Ligation-mediated pcr and pcr with partly degenerate, arbitrary primers both allow detection of clonal integration of retroviral vectors in human hematopoietic cells

B. Gentner, S. Fruehauf, S. Laufs, K. Kühlcke, W. J. Zeller, B. Schiedlmeier

Research output: Contribution to journalArticle

Abstract

Retroviral integration sites in the genome offer the unique opportunity to study the clonal development of hematopoiesis after transplantation and to track the multilineage progeny of transduced pluripotent stem cells. Traditional methods for investigating clonal integration such as inverse PCR do not reliably detect multiple clones in one PCR sample so that semisolid cultures and single-colony analyses are required. We wanted to study bulk cell populations of NOD/SCID mice chimeric bone marrows with regard to the number of different human NOD/SCID repopulating cell clones present 5-13 weeks after transplantation of these mice with retrovirally marked human CD34+ peripheral blood progenitor cells (PBPC). To this end we established a ligation-mediated PCR (LM-PCR) protocol consisting of digestion of DNA, thus creating a cell clone-specific restriction fragment length polymorphism (RFLP), isolation of fragments containing the LTR-human genomic DNA junction using magnetic beads, adaptor ligation, nested PCR, followed by direct sequencing of bands. The detection threshold for a clone was in the range of 5-10 ng DNA of transduced human cells in 1 μg mouse DNA background. Mixing studies allowed at least three different clones to be detected simultaneously in one LM-PCR. In four chimeric NOD/SCID mouse bone marrows we were able to detect up to 3 (3,2,1,0) different integration sites without unspecific mouse DNA background bands, suggesting that at least this number of primitive hematopoietic stem cells with engraftment potential had been transduced with the vector. 10, 2.5, 1.4, 1.2 % of all bone marrow cells were retrovirally marked in these mice, respectively. To complement the LM-PCR and comprehensively characterize clonal integration sites we established a more rapid twostep PCR with partly degenerate, arbitrary primers. This method was used for analysis of single colonies grown from retrovirally transduced PBPCs. The LTR-human genomic DNA junction could be amplified even from small colonies (

Original languageEnglish
JournalBlood
Volume96
Issue number11 PART I
Publication statusPublished - 2000

ASJC Scopus subject areas

  • Hematology

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