Multiple displacement amplification enables large-scale clonal analysis following retroviral gene therapy.
Analysis of the fate of retrovirally transduced cells after transplantation is often hampered by the scarcity of available DNA. We evaluated a promising method for whole-genome amplification, called multiple displacement amplification (MDA), with respect to even and accurate representation of retrovirally transduced genomic DNA. We proved that MDA is a suitable method to subsequently quantify engraftment efficiencies by quantitative real-time PCR by analyzing retrovirally transduced DNA in a background of untransduced DNA and retroviral integrations found in primary material from a retroviral transplantation model. The portion of these retroviral integrations in the amplified samples was 1.02-fold (range 0.2, to 2.1-fold) the portion determined in the original genomic DNA. Integration site analysis by ligation-mediated PCR (LM-PCR) is essential for the detection of retroviral integrations. The combination of MDA and LM-PCR showed an increase in the sensitivity of integration site analysis, as a specific integration site could be detected in a background of untransduced DNA, while the transduced DNA made up only 0.001%. These results show for the first time that MDA enables large-scale sensitive detection and reliable quantification of retrovirally transduced human genomic DNA and therefore facilitates follow-up analysis in gene therapy studies even from the smallest amounts of starting material.
Bleier
S
S
Molecular Oncology of Solid Tumors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany.
Maier
P
P
Allgayer
H
H
Wenz
F
F
Zeller
W J
WJ
Fruehauf
S
S
Laufs
S
S
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D005784:Gene Amplification; D015316:Genetic Therapy; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D012190:Retroviridae / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D017931:DNA Primers