Chiong Edmund, Dadbin Ali, Harris Loleta D, Sabichi Anita L, Grossman H Barton
Department of Urology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
J Urol. 2009 Jun;181(6):2737-48. doi: 10.1016/j.juro.2009.01.108. Epub 2009 Apr 17.
Cross-contamination of cell lines is a serious but often unrecognized problem. We describe the authentication of a panel of transitional cell carcinoma cell lines using the short tandem repeat profiling technique to detect cross-contamination.
Genomic DNA was isolated from UM-UC-1, UM-UC-2, UM-UC-3 (ATCC), UM-UC-6, UM-UC-9, UM-UC-10, UM-UC-11, UM-UC-13, UM-UC-14, UM-UC-16, T24 and KU7 cell lines. Short tandem repeat loci (D3S1358, D16S539, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317 and D7S820) and a segment of the X-Y homologous gene amelogenin were co-amplified by polymerase chain reaction. Profiling was done using POP-4TM performance optimized polymer 4 (Applied Biosystems) with an ABI Prism 310 genetic analyzer. DNA sequencing of TP53 and immunohistochemistry for p53 were performed in UM-UC-3 and UM-UC-3-GFP.
All cell lines had a unique short tandem repeat profile except UM-UC-2 and T24, which were virtually identical. T24 short tandem repeat profiles matched those of early passage number UM-UC-2. Stable transfection of the green fluorescence protein marker gene did not alter UM-UC-6, UM-UC-14 or KU7 profiles. However, the short tandem repeat profile for UM-UC-3-GFP was different from that of UM-UC-3. DNA sequencing showed a difference in TP53 between UM-UC-3 and UM-UC-3-GFP, confirming that UM-UC-3-GFP is not derived from UM-UC-3.
Short tandem repeat profiling provides a unique genetic signature of human cell lines that does not significantly change with passage or green fluorescence protein transduction. Using short tandem repeat profiling we noted that the cell line UM-UC-2 is T24. DNA fingerprinting using short tandem repeat profiling is an easy and reliable tool that can be used to verify cell lines.
细胞系的交叉污染是一个严重但常未被认识到的问题。我们描述了使用短串联重复序列分析技术对一组移行细胞癌细胞系进行鉴定以检测交叉污染的情况。
从UM-UC-1、UM-UC-2、UM-UC-3(美国典型培养物保藏中心)、UM-UC-6、UM-UC-9、UM-UC-10、UM-UC-11、UM-UC-13、UM-UC-14、UM-UC-16、T24和KU7细胞系中提取基因组DNA。通过聚合酶链反应共扩增短串联重复序列位点(D3S1358、D16S539、vWA、FGA、TH01、TPOX、CSF1PO、D5S818、D13S317和D seven S820)以及X-Y同源基因牙釉蛋白的一段序列。使用POP-4TM性能优化聚合物4(应用生物系统公司)和ABI Prism 310遗传分析仪进行分析。对UM-UC-3和UM-UC-3-GFP进行TP53的DNA测序和p53的免疫组织化学检测。
除UM-UC-2和T24几乎完全相同外,所有细胞系都有独特的短串联重复序列图谱。T24的短串联重复序列图谱与早期传代的UM-UC-2的图谱匹配。绿色荧光蛋白标记基因的稳定转染未改变UM-UC-6、UM-UC-14或KU7的图谱。然而,UM-UC-3-GFP的短串联重复序列图谱与UM-UC-3的不同。DNA测序显示UM-UC-3和UM-UC-3-GFP之间的TP53存在差异,证实UM-UC-3-GFP并非源自UM-UC-3。
短串联重复序列分析提供了人类细胞系独特的遗传特征,且该特征不会随传代或绿色荧光蛋白转导而显著改变。通过短串联重复序列分析我们发现细胞系UM-UC-2就是T24。使用短串联重复序列分析进行DNA指纹识别是一种可用于验证细胞系的简单且可靠的工具。