Real-Time PCR

An effective tool for measuring transduction efficiency in human hematopoietic progenitor cells

Anthony D. Villella, Jing Yao, Robert R. Getty, Beth E. Juliar, Constantin Yiannoutsos, Jennifer R. Hartwell, Shanbao Cai, Mohammed A. Sadat, Kenneth Cornetta, David A. Williams, Karen Pollok

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)483-491
Number of pages9
JournalMolecular Therapy
Volume11
Issue number3
DOIs
StatePublished - Mar 2005

Fingerprint

Hematopoietic Stem Cells
Real-Time Polymerase Chain Reaction
Sensitivity and Specificity
Polymerase Chain Reaction
Granulocyte Colony-Stimulating Factor
Fluorescence Microscopy
Bone Marrow Cells
Genetic Therapy
Linear Models
Stem Cells
Genes

Keywords

  • Gene therapy
  • Progenitor cell
  • Real-time PCR
  • Retrovirus
  • Transduction efficiency

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Real-Time PCR : An effective tool for measuring transduction efficiency in human hematopoietic progenitor cells. / Villella, Anthony D.; Yao, Jing; Getty, Robert R.; Juliar, Beth E.; Yiannoutsos, Constantin; Hartwell, Jennifer R.; Cai, Shanbao; Sadat, Mohammed A.; Cornetta, Kenneth; Williams, David A.; Pollok, Karen.

In: Molecular Therapy, Vol. 11, No. 3, 03.2005, p. 483-491.

Research output: Contribution to journalArticle

Villella, Anthony D. ; Yao, Jing ; Getty, Robert R. ; Juliar, Beth E. ; Yiannoutsos, Constantin ; Hartwell, Jennifer R. ; Cai, Shanbao ; Sadat, Mohammed A. ; Cornetta, Kenneth ; Williams, David A. ; Pollok, Karen. / Real-Time PCR : An effective tool for measuring transduction efficiency in human hematopoietic progenitor cells. In: Molecular Therapy. 2005 ; Vol. 11, No. 3. pp. 483-491.
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