Efficient Generation Of Chondroitin Synthase 3 Knock-out Mouse Model And Its Implications In Intervertebral Disc Degeneration

Background and ObjectiveLow back pain?LBP?is extremely common and has a high rate of disability,which seriously affects the life quality of patients.The economic cost of LBP is huge at each year,which puts a heavy burden on families and society.It is currently believed that the primary cause of LBP is intervertebral disc degeneration?IDD?.The main changes of IDD are the reduction of intervertebral disc water content and intervertebral space stenosis.The water content of the intervertebral disc is mainly determined by aggrecan,and the water retention capacity of the aggrecan is mainly dependent on chondroitin sulfate?CS?on its side chain.CS is mainly synthesized by chondroitin synthase?CHSY?.However,the role of CHSY in IDD has not yet been elucidated.Our previous results showed that the expression of Chsy3in human degenerated intervertebral disc was significantly decreased,and its expression decreased with increasing Pfirrmann grade scale.Due to the complex pathogenesis of IDD,there is currently no treatment that can effectively alleviate or reverse IDD.Therefore,animal models are extremely important for further study of IDD mechanisms and the exploration of new treatments.However,there is no internationally recognized IDD animal model that is effective,capable of being controlled and simulating the normal degeneration process.With the emergence and optimization of CRISPR-Cas9 technology and semi-cloning technology,the generation of knock-out mice has become simple and efficient.And knock-out mice are widely used in the study of human diseases.Therefore,this research intends to generate a Chsy3 knock-out mouse model and further studies its applications in IDD.Part I:Efficient generation of Chsy3 knock-out mouse modelMethods:?1?The CRISPR-Cas9 knock-out plasmid was designed and constructed based on the gene structure of Chsy3.?2?The DKO-AG-haESCs were transfected with lipofectamine 2000.After 12 hours of transfection,the mCherry-positive DKO-AG-haESCs were sorted using a flow cytometer and plated on 6-well plates.?3?The single cell clones were selected under a microscope and identified genetically.Then,the identified DKO-AG-haESCs were amplified on a 6-well plate and sorted using a flow cytometer regularly.?4?In vitro,the zygotes were developed to the 2-cell stage using ICAHCI technology,transplanted into uterus of maternal mouse to obtain Chsy3^+/-F0 mice,and then mated to F2 mice.?5?Genetic identification and potential off-targets analysis of Chsy3^-/-F2 mice were conducted.Results:?1?The CRISPR-Cas9 knock-out plasmid of pX330-mCherry-Chsy3-sgRNA was successfully constructed at the exon 1 of Chsy3.?2?After 12 hours of transfection,the ratio of mCherry-positive DKO-AG-haESCs was 8.5%,and then the mCherry-positive DKO-AG-haESCs were enriched using a flow cytometer.?3?Single-cell clones were selected,and a haploid cell line with-1 bp frameshift mutant knock-out of Chsy3 was obtained.?4?23 Chsy3^+/-F0 mice were successfully obtained using the ICAHCI technique,and then mated to F2 mice.?5?Chsy3^-/-F2 mice were successfully obtained with genotype of-1 bp frameshift mutation.The genotypes of H19-DMR and IG-DMR,two imprinted regions,are wild type.The top 20 CRISPR-Cas9 potential off-target sites were sequenced and identified without any off-targets.Conclusions:Using CRISPR-Cas9 gene editing technology and ICAHCI technology,we generated Chsy3^-/-F2 mice with-1 bp frameshift mutation efficiently and successfully.At the same time,we identified the top 20 CRISPR-Cas9 potential off-target sites and found no off-targets.Part ?:Phenotypic analysis of development and IDD in Chsy3 knock-out mouse modelMethods:?1?Under anesthesia,the body weight and body length of wild-type mice and Chsy3^-/-mice were measured to evaluate the development at 4 weeks,6 weeks,8 weeks,10 weeks,12 weeks,14 weeks,16 weeks,18 weeks,20 weeks,22 weeks,and 24 weeks.?2?The micro-CT examination of wild-type mice and Chsy3^-/-mice at 4 weeks and 8 weeks was performed to observe changes of vertebral bodies and intervertebral discs.?3?The MRI examination of wild-type mice and Chsy3^-/-mice at 4 weeks and 8 weeks was performed to observe signal intensity changes of intervertebral disc.?4?The expression of CHSY1,CHSY2,CHSY3 and AGGRECAN in nucleus pulposus of wild-type mice and Chsy3^-/-mice was conducted by immunohistochemistry?IHC?staining.?5?The expression of OSTN and ENPP1 in nucleus pulposus of wild-type mice and Chsy3^-/-mice was conducted by IHC staining.Results:?1?The body weight of wild-type mice and Chsy3^-/-mice showed no significant difference at each time point,but the body length of Chsy3^-/-mice was shorter than that of wild-type mice after 4 weeks.The difference was statistically significant.?2?On the sagittal CT images,endplate calcification was observed in Chsy3^-/-mice at 4 and 8 weeks.And the intervertebral disc height of Chsy3^-/-mice at 4 and 8 weeks was lower than that of wild-type mice,and the difference was statistically significant.?3?On the T2-weighted sagittal MRI,the IDD grade of Chsy3^-/-mice was significantly higher than that of wild-type mice at 4 and 8 weeks,while the signal intensity of intervertebral disc of Chsy3^-/-mice was significantly lower than that of wild-type mice at 4 and 8 weeks.?4?Compared with wild-type mice,the expression of CHSY1 in the nucleus pulposus of Chsy3^-/-mice was slightly down-regulated,the expression of AGGRECAN was obviously down-regulated,and CHSY3 was not expressed.?5?Compared with wild-type mice,the expression of OSTN in the nucleus pulposus of Chsy3^-/-mice was increased,while the expression of ENPP1 was decreased.Conclusions:With respect of development,the body weight of Chsy3^-/-mice was not significantly different from that of wild-type mice,but its body length was shorter than that of wild-type mice after 4 weeks.The expression of CHSY1 and AGGRECAN in Chsy3^-/-mice was down-regulated,the expression of OSTN in Chsy3^-/-mice was up-regulated,and the expression of ENPP1 in Chsy3^-/-mice was down-regulated,which led to a decrease in the height of the intervertebral disc,endplate calcification,a high grade of IDD,and a low signal intensity of the intervertebral disc.Part ?:Mechanism of IDD in Chsy3 knock-out mouse modelMethods:?1?The intervertebral disc tissues of wild-type mice and Chsy3^-/-mice at 8weeks were collected for primary cell culture.?2?The transcriptome expression levels of disc cells of wild-type mice and Chsy3^-/-mice were detected by high-throughput RNA-seq technology.?3?The expression of genes related to IDD was found using bioinformatics analysis of differentially expressed genes between wild-type mice and Chsy3^-/-mice.?4?The underlying mechanism of IDD was found by analyzing the differential expression of signaling pathways between wild-type mice and Chsy3^-/-mice.?5?The potential mechanism of IDD in Chsy3^-/-mice was verified by related experiments.Results:?1?Four disc cell lines of wild-type mice and Chsy3^-/-mice were successfully established by primary culture of intervertebral disc tissue.?2?The transcriptome gene expression levels in disc cells of Chsy3^-/-mice were significant different from wild-type mice by using high-throughput RNA-seq technology.?3?With GO and GSEA analysis,it was found that the expression of ossification-related genes and extracellular matrix degradation-related genes in disc cells of Chsy3^-/-mice were up-regulated,while the extracellular matrix synthesis-related genes were down-regulated.?4?Bioinformatics analysis revealed that the Hippo signaling pathway was significantly down-regulated in Chsy3^-/-mice,suggesting that the Hippo signaling pathway may be involved in IDD.?5?After the knock-out of Chsy3,the expression of YAP1 and its downstream of TEAD were down-regulated in the disc cell,further leading to the upregulation of ossification-related genes and extracellular matrix degradation-related genes,and the downregulation of extracellular matrix synthesis-related genes.And these changes eventually led to the IDD.Conclusions:Using high-throughput RNA-seq technology and bioinformatics analysis,we found that the expression of ossification-related genes and extracellular matrix degradation-related genes in the disc cells of Chsy3^-/-mice were up-regulated,while extracellular matrix synthesis-related genes were down-regulated,which led to the IDD.The down-regulation of Hippo signaling pathway after Chsy3 knock-out was the potential mechanism of IDD using bioinformatic analysis,which verified by relevant experiments.SummaryIn this study,the Chsy3^-/-mouse model was successfully and efficiently generated by using CRISPR-Cas9 technology and semi-cloning technology.The changes of intervertebral discs in Chsy3^-/-mice were evaluated in detail,and the mechanism of Chsy3 on IDD was analyzed by high-throughput RNA-seq technology.We found that the intervertebral disc of Chsy3^-/-mice underwent progressive degeneration,which was characterized by water content loss in the intervertebral disc,stenosis of the intervertebral space,and calcification of the cartilage endplate.The RNA-seq results showed that the transcriptome expression profiles of intervertebral disc cells in Chsy3^-/-mice were significantly different from those in wild-type mice.After Chsy3 knock-out,the expression of YAP1 and its downstream TEAD in Hippo signaling pathway were down-regulated,which promoted the expression of ossification-related genes and extracellular matrix degradation-related genes,and suppressed the expression of extracellular matrix synthesis-related genes in intervertebral disc cells.Together,these changes of genes expression led to the occurrence and development of IDD.