Retroviral vector integration occurs in preferred genomic targets of human bone marrow–repopulating cells by Stephanie Laufs, Bernhard Gentner, K. Zsuzsanna Nagy, Anna Jauch, Axel Benner, Sonja Naundorf, Klaus Kuehlcke, Bernhard Schiedlmeier, Anthony D. Ho, W. Jens Zeller, and Stefan Fruehauf Blood Volume 101(6):2191-2198 March 15, 2003 ©2003 by American Society of Hematology

Ligation-mediated PCR technique.Human genomic DNA was isolated. Ligation-mediated PCR technique.Human genomic DNA was isolated. A restriction enzyme digest (↓) was performed. Fragments containing LTR-genomic DNA junctions were marked with a biotinylated LTR-specific primer (GSP1-bio) and enriched by streptavidin-coated paramagnetic beads. Other DNA fragments were flushed away. An adapter-oligo-cassette was ligated to flanking DNA. Solid-phase nested PCR was performed (AP1/AP2, adapter-specific primers; GSP1/GSP5, LTR-specific primers). PCR bands were excised after gel electrophoresis and separately cloned, and clones with different insert lengths were sequenced. Stephanie Laufs et al. Blood 2003;101:2191-2198 ©2003 by American Society of Hematology

Retroviral integration patterns were detected by LM-PCR Retroviral integration patterns were detected by LM-PCR.(A) Three SF1m-transduced HT1080 cell line clones were analyzed. Retroviral integration patterns were detected by LM-PCR.(A) Three SF1m-transduced HT1080 cell line clones were analyzed. DNA from each clone was used in separate reactions (1, 2, 3) and mixed together in one reaction (1/2/3). The internal band (IB) originates from the 3′ LTR and is identical for all SF1m vector-transduced cells. (B) Five SF1m-transduced chimeric NOD/SCID mouse BMs analyzed with the optimized LM-PCR protocol. Whole chimeric BM DNA was digested withBsmAI. Nested LM-PCR products were analyzed by agarose gel electrophoresis. Numbers to the left of the blots denote fragment size in kilobases (kb). The control mouse received transplants of untransduced (mock-transduced) human cells. (C) Flanking sequences of clones isolated from mouse E3M7 (no. 7) are given. Stephanie Laufs et al. Blood 2003;101:2191-2198 ©2003 by American Society of Hematology

Chromosomal mapping of proviral sequences by FISH Chromosomal mapping of proviral sequences by FISH.FISH analysis using SF1m vector plasmid DNA as probe and subsequently performed M-FISH was established to detect proviral inserts in SF1m-transduced HT1080 cell line clones. Chromosomal mapping of proviral sequences by FISH.FISH analysis using SF1m vector plasmid DNA as probe and subsequently performed M-FISH was established to detect proviral inserts in SF1m-transduced HT1080 cell line clones. (A-B) M-FISH karyogram and metaphase spread of HT1080 clone N3 presenting a near tetraploid karyotype (n = 87) with following recurrent chromosomal aberrations: dup(5p), t(4;8), t(3;11), i(13q), and i(18p). (C) Same metaphase spread after FISH using the SF1m vector plasmid DNA probe shows hybridization signals on the human wild-type MDR1 gene locus 7q21 and on chromosome 1q41. (D-F) Chromosomal localization of the SF1m vector plasmid DNA probe in HT1080 cell line clones N2 (12q14), N3 (1q41), and N4 (20q11), respectively. Additional signals are present on the human wild-type MDR1 gene locus (7q21). Stephanie Laufs et al. Blood 2003;101:2191-2198 ©2003 by American Society of Hematology

Chromosomal distribution of SF1m retroviral vector integrations Chromosomal distribution of SF1m retroviral vector integrations.LM-PCR sequences (141) from NOD/SCID mouse BMs were unambiguously assigned to human DNA clones mapped to chromosomes. Chromosomal distribution of SF1m retroviral vector integrations.LM-PCR sequences (141) from NOD/SCID mouse BMs were unambiguously assigned to human DNA clones mapped to chromosomes. The retrovirally transduced PBPCs transplanted to the mice originated from 3 donors and integration sites are marked accordingly: + represents donor 1; §, donor 2; #, donor 3. Numbers in boldface indicate multiple integrations in 1 chromosomal region. Stephanie Laufs et al. Blood 2003;101:2191-2198 ©2003 by American Society of Hematology