Linked Life Data

18049469

Source:http://linkedlifedata.com/resource/pubmed/id/18049469

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
12
pubmed:dateCreated
2007-11-30
pubmed:abstractText
Integrating vector systems used in clinical gene therapy have proven their therapeutic potential in the long-term correction of immunodeficiencies. The integration loci of such vectors in the cellular genome represent a molecular marker unique for each transduced cell and its clonal progeny. To gain insight into the physiology of gene-modified hematopoietic repopulation and vector-related influences on clonal contributions, we have previously introduced a technology--linear amplification-mediated (LAM) PCR--for detecting and sequencing unknown DNA flanking sequences down to the single cell level (Supplementary Note online). LAM-PCR analyses have enabled qualitative and quantitative measurements of the clonal kinetics of hematopoietic regeneration in gene transfer studies, and uncovered the clonal derivation of non-leukemogenic and leukemogenic insertional side effects in preclinical and clinical gene therapy studies. The reliability and robustness of this method results from the initial preamplification of the vector-genome junctions preceding nontarget DNA removal via magnetic selection. Subsequent steps are carried out on a semisolid streptavidin phase, including synthesis of double complementary strands, restriction digest, ligation of a linker cassette onto the genomic end of the fragment and exponential PCR(s) with vector- and linker cassette-specific primers. LAM-PCR can be adjusted to all unknown DNA sequences adjacent to a known DNA sequence. Here we describe the use of LAM-PCR analyses to identify 5' long terminal repeat (LTR) retroviral vector adjacent genomic sequences.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
1548-7105
pubmed:author
pubmed:issnType
Electronic
pubmed:volume
4
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1051-7
pubmed:dateRevised
2008-4-29
pubmed:meshHeading
pubmed:year
2007
pubmed:articleTitle
High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR).
pubmed:affiliation
Department of Translational Oncology, National Center for Tumor Diseases, German Cancer Research Center, Im Neuenheimer Feld 350, 69120 Heidelberg, Germany. manfred.schmidt@nct-heidelberg.de
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't, Research Support, N.I.H., Extramural