| 赵俐婷,王乃宁,贺芳,等.CRISPR/Cas 9介导的基质 Gla蛋白基因敲除对破骨细胞分化的影响[J].安徽医药,2019,23(12):2361-2365. |
| CRISPR/Cas 9介导的基质 Gla蛋白基因敲除对破骨细胞分化的影响 |
| The effect of CRISPR/Cas 9?mediated MGP knockout on osteoclast differentiation |
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| DOI：10.3969/j.issn.1009?6469.2019.12.007 |
| 中文关键词: 基因敲除技术 骨钙素 破骨细胞 转染 CRISPR/Cas 9 RAW264.7细胞(小鼠单核巨噬细胞白血病细胞) |
| 英文关键词: Gene knockout techniques Osteocalcin Osteoclasts Transfection CRISPR/Cas 9 RAW264.7 cells(Leukemia cells in mouse mononuclear macrophage) CRISPR/Cas 9(Clustered Regularly InterspacedShort Palindromic Repeats and CRISPR ? AssociatedProteins) |
| 基金项目:国家自然科学基金项目(81670806，81972133，81300716)；中央高校基本科研业务费(xzy01201909)；陕西省自然科学基础研究计划(2017JM8015) |
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| 中文摘要: |
| 目的利用 CRISPR/Cas 9基因编辑技术，构建基质 Gla蛋白(MGP)基因敲除的 RAW264.7巨噬细胞系，明确 MGP基因在破骨细胞分化过程中的作用。方法利用在线软件分析并筛选出 3个针对 MGP基因外显子区的单链向导 RNA(gRNA)人工合成 gRNA寡核苷酸序列，并将其插入线性化的 LentiCRISPRv2质粒中，构建成 LentiCRISPRv2?MGP?gRNA重组质粒。将重组，质粒与 psPAX2、VSVG包装质粒一同转染 293?T细胞，包装并收集重组慢病毒，将其感染 RAW264.7细胞。经嘌呤霉素筛选得到稳定 RAW264.7细胞系，实时荧光定量多聚核苷酸链式反应(qPCR)、蛋白质印迹法(Western Blot)验证 MGP mRNA及蛋白表达水平。 qPCR检测破骨细胞分化标志分子的表达情况。结果成功构建了 LentiCRISPRv2?MGP?gRNA重组质粒，成功包装了含有 MGP?gRNA的重组慢病毒，筛选得到了稳定低表达 MGP的 RAW264.7细胞系。 qPCR及蛋白质印迹法检测显示，MGP mRNA、蛋白表达显著性下调(P＜0.05)。 qPCR结果显示，最具有代表性的 LentiCRISPRv2?MGP?gRNA1细胞破骨标志分其子 Itgb3(2.29±0.17)、 Acp5(2.86±0.15)、 Ctsk(2.07±0.13)的 mRNA水平均显著上调(P＜0.05)。结论利用 CRISPR/Cas 9基因编辑技术成功构建了 MGP基因敲除的 RAW264.7细胞系，敲除 MGP后 RAW264.7细胞破骨分化能力增强。 |
| 英文摘要: |
| Objective To construct matrix Gla protein(MGP)knockout RAW264.7 cell line by CRISPR/Cas 9 technology and to make clear the role of MGP in osteoclast differentiation.Methods Three single?stranded guide RNAs(gRNAs)targeting MGPgene exons were screened using the online tool before synthesized gRNAs were inserted into the linear LentiCRISPRv2 plasmid re?spectively，and the LentiCRISPRv2?MGP?gRNA recombinant plasmids were constructed.Then the recombinant plasmids，together with psPAX2 and VSVG packaging plasmids，were transfected into 293?T cells.The lentivirus was packaged，collected and recom? bined to infect RAW264.7 cells.Stable RAW264.7 cell lines were screened by puromycin.The mRNA and protein levels of MGPwere detected by real?time fluorescent quantitative polynucleotide chain reaction(qPCR)and Western Blot.The markers of osteo? clast differentiation were examined by qPCR.Results LentiCRISPRv2?MGP?gRNA plasmids were successfully constructed，and the recombinant lentivirus were successfully packaged.Stable MGP?deficient RAW264.7 cells were screened.qPCR and Western Blot re?sults revealed that the expressions of MGP mRNA and protein were significantly down?regulated(P＜0.05).qPCR results showed that the mRNA levels of the most representative osteoclastic markers Itgb3(2.29±0.17)，Acp5(2.86±0.15)，Ctsk(2.07±0.13)in LentiCRISPRv2?MGP?gRNA1 were significantly up?regulated(P＜0.05).Conclusion The stable MGP knockout RAW264.7 cell line were successfully constructed by CRISPR/Cas 9 technology.Osteoclast differentiation of RAW264.7 cells was accelerated afterMGP knockout. |
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