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用于绘制参与治疗抗性的基因中病毒整合位点的新方案。

Novel protocol for mapping virus integration sites in genes involved in therapy resistance.

作者信息

Massimino Michele, Huang Zhangzan, Helmijr Jean, Muritti Camilla, Uijtdewillegen Anna, Vigneri Paolo, Martens John W M, Jansen Maurice P H M

机构信息

Department of Medical Oncology, Erasmus MC Cancer Institute, University Medical Centre Rotterdam, Dr. Molewaterplein 40, 3015 GD, Rotterdam, The Netherlands.

Department of General Surgery and Medical-Surgical Specialties, University of Catania, Catania, Italy.

出版信息

Sci Rep. 2025 Jul 1;15(1):21841. doi: 10.1038/s41598-025-05160-4.

DOI:10.1038/s41598-025-05160-4
PMID:40592883
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12215797/
Abstract

Retroviral transduction of cancer cell lines has been used to find genes related to therapy resistance. Characterization of virus integration sites (VIS) pinpointing these genes has been cumbersome. This study defines a sequencing-based protocol to rapidly characterize genomic loci near VIS. The protocol selectively amplifies VIS-genome junctions using the retroviral vector neomycin (NEO) gene, Genomic Walker Adapter approach, linker-mediated NEO-PCR (LM-NEO-PCR) or biotinylated NEO-capture, followed by long terminal repeats PCR (LTR-PCR). LTR-genome junctions were sequenced (NGS), reads mapped, quantified, and linked to genes. The protocol was tested on DNA from single clones holding twenty reported VIS loci and on multiplex and diluted clone DNA samples. Our VIS-NGS protocol enriched significantly (p < 0.02) more loci at high reads coverage in samples with VIS compared to negative controls. The protocol found seventeen reported VIS loci (85%) in single clone DNAs, of which fifteen loci (88%) were also detected in multiplex samples. Six loci were evaluated for all dilutions, with three loci detected at lowest 1% clone proportion. The protocol can be conducted in two weeks and successfully found almost all VIS loci in single VIS clones and detected half of the evaluated loci at low clone proportion.

摘要

逆转录病毒转导癌细胞系已被用于寻找与治疗抗性相关的基因。确定这些基因的病毒整合位点（VIS）的特征一直很麻烦。本研究定义了一种基于测序的方案，以快速确定VIS附近的基因组位点。该方案使用逆转录病毒载体新霉素（NEO）基因、基因组步移衔接子方法、衔接子介导的NEO-PCR（LM-NEO-PCR）或生物素化的NEO捕获选择性扩增VIS-基因组连接点，随后进行长末端重复序列PCR（LTR-PCR）。对LTR-基因组连接点进行测序（NGS）、读取映射、定量，并与基因相关联。该方案在含有20个已报道的VIS位点的单克隆DNA以及多重和稀释的克隆DNA样本上进行了测试。与阴性对照相比，我们的VIS-NGS方案在具有VIS的样本中，在高读取覆盖率下显著富集了更多的位点（p < 0.02）。该方案在单克隆DNA中发现了17个已报道的VIS位点（85%），其中15个位点（88%）也在多重样本中被检测到。对所有稀释度的6个位点进行了评估，在最低1%的克隆比例下检测到3个位点。该方案可在两周内完成，并成功在单个VIS克隆中发现了几乎所有的VIS位点，且在低克隆比例下检测到了一半的评估位点。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3d2f/12215797/51cff1a69296/41598_2025_5160_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3d2f/12215797/acb0f29ca555/41598_2025_5160_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3d2f/12215797/031832212c6b/41598_2025_5160_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3d2f/12215797/144d407ccecb/41598_2025_5160_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3d2f/12215797/c6838a3e9536/41598_2025_5160_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3d2f/12215797/30c5c1aa0b5f/41598_2025_5160_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3d2f/12215797/51cff1a69296/41598_2025_5160_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3d2f/12215797/acb0f29ca555/41598_2025_5160_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3d2f/12215797/031832212c6b/41598_2025_5160_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3d2f/12215797/144d407ccecb/41598_2025_5160_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3d2f/12215797/c6838a3e9536/41598_2025_5160_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3d2f/12215797/30c5c1aa0b5f/41598_2025_5160_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3d2f/12215797/51cff1a69296/41598_2025_5160_Fig6_HTML.jpg

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