全部 标题 作者
关键词 摘要

OALib Journal期刊
ISSN: 2333-9721
费用:99美元

查看量下载量

相关文章

更多...

建立CRISPR/Cas9慢病毒系统高效敲除人源AIP1基因

DOI: 10.13560/j.cnki.biotech.bull.1985.2015.08.032, PP. 219-224

Keywords: CRISPR/Cas9,慢病毒,AIP1,稳定细胞株

Full-Text   Cite this paper   Add to My Lib

Abstract:

旨在构建AIP1基因CRISPR/Cas9敲除体系,获得高效、稳定、永久性AIP1敲除的人胚肾细胞株(293T)。针对AIP1的外显子设计3个20bp的sgRNA(sp1、sp2和sp3)。与PX458载体连接,构建PX458-sgRNA敲除表达载体。T7E1实验评估敲除效率。将敲除效率最高的sgRNA与lentiCRISPRv2慢病毒载体连接,构建lentiCRISPRv2-sgRNA敲除表达载体,将阳性重组质粒导入病毒包装细胞293T中,收集病毒上清液转染293T细胞。对敲除成功的293T细胞通过有限稀释法构建敲除AIP1基因的稳定细胞株。Westernblot测定转染后293T细胞中AIP1蛋白的表达量。结果显示,测序证实AIP1的3个靶序列分别正确插入PX458表达载体中,T7E1实验证实AIP1sgRNAsp2靶向敲除效率最高;成功构建lentiCRISPRv2-sgRNAsp2AIP1敲除表达载体,并包装病毒,感染293T细胞;Westernblot证实获得稳定的AIP1基因表达缺失的293T细胞株。建立了能稳定敲除AIP1基因的CRISPR/Cas9慢病毒系统,成功获得AIP1敲除的293T稳定细胞株。

References

[1]  Chen H, Pong R, Wang Z, et al. Differential regulation of the human gene DAB2IP in normal and malignant prostatic epithelia:Cloning and characterization[J]. Genomics, 2002, 79(4):573-581.
[2]  Chen H, Toyooka S, Gazdar AF, et al. Epigenetic regulation of a novel tumor suppressor gene(hDAB2IP)in prostate cancer cell lines[J]. J Biol Chem, 2003, 278(5):3121-3130.
[3]  Yano M, Toyooka S, Tsukuda K, et al. Aberrant promoter methylation of human DAB2 interactive protein(hDAB2IP)gene in lung cancers[J]. International Journal of Cancer, 2005, 113(1):59-66.
[4]  Yano M, Toyooka S, Tsukuda K, et al. Aberrant promoter methylation of human DAB2 interactive protein(hDAB2IP)gene in lung cancers[J]. Int J Cancer, 2005, 113(1):59-66.
[5]  Dote H, Toyooka S, Tsukuda K, et al. Aberrant promoter methylation in human DAB2 interactive protein(hDAB2IP)gene in gastrointestinal tumour[J]. Br J Cancer, 2005, 92(6):1117-1125.
[6]  Yang H, Wang H, Shivalila CS, et al. One-Step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering[J]. Cell, 2013, 154(6):1370-1379.
[7]  Carroll D. Staying on target with CRISPR-Cas[J]. Nat Biotechnol, 2013, 31(9):807-809.
[8]  Yu L, Qin L, Zhang H, et al. AIP1 prevents graft arteriosclerosis by inhibiting interferon-gamma-dependent smooth muscle cell proliferation and intimal expansion[J]. Circ Res, 2011, 109(4):418-427.
[9]  Min W, Pober JS. AIP1 in graft arteriosclerosis[J]. Trends Cardiovasc Med, 2011, 21(8):229-233.
[10]  Min J, Zaslavsky A, Fedele G, et al. An oncogene-tumor suppressor cascade drives metastatic prostate cancer by coordinately activating Ras and nuclear factor-κB[J]. Nature Medicine, 2010, 16(3):286-294.
[11]  Xie D, Gore C, Liu J, et al. Role of DAB2IP in modulating epithelial-to-mesenchymal transition and prostate cancer metastasis[J]. Proc Natl Acad Sci USA, 2010, 107(6):2485-2490.
[12]  Qiu G, Xie H, Wheelhouse N, et al. Differential expression of hDAB2IPA and hDAB2IPB in normal tissues and promoter methylation of hDAB2IPA in hepatocellular carcinoma[J]. Journal of Hepatology, 2007, 46(4):655-663.
[13]  Harms DW, Quadros RM, Seruggia D, et al. Mouse genome editing using the CRISPR/Cas system[J]. Curr Protoc Hum Genet, 2014, 83:15-17.
[14]  Fu Y, Foden JA, Khayter C, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells[J]. Nature Biotechnology, 2013, 31(9):822-826.
[15]  Gretarsdottir S, Baas AF, Thorleifsson G, et al. Genome-wide association study identifies a sequence variant within the DAB2IP gene conferring susceptibility to abdominal aortic aneurysm[J]. Nature Genetics, 2010, 42(8):692-697.

Full-Text

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133