title: Real-Time PCR: an Effective Tool for Measuring Transduction Efficiency in Human Hematopoietic Progenitor Cells.
abstract: Accurate measurement of gene transfer into hematopoietic progenitor cells is an essential prerequisite for assessing the utility of gene therapy approaches designed to correct hematologic defects. We developed a reliable method to measure transduction efficiency at the level of the progenitor cell with real-time polymerase chain reaction (PCR) analysis of individual progenitor-derived colonies. We hypothesized that this method would demonstrate better sensitivity and specificity than are currently achievable with conventional PCR. An oncoretroviral vector containing the enhanced green fluorescent protein was used to transduce human CD34+cells derived from bone marrow or granulocyte-colony-stimulating factor-mobilized peripheral blood. Progenitor assays were set up and colonies plucked after visualization by fluorescence microscopy. By analyzing microscopically identified fluorescent samples and nontransduced samples, we calculated an overall sensitivity and specificity of 90.2 and 95.0%, respectively. Real-time PCR had higher specificity and sensitivity than conventional PCR as analyzed by generalized linear models (P = 0.002 and P = 0.019, respectively). In conclusion, we found real-time PCR to have superior sensitivity and specificity compared to conventional PCR in determining transduction efficiency of hematopoietic progenitor cells.
authors.affiliation: Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.
authors.forename: Anthony D
authors.initials: AD
authors.lastname: Villella
authors.affiliation: Medical and Molecular Genetics, Indiana University Cancer Center, Indianapolis, IN 46202, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Yao
authors.affiliation: Medical and Molecular Genetics, Indiana University Cancer Center, Indianapolis, IN 46202, USA.
authors.forename: Robert R
authors.initials: RR
authors.lastname: Getty
authors.affiliation: Biostatistics and Data Management Core, Indiana University Cancer Center, Indianapolis, IN 46202, USA.
authors.forename: Beth E
authors.initials: BE
authors.lastname: Juliar
authors.affiliation: Biostatistics and Data Management Core, Indiana University Cancer Center, Indianapolis, IN 46202, USA.
authors.forename: Constantin
authors.initials: C
authors.lastname: Yiannoutsos
authors.affiliation: Herman B. Wells Center for Pediatric Research, Indiana University, Indianapolis, IN 46202, USA.
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Hartwell
authors.affiliation: Herman B. Wells Center for Pediatric Research, Indiana University, Indianapolis, IN 46202, USA.
authors.forename: Shanbao
authors.initials: S
authors.lastname: Cai
authors.affiliation: Herman B. Wells Center for Pediatric Research, Indiana University, Indianapolis, IN 46202, USA.
authors.forename: Mohammed A
authors.initials: MA
authors.lastname: Sadat
authors.affiliation: Medical and Molecular Genetics, Indiana University Cancer Center, Indianapolis, IN 46202, USA.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Cornetta
authors.affiliation: Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA. Electronic address: David.Williams@cchmc.org.
authors.forename: David A
authors.initials: DA
authors.lastname: Williams
authors.affiliation: Herman B. Wells Center for Pediatric Research, Indiana University, Indianapolis, IN 46202, USA. Electronic address: kpollok@iupui.edu.
authors.forename: Karen E
authors.initials: KE
authors.lastname: Pollok
keywords: gene therapy; progenitor cell; real-time PCR; retrovirus; transduction efficiency