Evaluating a ligation-mediated PCR and pyrosequencing method for the detection of clonal contribution in polyclonal retrovirally transduced samples.

ABSTRACT: Retroviral gene transfer has proven therapeutic potential in clinical gene therapy trials but may also cause abnormal cell growth via perturbation of gene expression in the locus surrounding the insertion site. By establishing clonal marks, retroviral insertions are also used to describe the regenerative potential of individual cells. Deep sequencing approaches have become the method of choice to study insertion profiles in preclinical models and clinical trials. We used a protocol combining ligation-mediated polymerase chain reaction (LM-PCR) and pyrosequencing for insertion profiling and quantification in cells of various tissues transduced with various retroviral vectors. The presented method allows simultaneous analysis of a multitude of DNA-barcoded samples per pyrosequencing run, the

SUBMITTER: Brugman MH 

PROVIDER: S-EPMC3732125 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature