Rapid detection of retroviral vector integration sites in colony-forming human peripheral blood prgenitor cells using PCR with arbitrary primers

B. Gentner, S. Laufs, K. Z. Nagy, W. J. Zeller, S. Fruehauf

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

We have developed a highly sensitive polymerase chain reaction (PCR)-based technique termed two-step PCR, which uses arbitrary primers to identify proviral integration sites in retrovirally marked human colony-forming cells. The two-step PCR was established on cell line clones transduced with the SF1m retroviral vector and independently validated by demonstrating identical integration sites with ligation-mediated PCR, a different technique requiring restriction enzyme digestion and adapter ligation for amplifying unknown DNA flanking the provirus. Two-step PCR was performed on peripheral blood progenitor cell (PBPC) colonies that contained as few as 75 cells, which was estimated by quantitative real-time PCR. We were able to amplify and directly sequence proviral integration sites in 35 % of PBPC colonies (25/72, five donors). Identity to the vector long-terminal repeat was confirmed and flanking DNA was found to match with human database sequences, reaffirming specificity. Two-step PCR is a valuable new tool for rapid analysis of genomic target sites for viral vectors, and will aid significantly in understanding clonal development of hematopoiesis and other cell types. Our protocol has the potential for general applicability as the arbitrary primers described here bind to genomic DNA and are thus independent of the vector backbone used.

Original languageEnglish
Pages (from-to)789-794
Number of pages6
JournalGene Therapy
Volume10
Issue number9
DOIs
Publication statusPublished - May 2003

Fingerprint

Blood Cells
Polymerase Chain Reaction
Ligation
DNA
Stem Cells
Proviruses
Terminal Repeat Sequences
Hematopoiesis
Real-Time Polymerase Chain Reaction
Digestion
Clone Cells
Databases
Cell Line
Enzymes

Keywords

  • Gene therapy
  • Integration site analysis
  • Peripheral blood progenitor cells
  • Two-steps PCR

ASJC Scopus subject areas

  • Genetics

Cite this

Rapid detection of retroviral vector integration sites in colony-forming human peripheral blood prgenitor cells using PCR with arbitrary primers. / Gentner, B.; Laufs, S.; Nagy, K. Z.; Zeller, W. J.; Fruehauf, S.

In: Gene Therapy, Vol. 10, No. 9, 05.2003, p. 789-794.

Research output: Contribution to journalArticle

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