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Related Experiment Video

Updated: Jun 8, 2026

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Published on: August 6, 2014

High-throughput, sensitive quantification of repopulating hematopoietic stem cell clones.

Sanggu Kim¹, Namshin Kim, Angela P Presson

¹Department of Microbiology,University of California, David Geffen School of Medicine, Los Angeles, CA 90095, USA.

Journal of Virology

|September 17, 2010

Summary

This study introduces a new quantitative method for analyzing vector integration sites (VIS) in gene therapy. The improved assay accurately measures hematopoietic stem and progenitor cell (HPSC) clone repopulation, enhancing safety assessments for gene therapies.

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Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

Published on: July 8, 2012

Area of Science:

Biotechnology
Molecular Biology
Genetics

Background:

Retroviral vector gene therapy offers potential for treating genetic diseases but carries risks of oncogenesis from vector insertions.
Current vector integration site (VIS) sequencing methods for clonal analysis are often semiquantitative, limiting precise assessment of hematopoietic progenitor/stem cell (HPSC) repopulation.

Purpose of the Study:

To develop and validate a novel, high-throughput, and quantitative system for accurate clonality analysis using 454 pyrosequencing.
To improve the detection and quantification of repopulating HPSC clones in gene therapy applications.

Main Methods:

Developed a bidirectional vector integration site (VIS) PCR method to analyze both 5' and 3' vector-host junctions.
Optimized conditions for quantitative VIS sequencing and validated the assay in a nonhuman primate model.
Assessed assay reliability and sensitivity by comparing results with clone-specific real-time PCR.

Main Results:

The novel assay enabled high-throughput and sensitive assessment of hundreds of repopulating HPSC clones.
A strong correlation was observed between the developed quantitative VIS sequencing assay and clone-specific real-time PCR for the majority of tested clones.
The bidirectional VIS PCR method improved vector integration site detection.

Conclusions:

The developed system provides accurate and sensitive clonality analysis for gene therapy and stem cell research.
This quantitative VIS sequencing assay is valuable for monitoring HPSC repopulation and assessing the safety of gene therapies.
The assay facilitates a deeper understanding of clonal dynamics in stem cell research and cancer studies.