Please wait a minute...
浙江大学学报（医学版）
J Zhejiang Univ (Med Sci)  2018, Vol. 47 Issue (5): 520-524    DOI: 10.3785/j.issn.1008-9292.2018.10.12
    
Establishment of cell lines for quality control of prenatal genetic diagnosis by SV40LT gene transfection
WENG Binghuan1(),XU Wei2,3,SU Lan1,SHEN Min1,LI Rong3,XU Xiaopeng2,LI Lanjuan2,3,*()
1. Key Laboratory of Reproductive Genetics of the Ministry of Education, Women's Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China
2. State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China
3. Shulan(Hangzhou) Hospital, Hangzhou 310004, China
Download: HTML( 8 )   PDF(978KB)
Export: BibTeX | EndNote (RIS)      

Abstract  

Objective: To establish a cell lines for quality control of prenatal genetic diagnosis. Methods: The recombined SV40LTag-pcDNA3.1(-) vector was constructed and transfected by lipidosome into human amniotic fluid cells with common aneuploidy. Positive clones were screened by G418, and the immortality of transfected cell line was identified. Results: Cell line with karyotype of 46, XY, t(8;19)(q24.3;q13.1) from primary amniotic fluid cells was established. Karyotype analytical results indicated that the cell line at its 15th generation maintained the same karyotype of its primary cell. Conclusion: Gene SV40LT can lead to immortality of amniotic fluid cells, which contributes to preparing cell lines for internal and external quality control in prenatal genetic diagnosis.

Key words： Simian virus 40/immunology      Simian virus 40/genetics      Liposomes      Transfection      Amniotic fluid/cytology      Genetic vectors      Quality control      Cells, cultured     
Received: 26 January 2018      Published: 23 January 2019
CLC:  R394  
  R722.11  
  R446  
Corresponding Authors: LI Lanjuan     E-mail: 5503006@zju.edu.cn;ljli@zju.edu.cn
Service
E-mail this article
Add to my bookshelf
Add to citation manager
E-mail Alert
RSS
Articles by authors
WENG Binghuan
XU Wei
SU Lan
SHEN Min
LI Rong
XU Xiaopeng
LI Lanjuan
Cite this article:

WENG Binghuan,XU Wei,SU Lan,SHEN Min,LI Rong,XU Xiaopeng,LI Lanjuan. Establishment of cell lines for quality control of prenatal genetic diagnosis by SV40LT gene transfection. J Zhejiang Univ (Med Sci), 2018, 47(5): 520-524.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2018.10.12     OR     http://www.zjujournals.com/med/Y2018/V47/I5/520


脂质体介导SV40LT基因转染构建羊水染色体核型分析质控细胞系研究

目的: 研究羊水染色体核型分析质控细胞系的构建方法。方法: 以T4 DNA连接经BamHⅠ单酶切的SV40LTag-pcDNA和pcDNA3.1（-）DNA，重组SV40LTag-pcDNA3.1（-）克隆，以脂质体介导法将重组克隆转染至染色体结构异常的羊水细胞，以G418筛选阳性克隆，观察细胞系的传代生长特性及其作为染色体核型分析质量评估的可行性。结果: 构建了染色体核型为46，XY，t（8；19）（q24.3；q13.1）的羊水细胞系，传至第15代的细胞系经染色体核型分析，其核型与原代细胞一致。结论: 染色体结构异常的羊水细胞转染SV40LT基因后可转化为无限扩增且染色体核型稳定的细胞系，从而制成羊水细胞染色体核型分析的质控细胞系。


关键词： 猴病毒40/免疫学,  猴病毒40/遗传学,  脂质体,  转染,  羊水/细胞学,  遗传载体,  质量控制,  细胞, 培养的 
Fig 1 Positive clone of amniotic cells transfected with SV40LT gene under inverted microscope
Fig 2 Morphology of the amniotic cells from the 15th generation after transfected with SV40LT gene under inverted microscope
Fig 3 Chromosome karyogram of amniotic cells after transfected with SV40LT gene
Fig 4 Fluorescence in situ hybridization test results of chromosome aneuploidy in amniotic cells after transfected with SV40LT gene
[1]   KIRCHHOFF C , ARAKI Y , HUHTANIEMI I et al. Immortalization by large T-antigen of the adult epididymal duct epithelium[J]. Mol Cell Endocrinol, 2004, 216 (1-2): 83- 94
doi: 10.1016/j.mce.2003.10.073
[2]   CAMPISI J . Replicative senescence:an old lives' tale?[J]. Cell, 1996, 84 (4): 497- 500
doi: 10.1016/S0092-8674(00)81023-5
[3]   REDDEL R R , KE Y , GERWIN B I et al. Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes[J]. Cancer Res, 1988, 48 (7): 1904- 1909
[4]   TEVETHIA M J , OZER H L . SV40-mediated immortalization[J]. Methods Mol Biol, 2001, 165:185- 99
[5]   SCHOLTE B J , KANSEN M , HOOGEVEEN A T et al. Immortalization of nasal polyp epithelial cells from cystic fibrosis patients[J]. Exp Cell Res, 1989, 182 (2): 559- 571
doi: 10.1016/0014-4827(89)90259-0
[6]   REDDEL R R , KE Y , GERWIN B I et al. Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes[J]. Cancer Res, 1988, 48 (7): 1904- 1909
[7]   CHANG P L , GUNBY J L , TOMKINS D J et al. Transformation of human cultured fibroblast with plasmids carrying dominant selection marks and immortalizing potential[J]. Exp Cell Res, 1986, 167 (2): 407- 416
doi: 10.1016/0014-4827(86)90181-3
[8]   王薇, 陈园园, 陈希 et al. 我国产前染色体核型分析的室间质评调查结果分析[J]. 中华医学遗传学杂志, 2014, 31 (4): 483- 486
WANG Wei , CHEN Yuanyuan , CHEN Xi et al. Result survey analysis of prenatal chromosome karyotyping in an external quality assessment program[J]. Chinese Journal of Medical Genetics, 2014, 31 (4): 483- 486
doi: 10.3760/cma.j.issn.1003-9406.2014.04.016
[9]   申子瑜, 李少男, 王治国 . 我国政府对临床实验室质量保证的管理行为[J]. 中华检验医学杂志, 2006, 29 (1): 6- 8
SHEN Ziyu , LI Shaonan , WANG Zhiguo . The government regulation of clinical laboratory quality in China[J]. Chinese Journal of Laboratory Medicine, 2006, 29 (1): 6- 8
doi: 10.3760/j:issn:1009-9158.2006.01.003
[10]   IZQUIERDO L A , BERKSHIRE S . Access, quality and costs of prenatal diagnosis[J]. Bol Asoc Med P R, 2010, 102 (4): 25- 29
[11]   RAMSDEN S C , MANN K , MCCONNELL C et al. External quality assessment of rapid prenatal detection of numerical chromosomal aberrations using molecular genetic techniques:3 years experience[J]. Prenat Diagn, 2007, 27 (5): 404- 408
doi: 10.1002/(ISSN)1097-0223
[12]   HASTINGS R . Quality control in FISH as part of a laboratory's quality management system[J]. Methods Mol Biol, 2010, 659:249- 259
doi: 10.1007/978-1-60761-789-1
[13]   REDDEL R R , KE Y , GERWIN B I et al. Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes[J]. Cancer Res, 1988, 48 (7): 1904- 1909
[1] CHEN Dahua, LI Youming. Construction of all-in-one CRISPR/Cas9 vector system targeting miR-101a gene in mouse hepatic cell line AML12[J]. J Zhejiang Univ (Med Sci), 2017, 46(4): 427-432.
[2] JIANG Yiqian, GUO Qingmin, GU Jianzhong, XU Xiaoping, AN Suhong, SU Fang, BAO Yanhong, HUANG Changxin, GUAN Xiaoxiang. Effect of microRNA-29b on proliferation and migration of breast cancer cells and its molecular mechanism[J]. J Zhejiang Univ (Med Sci), 2017, 46(4): 349-356.
[3] WANG Hebin,LI Yang,LIU Xingang,ZHOU Jun,WANG Qingqing,TANG Guping. Preparation, characterization and cytology study of Pluronic-PEI micelles[J]. J Zhejiang Univ (Med Sci), 2017, 46(2): 127-133.
[4] LI Wenlong,QU Haibin. Application progress on near infrared spectroscopy in quality control and process monitoring of traditional Chinese medicine[J]. J Zhejiang Univ (Med Sci), 2017, 46(1): 80-88.
[5] WANG Jing, HU Yan, TAN Bi-qin, WANG Jia-jia, ZHAO Meng-ting, WENG Qin-jie, ZHU Di-feng, WANG Hui-ying. Construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice[J]. J Zhejiang Univ (Med Sci), 2015, 44(5): 511-516.
[6] WANG Kan, et al. Highlights from 1st AGA professional conference in China[J]. J Zhejiang Univ (Med Sci), 2014, 43(6): 728-730.
[7] DU Xiao， WANG Yi-han， WANG Zi-qiang， CHENG Zhong， LI Yang， HU Jian-kun， CHEN Zhi-xin， ZHOU Zong-guang. Downregulation of Notch1 by small interfering RNA enhances chemosensitivity to gemcitabine in pancreatic cancer cells through activating apoptosis activity[J]. J Zhejiang Univ (Med Sci), 2014, 43(3): 313-318.
[8] YANG You-ping, DING Yan, WANG Ji-rong, ZENG Ling-hui, LIN Hong-xia, ZHU Yang-li, WU Hong-wei, WANG Ruo-yan, ZHANG Jian-min, YING Rong-biao. Construction and identification of lentiviral vector containing human ILK-shRNA and mda7 gene[J]. J Zhejiang Univ (Med Sci), 2014, 43(2): 193-199.
[9] HE Xing，LI Hua，SHAO Yan，HU Ying，GU Zichun，CHEN Li，MA Jingjing，LAN Zhiyong. Comparison of three different methods in transfection of plasmid pGPU6/GFP/Neo to chicken embryo fibroblasts[J]. J Zhejiang Univ (Med Sci), 2013, 42(6): 660-665.
[10] HA Xiaoqin，JIANG Donghong，DENG Zhiyun，DONG Juzi，ZHAO Yong，PENG Junhua，YANG Zhihua . Construction of recombinant adenovirus vector pAdxsi-GFP-HIF containing hypoxia inducible factor gene and its expression in endothelial cells[J]. J Zhejiang Univ (Med Sci), 2013, 42(6): 654-659.
[11] LIN Hu，QU Chenxi，YU Yijie，TANG Yining，SUN Xiaoyi. Preparation of freeze-dried long-circulation oridonin liposomes and their pharmacokinetics in rats[J]. J Zhejiang Univ (Med Sci), 2013, 42(6): 638-643.
[12] HE Ping, TAN Li, HU Wei-Wei, DAI Hai-Bin, HU Yong-Zhou, CHEN Zhong. Identification of a HEK-293 cell line containing stably-transfected H3R gene and screening for novel non-imidazole histamine H3 receptor antagonists[J]. J Zhejiang Univ (Med Sci), 2013, 42(3): 276-282.
[13] . Characteristics of cationic polymers PEI-CyD，PEI-PHPA，PEE-PHPA and PEI25kD in vitro and in vivo[J]. J Zhejiang Univ (Med Sci), 2012, 41(6): 620-630.
[14] . Establishment of a transgenic cell line with stable expression of human CD14[J]. J Zhejiang Univ (Med Sci), 2012, 41(5): 506-511.
[15] . Ubiquitination of recombinant adeno-associated viral vector and its application[J]. J Zhejiang Univ (Med Sci), 2012, 41(5): 586-591.