Study of Reference Materials for Quantitative Analysis of Gene Copy Numbers of Lentiviral Vectors
10.19756/j.issn.0253-3820.241229
- VernacularTitle:慢病毒载体基因拷贝数定量分析用标准物质研究
- Author:
Yin-Bo HUO
1
;
Jia-Qi YANG
;
Qing TAO
;
Wen LIANG
;
Li XU
;
Lan-Ying LI
;
Xiao-Lei ZUO
;
Juan YAN
;
Min DING
;
Ai-Wen MA
;
Gang LIU
Author Information
1. 上海市计量测试技术研究院,国家市场监督管理总局生物分析计量溯源重点实验室,上海 201203
- Keywords:
Lentiviral vector;
Vector copy number;
Cell reference material;
Digital polymerase chain reaction
- From:
Chinese Journal of Analytical Chemistry
2025;53(9):1555-1565
- CountryChina
- Language:Chinese
-
Abstract:
Lentiviral vectors(LVs)are key gene delivery tools for integrating target genes into the host genome,but they may also pose risks of insertional mutagenesis.The vector copy number(VCN)in cells is critical for determining the safety of gene modification.However,the reliability and accuracy of its quantification process are influenced by multiple factors.Developing cell reference materials with specific vector copy numbers represents a viable approach to enhance the reliability and consistency of measurement results,enabling quality control of the quantification process and traceability of outcomes.However,the preparation of such reference materials faces challenges in cell sample design,preparation protocols,and advanced quantification techniques.In this study,T lymphocyte cell line Jurkat-based reference materials with LV gene copy numbers of 1 and 2 copy/cell were developed.A high-precision duplex digital polymerase chain reaction(dPCR)method was established to quantify the LV gene and endogenous genes simultaneously.Additionally,the results of dPCR were cross-validated through next-generation sequencing and flow cytometric analysis.Ultimately,confocal microscopy characterization results showed that the developed cell reference materials had intact morphology.The quantification result of VCN-1 was(1.07±0.11)copy/cell,and that of VCN-2 was(2.09±0.21)copy/cell.These cell reference materials demonstrated compliance with stability and homogeneity requirements,and could be applied for quality control throughout the VCN measurement workflow and metrological traceability,improving the accuracy,comparability,and validity of copy number measurements.
