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细胞系鉴定

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发表于 2017-9-14 19:53:30 | 显示全部楼层 |阅读模式
什么是细胞系鉴定
细胞系鉴定即通过STR(短串联重复)信息所建立细胞系的遗传特性。一旦细胞系的遗传特性被确立,细胞系就应该定期的检测,以防止出现细胞系被误认或交叉污染的情况。
由于细胞系发生交叉污染或身份误认,可以导致实验数据的无效和数据的误导。NIH已发出公告,讨论细胞系鉴定的必要性,并且越来越的的期刊要求文章发表前需提供细胞鉴定材料。
何时需要细胞系鉴定
当建立或获得一个新的细胞系时
在细胞冻存之前,或细胞已连续培养2个月时
在开始系列实验或发表文章之前
细胞系表现不稳定或结果与预期差别较大时
当实验室使用超过一种细胞系时,所有细胞系最好先鉴定以排除交叉污染的可能
如何判断细胞系是否正确
收到细胞系鉴定结果以及STR信息,可以自行和参考数据库进行比对。如果细胞样本的每个STR位点和参考细胞STR位点都能重合匹配,即说明该细胞系正确,反之则说明你的细胞系可能被错误标记,交叉污染或发生遗传不稳定。一般说来,匹配度≥80%即可认为该细胞系正确,匹配度<80%则说明该细胞系的来源需要被怀疑。
如果细胞系被鉴定出发生交叉污染或错误标记,则需要拿更早代数的细胞进行鉴定,从而找到问题的起源或者直接从可靠的机构购买一株新的细胞系。
如何确定细胞系发生交叉污染
如果存在细胞系之间发生交叉污染,那么在进行STR位点检测的时候,多个位点会出现两个以上的峰。峰高值表示该等位基因的信号强度,理论上峰值与样本的DNA浓度有直接的关系。因此,存在交叉污染的混合细胞系中,其中一个位点中某些峰会明显高于其他的峰,其中大峰是属于混合细胞系中那个主要细胞系,而小峰则属于那个次要细胞系。
值得注意的是,当发生两个或以上细胞混合的时候,检测结果看起来非常像细胞系的“遗传不稳定”——当一个细胞系存在亚群时,尤其是一些癌细胞系,由于遗传不稳定的存在,在一些位点的STR特性会不同。因此经验丰富的分子专家是非常重要的,他能够帮助分析复杂的电泳图谱(STR峰图),从而判断是否由于发生细胞系交叉污染而导致多等位基因情况。上海翼和生物,作为老牌的遗传技术服务公司,十年以上基因分型经验,可以帮您解决你的疑惑。
结果如何进行数据库比对
Cell Line Integrated Molecular Authentication (CLIMA)
http://bioinformatics.istge.it/clima/ 
American Type Culture Collection (ATCC)
https://www.atcc.org/Culturesand ... bid/174/Default.asx
Japanese Collection of Research Bioresource (JCRB)
http://cellbank.nibio.go.jp/cellbank_e.html
German Collection of Microorganisms and Cell Cultures (DSMZ)
http://www.dsmz.de/human_and_ani ... ?contentleft_id=101
细胞样本要求
每种细胞需单独收集在1.5ml离心管中,细胞数目在0.5 x 10^6至1 x 10^6之间。细胞需要离心沉淀,并且竟可能去除上清培养基,沉淀冰冻保存。细胞样本需保持冰冻状态直到基因组DNA提取完毕,并且在1.5ml管壁外标明细胞数目。



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发表于 2017-9-17 17:26:31 | 显示全部楼层
STR profiles of cell lines
Cell line authentication
Concerns around the identity of cancer cell lines used in scientific research have been increasing over several years and was the topic of a recent editorial in Nature (-, 2009). Cross-contamination has even been shown to be present in such widely used and supposedly well characterised groups of cell lines as the NCI60 set. For instance the NCI60 cell lines OVCAR-8 and NCI-ADR-RES have been shown to be over 97% identical using the Affymetrix SNP6.0 array in this laboratory - this result has been confirmed by multiple laboratories around the world and was recently reported in the scientific literature (Liscovitch and Ravid, 2007). Two other such pairing are also present within the NCI60 series of lines - both M14/MDA-MB-435 (Rae et al, 2007) and U251MG/SNB-19 have identities over 94% when compared using the SNP6 array.

Many of the cell line repositories are now providing short tandem repeat (STR) profiles of the lines they hold allowing identity of lines within the scientific community to be confirmed by a simple assay. We are currently confirming the identity of our cancer cell line set against those provided by the repositories, where possible. Each of the cell lines within our core set is being tested using a panel of 16 STRs (AmpFLSTR Identifiler KIT, ABI), which includes the 9 currently used by most of the cell line repositories (ATCC, Riken, JCRB and DSMZ). We are also providing a single nucleotide polymorphism (SNP) profile based on a panel of 63 SNPs assayed using the Sequenom Genetic Analyser which we use for in-house identity checking whenever a cell line is propagated.

The provision of STR profiles by the cell line repositories and of our in-house cell lines is ongoing and will be updated when appropriate.

Prior to accessing the STR or SNP datasets a Data Access Agreement must be completed: http://www.sanger.ac.uk/genetics/CGP/Archive

The username and password provided can be used to download the STR and SNP profiles for each cell line at the CGP Data Archive: http://www.sanger.ac.uk/research ... enome/archive/#t_cl

References
Identity crisis.
No authors listed
Nature 2009;457;7232;935-6
PUBMED: 19225471; DOI: 10.1038/457935b
MDA-MB-435 cells are derived from M14 melanoma cells--a loss for breast cancer, but a boon for melanoma research.
Rae JM, Creighton CJ, Meck JM, Haddad BR and Johnson MD
Breast cancer research and treatment 2007;104;1;13-9
PUBMED: 17004106; DOI: 10.1007/s10549-006-9392-8
A case study in misidentification of cancer cell lines: MCF-7/AdrR cells (re-designated NCI/ADR-RES) are derived from OVCAR-8 human ovarian carcinoma cells.
Liscovitch M and Ravid D
Cancer letters 2007;245;1-2;350-2
PUBMED: 16504380; DOI: 10.1016/j.canlet.2006.01.013
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