慢病毒介导的CRISPR/Cas9敲除TGF-β1基因对鹿茸软骨细胞增殖的影响
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摘要
为探究TGF-β1基因在鹿茸快速生长期的作用,利用慢病毒介导的CRISPR/Cas9基因编辑技术,设计并构建3条靶向梅花鹿TGF-β1基因第1外显子的gRNA寡核苷酸序列,在人胚肾细胞293T内进行慢病毒包装,通过neo抗性筛选稳转细胞株。提取稳转细胞株总蛋白,经BCA法测定总蛋白质量浓度,Western Blot检测TGF-β1蛋白的相对表达水平;MTT法检测TGF-β1基因敲除对鹿茸软骨细胞体外增殖的影响。结果表明:成功构建了靶向梅花鹿TGF-β1的CRISPR/Cas9基因敲除载体p BOBI-gRNA2。TGF-β1基因的缺失抑制了鹿茸软骨细胞的体外增殖。
To investigate the role of TGF-β1 gene in the rapid growth stage of antler,we used lentivirus-mediated CRISPR/Cas9 gene editing technology to design and construct three gRNA oligonucleotide sequences targeting exon 1 of the TGF-β1 gene in sika deer. Lentiviral packaging was performed in 293 T cells and the stable cell line was obtained by neo resistance screening. The total protein of the stable cell line was extracted and the concentration was measured by BCA,the relative expression level of TGF-β1 protein was detected by Western Blot,and the effect of TGF-β1 gene knockout on the proliferation of antler cartilage cells were detected by MTT. The CRISPR/Cas9 gene knockout vector targeting sika deer TGF-β1 was successfully constructed as pB OBI-gRNA2. The loss of TGF-β1 gene inhibited the proliferation of antler cartilage cells in vitro.
To investigate the role of TGF-β1 gene in the rapid growth stage of antler,we used lentivirus-mediated CRISPR/Cas9 gene editing technology to design and construct three gRNA oligonucleotide sequences targeting exon 1 of the TGF-β1 gene in sika deer. Lentiviral packaging was performed in 293 T cells and the stable cell line was obtained by neo resistance screening. The total protein of the stable cell line was extracted and the concentration was measured by BCA,the relative expression level of TGF-β1 protein was detected by Western Blot,and the effect of TGF-β1 gene knockout on the proliferation of antler cartilage cells were detected by MTT. The CRISPR/Cas9 gene knockout vector targeting sika deer TGF-β1 was successfully constructed as pB OBI-gRNA2. The loss of TGF-β1 gene inhibited the proliferation of antler cartilage cells in vitro.
引文
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[23]杨强,徐盼,蒋凯,等.慢病毒介导的CRISPR/Cas9技术编辑PFF细胞BMPR-IB基因及BMPs信号通路重要基因表达分析[J].中国农业科学,2018,51(7):1378-1389.
[2]孙红梅,金美伶,路晓,等.鹿作为生物医学模型的研究进展[J].特产研究,2015,37(2):52-56.
[3]张伟,褚文辉,李春义.鹿茸成骨过程及其相关调控机制研究进展[J].中国农学通报,2015,31(8):6-11.
[4]李春义.鹿茸完全再生机制研究进展[J].农业生物技术学报,2017,25(1):1-10.
[5]路晓,孙红梅,张伟,等.以鹿茸为模型探索软骨组织发生[J].中国农业科技导报,2015,17(4):71-77.
[6]吴炎,牛永梅,司博,等.鹿茸细胞生长因子的研究进展[J].黑龙江畜牧兽医,2015(19):61-63.
[7] ROMAN-BLAS J A,STOKES D G,JIMENEZ S A. Modulation of TGF-beta signaling by proinflammatory cytokines in articular chondrocytes[J]. Osteoarthritis Cartilage,2007,15(12):1367-1377.
[8]殷诗舒,黄生强.CRISPR/Cas9基因编辑技术的研究进展[J].猪业科学,2016,33(11):108-109.
[9] WEI C,LIU J,YU Z,et al. TALEN or Cas9-rapid,efficient and specific choices for genome modifications[J]. Journal of Genetics and Genomics,2013,40(6):281-289.
[10] CHO S W,KIM S,KIM J S,et al. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease[J].Nature Biotechnology,2013,31(3):230-232.
[11]孙玉章,孙明军,代永联,等.CRISPR/Cas9介导的基因组编辑技术及应用研究进展[J].中国动物检疫,2016,33(12):64-68.
[12]吴卫军,赵婕青,周振华,等.CRISPR-Cas9技术在基因调节中的研究进展[J].药物生物技术,2016,23(6):544-547.
[13] DOUDNA J A,CHARPENTIER E. The new frontier of genome engineering with CRISPR-Cas9[J]. Science,2014,346. doi:10.1126/science.1258096.
[14]张璐,韩玉帅,郭斌,等.梅花鹿鹿茸间充质层与前成软骨层细胞的培养及SB-431542对其增殖的影响[J].中国农学通报,2011,27(11):35-38.
[15] ZHEN G,WEN C,JIA X,et al. Inhibition of TGF-βsignaling in mesenchymal stem cells of subchondral bone attenuates osteoarthritis[J].Nature Medicine,2013,19(6):704-712.
[16]王旭东,战忠利.TGF-β及其受体与肿瘤的研究进展[J].中国肿瘤临床,2005,32(17):1016-1020.
[17]王秋实,李平.TGF-β1受体1和2在TGF-β1调节细胞增殖中的作用[J].中国生物化学与分子生物学报,2017,33(2):122-127.
[18] HUANG S S,HUANG J S. TGF-beta control of cell proliferation[J]. J Cell Biochem,2005,96(3):447-462.
[19] HELDIN C H,MOUSTAKAS A. Signaling receptors for TGF-βfamily members[J]. Cold Spring Harb Perspect Bio1,2016,8(8).doi:10.1101/cshperspect.a022053.
[20]何治尧,秦舟,门可,等.CRISPR/Cas9技术在生物医学领域的非病毒及病毒载体[J].中国科学:生命科学,2017,47(11):1141-1150.
[21]肖婧,张宗德,申阿东.CRISPR-Cas9系统在真核细胞领域内的研究进展[J].标记免疫分析与临床,2016,23(10):1218-1225.
[22]庞中兵,王伟,刘赛宝,等.利用重组慢病毒载体共表达Cas9-sgRNA构建TLR4基因敲除DF1细胞系[J].中国兽医科学,2018,48(8):958-964.
[23]杨强,徐盼,蒋凯,等.慢病毒介导的CRISPR/Cas9技术编辑PFF细胞BMPR-IB基因及BMPs信号通路重要基因表达分析[J].中国农业科学,2018,51(7):1378-1389.