Real-Time PCR: an Effective Tool for Measuring Transduction Efficiency in Human Hematopoietic Progenitor Cells
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 w...
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Published in | Molecular therapy Vol. 11; no. 3; pp. 483 - 491 |
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Main Authors | , , , , , , , , , , |
Format | Journal Article |
Language | English |
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Elsevier Limited
01.03.2005
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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. |
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AbstractList | 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. 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. |
Author | Getty, Robert R Juliar, Beth E Williams, David A Pollok, Karen E Cornetta, Kenneth Sadat, Mohammed A Yao, Jing Yiannoutsos, Constantin Cai, Shanbao Villella, Anthony D Hartwell, Jennifer R |
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Cites_doi | 10.1089/hum.1996.7.3-343 10.1002/sim.4780111406 10.1038/sj.gt.3300951 10.1089/10430349950016898 10.1128/JCM.39.8.2904-2910.2001 10.1038/nm0896-876 10.1093/clinchem/46.3.324 10.1089/10430340152677430 10.1128/JVI.69.12.7430-7436.1995 10.1038/sj.gt.3301112 10.1038/sj.gt.3300948 10.1038/sj.bmt.1702720 10.1128/JCM.41.2.592-600.2003 10.1182/blood.V90.9.3304 10.1186/1471-2180-2-17 10.1128/JVI.65.5.2220-2224.1991 |
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Keywords | gene therapy retrovirus transduction efficiency real-time PCR progenitor cell |
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References | Croop, J. M. 2000; 26 Cossett, F. L. 1995; 69 Mygind, T. 2002; 2 Sanburn, N., Cornetta, K. 1999; 6 Klein, D. 2000; 7 Pollok, K. 2001; 12 Rantakokko-Jalava, K., Jalava, J. 2001; 39 Miller, A. 1991; 65 Becker, K. 1999; 10 Stipecke, R. 1999; 6 Bierhuizen, M. F. A. 1997; 90 Gerard, C. J. 1996; 7 Apfalter, P. 2003; 41 Ke, D. 2000; 46 Zeger, S. L., Liang, K. Y. 1992; 11 Hanenberg, H. 1996; 2 Hanenberg (10.1016/j.ymthe.2004.10.017_bib14) 1996; 2 Becker (10.1016/j.ymthe.2004.10.017_bib6) 1999; 10 Mygind (10.1016/j.ymthe.2004.10.017_bib5) 2002; 2 Ke (10.1016/j.ymthe.2004.10.017_bib4) 2000; 46 Stipecke (10.1016/j.ymthe.2004.10.017_bib1) 1999; 6 Croop (10.1016/j.ymthe.2004.10.017_bib10) 2000; 26 Pollok (10.1016/j.ymthe.2004.10.017_bib15) 2001; 12 Apfalter (10.1016/j.ymthe.2004.10.017_bib2) 2003; 41 Klein (10.1016/j.ymthe.2004.10.017_bib7) 2000; 7 Zeger (10.1016/j.ymthe.2004.10.017_bib16) 1992; 11 Sanburn (10.1016/j.ymthe.2004.10.017_bib8) 1999; 6 Cossett (10.1016/j.ymthe.2004.10.017_bib13) 1995; 69 Miller (10.1016/j.ymthe.2004.10.017_bib12) 1991; 65 Gerard (10.1016/j.ymthe.2004.10.017_bib9) 1996; 7 Rantakokko-Jalava (10.1016/j.ymthe.2004.10.017_bib3) 2001; 39 Bierhuizen (10.1016/j.ymthe.2004.10.017_bib11) 1997; 90 |
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Clin. Microbiol. doi: 10.1128/JCM.41.2.592-600.2003 contributor: fullname: Apfalter – volume: 90 start-page: 3304 year: 1997 ident: 10.1016/j.ymthe.2004.10.017_bib11 article-title: Enhanced green fluorescent protein as selectable marker of retroviral-mediated gene transfer in immature hematopoietic bone marrow cells publication-title: Blood doi: 10.1182/blood.V90.9.3304 contributor: fullname: Bierhuizen – volume: 2 start-page: 17 year: 2002 ident: 10.1016/j.ymthe.2004.10.017_bib5 article-title: Determination of PCR efficiency in Chelex-100 purified clinical samples and comparison of real-time quantitative PCR and conventional PCR for detection of Chlamydia pneumoniae publication-title: BMC Microbiol. doi: 10.1186/1471-2180-2-17 contributor: fullname: Mygind – volume: 65 start-page: 2220 year: 1991 ident: 10.1016/j.ymthe.2004.10.017_bib12 article-title: Construction and properties of retrovirus packaging cell based on gibbon ape leukemia virus publication-title: J. Virol. doi: 10.1128/JVI.65.5.2220-2224.1991 contributor: fullname: Miller |
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SubjectTerms | Bone marrow Efficiency Gene therapy Generalized linear models Genetic testing Genomes Granulocytes Hematology Medical research Methods Pediatrics Polymerase chain reaction Proteins Statistical analysis Stem cells |
Title | Real-Time PCR: an Effective Tool for Measuring Transduction Efficiency in Human Hematopoietic Progenitor Cells |
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