Comparative clonal analysis of reconstitution kinetics after transplantation of hematopoietic stem cells gene marked with a lentiviral SIN or a γ-retroviral LTR vector
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Comparative clonal analysis of reconstitution kinetics after transplantation of hematopoietic stem cells gene marked with a lentiviral SIN or a γ-retroviral LTR vector. / Cornils, Kerstin; Bartholomae, Cynthia C; Thielecke, Lars; Lange, Claudia; Arens, Anne; Glauche, Ingmar; Mock, Ulrike; Riecken, Kristoffer; Gerdes, Sebastian; von Kalle, Christof; Schmidt, Manfred; Roeder, Ingo; Fehse, Boris.
in: EXP HEMATOL, Jahrgang 41, Nr. 1, 01.01.2013, S. 28-38.e3.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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T1 - Comparative clonal analysis of reconstitution kinetics after transplantation of hematopoietic stem cells gene marked with a lentiviral SIN or a γ-retroviral LTR vector
AU - Cornils, Kerstin
AU - Bartholomae, Cynthia C
AU - Thielecke, Lars
AU - Lange, Claudia
AU - Arens, Anne
AU - Glauche, Ingmar
AU - Mock, Ulrike
AU - Riecken, Kristoffer
AU - Gerdes, Sebastian
AU - von Kalle, Christof
AU - Schmidt, Manfred
AU - Roeder, Ingo
AU - Fehse, Boris
N1 - Copyright © 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.
PY - 2013/1/1
Y1 - 2013/1/1
N2 - Retroviral gene marking has been used successfully in preclinical and clinical transplantation settings. Highly sensitive techniques for vector insertion-site determination, such as linear amplification-mediated polymerase chain reaction (LAM-PCR) in conjunction with next-generation sequencing, have been introduced to assess the composition of gene-marked hematopoiesis at a single-cell level. Here we used these novel techniques for directly comparing clonal reconstitution kinetics in mice transplanted with bone-marrow-derived stem cells genetically marked with either a standard, spleen focus-forming virus long terminal repeat (LTR)-driven γ-retroviral, or a lentiviral self-inactivating vector containing an identical but internal spleen focus-forming virus-derived enhancer/promoter. We observed that the use of the lentiviral self-inactivating vector for gene marking was associated with a broader repertoire of differently marked hematopoietic clones. More importantly, we found a significantly higher probability of insertions in growth-promoting, clonal-dominance-associated genes in the spleen focus-forming virus LTR-driven γ-retroviral vector at later time points of analysis. Based on our data, we suggest that the combined use of LAM-PCR and next-generation sequencing represents a potent tool for the analysis of clonal reconstitution kinetics in the context of gene marking with integrated vectors. At the same time, our findings prove that the use of multiple restriction enzymes for LAM-PCR is indispensable to detect most or ideally all individual stem cell clones contributing to hematopoiesis. We have also found that techniques such as quantitative PCR can be helpful to retrospectively analyze reconstitution kinetics for individual hematopoietic stem cell clones. Finally, our results confirm the notion that marking with lentiviral self-inactivating vectors is associated with a lower risk of genotoxicity as compared with γ-retroviral LTR vectors.
AB - Retroviral gene marking has been used successfully in preclinical and clinical transplantation settings. Highly sensitive techniques for vector insertion-site determination, such as linear amplification-mediated polymerase chain reaction (LAM-PCR) in conjunction with next-generation sequencing, have been introduced to assess the composition of gene-marked hematopoiesis at a single-cell level. Here we used these novel techniques for directly comparing clonal reconstitution kinetics in mice transplanted with bone-marrow-derived stem cells genetically marked with either a standard, spleen focus-forming virus long terminal repeat (LTR)-driven γ-retroviral, or a lentiviral self-inactivating vector containing an identical but internal spleen focus-forming virus-derived enhancer/promoter. We observed that the use of the lentiviral self-inactivating vector for gene marking was associated with a broader repertoire of differently marked hematopoietic clones. More importantly, we found a significantly higher probability of insertions in growth-promoting, clonal-dominance-associated genes in the spleen focus-forming virus LTR-driven γ-retroviral vector at later time points of analysis. Based on our data, we suggest that the combined use of LAM-PCR and next-generation sequencing represents a potent tool for the analysis of clonal reconstitution kinetics in the context of gene marking with integrated vectors. At the same time, our findings prove that the use of multiple restriction enzymes for LAM-PCR is indispensable to detect most or ideally all individual stem cell clones contributing to hematopoiesis. We have also found that techniques such as quantitative PCR can be helpful to retrospectively analyze reconstitution kinetics for individual hematopoietic stem cell clones. Finally, our results confirm the notion that marking with lentiviral self-inactivating vectors is associated with a lower risk of genotoxicity as compared with γ-retroviral LTR vectors.
KW - Animals
KW - Clone Cells
KW - Gammaretrovirus
KW - Genetic Vectors
KW - Hematopoiesis
KW - Hematopoietic Stem Cell Transplantation
KW - Hematopoietic Stem Cells
KW - Kinetics
KW - Lentivirus
KW - Mice
KW - Polymerase Chain Reaction
KW - Terminal Repeat Sequences
KW - Transduction, Genetic
U2 - 10.1016/j.exphem.2012.09.003
DO - 10.1016/j.exphem.2012.09.003
M3 - SCORING: Journal article
C2 - 22989760
VL - 41
SP - 28-38.e3
JO - EXP HEMATOL
JF - EXP HEMATOL
SN - 0301-472X
IS - 1
ER -