Komosa Martin, Root Heather, Meyn M Stephen
Genetics and Genome Biology Program, The Hospital for Sick Children, Toronto, Ontario, M5G 0A4, Canada.
Genetics and Genome Biology Program, The Hospital for Sick Children, Toronto, Ontario, M5G 0A4, Canada Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada Department of Paediatrics, University of Toronto, Toronto, Ontario, M5S 1A8, Canada Department of Molecular Genetics, University of Toronto, Toronto, Ontario, M5S 1A8, Canada
Nucleic Acids Res. 2015 Feb 27;43(4):2152-63. doi: 10.1093/nar/gkv091. Epub 2015 Feb 8.
Current methods for characterizing extrachromosomal nuclear DNA in mammalian cells do not permit single-cell analysis, are often semi-quantitative and frequently biased toward the detection of circular species. To overcome these limitations, we developed Halo-FISH to visualize and quantitatively analyze extrachromosomal DNA in single cells. We demonstrate Halo-FISH by using it to analyze extrachromosomal telomere-repeat (ECTR) in human cells that use the Alternative Lengthening of Telomeres (ALT) pathway(s) to maintain telomere lengths. We find that GM847 and VA13 ALT cells average ∼80 detectable G/C-strand ECTR DNA molecules/nucleus, while U2OS ALT cells average ∼18 molecules/nucleus. In comparison, human primary and telomerase-positive cells contain <5 ECTR DNA molecules/nucleus. ECTR DNA in ALT cells exhibit striking cell-to-cell variations in number (<20 to >300), range widely in length (<1 to >200 kb) and are composed of primarily G- or C-strand telomere-repeat DNA. Halo-FISH enables, for the first time, the simultaneous analysis of ECTR DNA and chromosomal telomeres in a single cell. We find that ECTR DNA comprises ∼15% of telomere-repeat DNA in GM847 and VA13 cells, but <4% in U2OS cells. In addition to its use in ALT cell analysis, Halo-FISH can facilitate the study of a wide variety of extrachromosomal DNA in mammalian cells.
目前用于表征哺乳动物细胞中染色体外核DNA的方法不允许进行单细胞分析，通常是半定量的，并且常常偏向于检测环状物种。为了克服这些局限性，我们开发了Halo-FISH来可视化和定量分析单细胞中的染色体外DNA。我们通过使用Halo-FISH分析使用端粒替代延长（ALT）途径来维持端粒长度的人类细胞中的染色体外端粒重复序列（ECTR）来证明其可行性。我们发现GM847和VA13 ALT细胞平均每个细胞核约有80个可检测到的G/C链ECTR DNA分子，而U2OS ALT细胞平均每个细胞核约有18个分子。相比之下，人类原代细胞和端粒酶阳性细胞每个细胞核中含有的ECTR DNA分子少于5个。ALT细胞中的ECTR DNA在数量上表现出显著的细胞间差异（<20至>300），长度范围广泛（<1至>200 kb），并且主要由G链或C链端粒重复DNA组成。Halo-FISH首次实现了在单个细胞中同时分析ECTR DNA和染色体端粒。我们发现ECTR DNA在GM847和VA13细胞中占端粒重复DNA的约15%，但在U2OS细胞中<4%。除了用于ALT细胞分析外，Halo-FISH还可以促进对哺乳动物细胞中多种染色体外DNA的研究。
