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Efficient Characterization of Retro‐, Lenti‐, and Foamyvector‐Transduced Cell Populations by High‐Accuracy Insertion Site Sequencing
Author(s) -
SCHMIDT MANFRED,
GLIMM HANNO,
WISSLER MANUELA,
HOFFMANN GESA,
OLSSON KARIN,
SELLERS STEPHANIE,
CARBONARO DENISE,
TISDALE JOHN F.,
LEURS CORDULA,
HANENBERG HELMUT,
DUNBAR CYNTHIA E.,
KIEM HANSPETER,
KARLSSON STEFAN,
KOHN DONALD B.,
WILLIAMS DAVID,
KALLE CHRISTOF
Publication year - 2003
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1111/j.1749-6632.2003.tb03239.x
Subject(s) - insertional mutagenesis , biology , genomic dna , dna sequencing , dna , computational biology , genetic enhancement , gene , mutagenesis , transgene , exogenous dna , polymerase chain reaction , microbiology and biotechnology , genetics , mutation , genome
A bstract : The identification of unknown genomic flanking DNA sequences can be used for the molecular monitoring of retro‐, lenti‐ and foamyviral integration, transgenes in early embryogenesis, insertional mutagenesis, cell fate, and stem cell plasticity. Most existing methods reflect shortcomings in sensitivity and or specificity, thus limiting genomic sequencing of unknown flanking DNA to clonal preparations. The application of linear amplification‐mediated PCR (LAM‐PCR), a recently developed direct sequencing technique for flanking DNA, should circumvent current limitations in different research fields. This technique combines preamplification of target DNA with a unique succession of enzymatic reactions on solid‐phase. Using LAM‐PCR, we show the previously unfeasible in vivo retro‐, lenti‐ and foamyvirus integration site analysis in primate peripheral blood hematopoietic cells and human xenograft hematopoiesis. In light of two severe adverse events that occurred in a clinical SCID‐X1 gene therapy trial, in vivo monitoring of the reinfused transduced cell pool by integration site analysis will be an important component of each gene transfer and therapy study aimed at clinical use.