sequencing.In order to detect the role of the C9orf72 gene in regulating cellular inflammation,C9orf72+/-U937 model was stimulated in vitro with LPS at a final concentration of 1 μg/mL and then the expression level of cytokine IL-1 β,IL-6,IL-8,IL-10 and TNF-α cytokine mRNA was detected by Real-Time PCR.The results showed that the expression level of IL-6,IL-8,TNF-α and IL-10 cytokine mRNA were significantly increased in the C9orf72+/-U937 cell model compared with normal U937 cells,indicating that C9orf72 gene plays an important role in regulating the inflammation of U937 cells.The cell model also laid a foundation for the study of the function of the C9orf72 gene and the exploration of the possible mechanism of C9ALS/FTD disease resulting from haploinsufficiency of C9orf72 protein.4.Establishing the HEK293 cell model of C9orf72 GGGGCC hexanucleotide repeats KI:We synthesized(GGGGCC)6 DNA primers with restriction enzyme sites at both ends and obtained about 200 interrupted GGGGCC repeats by restriction enzyme ligation.Then,a targeting vector co-expressing sgRNA and Cas9,which can generate DSB in the first intron region of the C9orf72 gene was constructed.At the same time,we constructed the donor vector mediating a specific integration of(GGGGCC)200 repeats into the first intron region of the C9orf72 by up-stream homologous arm and down-stream homologous arm.Then the targeting vector and donor vector was co-transformed into HEK293 cells.After screening with drug-resistance,we initially obtained the cell model in which(GGGGCC)10-70 repeats have been specifically integrated into the first intron region of C9orf72,which was confirmed by PCR and sequencing.The establishment of this cell model laid a foundation for the study of the mechanism of the diseases resulted from GGGGCC repeats and the establishment of animal model with C9orf72 GGGGCC hexanucleotide repeat expansion by CRISPR/Cas9.In summary,in this study,we successfully constructed three different cell models for C9orf72 gene study which could lay an important foundation for our lab to subsequently uncover the molecular mechanism of ALS and FTD caused by C9orf72 GGGGCC hexanucleotide repeat expansion and had a great significance and application values.