High efficiency transduction of human CD34' progenitors on fibronectin CH-296 verified by clonal integration analysis

M. A. Dao, K. Hashino, L. Kato, D. B. Kohn, P. A. Williams, Jan Nolta

Research output: Contribution to journalArticle

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Abstract

The recombinant fibronectin fragment CH-296 has been previously demonstrated to co-localize retroviral vector particles and hematopoietic progenitors, leading to efficient transduction of human hematopoietic progenitors (Hanenberg et al.. Expl. Hem. 23:19a. 1995). We further studied retroviral-mediated gene transfer into human CD34+ progenitors on CH-296 by clonal integration analysis of individual transduced colonies. CD34+ cells (>95% pure) from normal donors were transduced by the high liter retroviral vector. LN, packaged in the GALV pseudotype cell line PG13 (Miller ft al, J. Virol. 65: 2220, 1994). Cells were transduced on plates coated with CH-296. irradiated allogenek stroma, or BSA. Vector supernatant, prepared al 32°C, was added to the plates at 24 hour intervals for 1, 2, or 3 days, in the presence of IL-3, IL-6, and SCF. Following transduction, methylcellulose-based colony-forming assays were plated +/-G418, and the remainder of the cells from each condition were transplanted into immune deficient mice to allow subsequent analysis of the transduction extent of more primitive hematopoietic progenitors. Transduction on the fibronectin domain was significantly higher than that obtained using the other conditions. A total of 515/ 1,112 G418 CFU (46.3%) were obtained from cells transduced on CH-296, as opposed to 56/ 1,128 (4.9%) on BSA coated plates, and 337/ 2,010 (16.7%) on stromal support. Two transductions at 24 hour intervals proved to be optimal. To verify the high extents of transduction on CH-296, individual, well-isolated G418 colonies were plucked and the genomic DNA was extracted and studied. PCR for the neo gene, carried by me LN vector, confirmed that each colony that had attained a size of at least 100 cells was derived from a progenitor cell that carried an integrated provins DNA from each colony was then subjected to clonal integration analysis by inverse PCR (Noita ft at., Proc- Nail. Acad. Sei. USA, Vol. 93, in press 1996). Unique proviral integrants were found for each colony, demonstrating dut the observed results were not due to several G4l g-resistant colonies rescuing or contaminating other clones in the same plate. Retroviral - mediated transduction on plates coated with the CH-296 fragment of fibronectin represents a major improvement over previously established human hematopoietic progenitor transduction protocols.

Original languageEnglish (US)
Pages (from-to)1057
Number of pages1
JournalExperimental Hematology
Volume24
Issue number9
StatePublished - 1996
Externally publishedYes

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Fibronectins
Gibbon ape leukemia virus
Polymerase Chain Reaction
Methylcellulose
Interleukin-3
DNA
Nails
Genes
Interleukin-6
Stem Cells
Clone Cells
Cell Line
antibiotic G 418

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Genetics
  • Hematology
  • Oncology
  • Transplantation

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High efficiency transduction of human CD34' progenitors on fibronectin CH-296 verified by clonal integration analysis. / Dao, M. A.; Hashino, K.; Kato, L.; Kohn, D. B.; Williams, P. A.; Nolta, Jan.

In: Experimental Hematology, Vol. 24, No. 9, 1996, p. 1057.

Research output: Contribution to journalArticle

Dao, M. A. ; Hashino, K. ; Kato, L. ; Kohn, D. B. ; Williams, P. A. ; Nolta, Jan. / High efficiency transduction of human CD34' progenitors on fibronectin CH-296 verified by clonal integration analysis. In: Experimental Hematology. 1996 ; Vol. 24, No. 9. pp. 1057.
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abstract = "The recombinant fibronectin fragment CH-296 has been previously demonstrated to co-localize retroviral vector particles and hematopoietic progenitors, leading to efficient transduction of human hematopoietic progenitors (Hanenberg et al.. Expl. Hem. 23:19a. 1995). We further studied retroviral-mediated gene transfer into human CD34+ progenitors on CH-296 by clonal integration analysis of individual transduced colonies. CD34+ cells (>95{\%} pure) from normal donors were transduced by the high liter retroviral vector. LN, packaged in the GALV pseudotype cell line PG13 (Miller ft al, J. Virol. 65: 2220, 1994). Cells were transduced on plates coated with CH-296. irradiated allogenek stroma, or BSA. Vector supernatant, prepared al 32°C, was added to the plates at 24 hour intervals for 1, 2, or 3 days, in the presence of IL-3, IL-6, and SCF. Following transduction, methylcellulose-based colony-forming assays were plated +/-G418, and the remainder of the cells from each condition were transplanted into immune deficient mice to allow subsequent analysis of the transduction extent of more primitive hematopoietic progenitors. Transduction on the fibronectin domain was significantly higher than that obtained using the other conditions. A total of 515/ 1,112 G418 CFU (46.3{\%}) were obtained from cells transduced on CH-296, as opposed to 56/ 1,128 (4.9{\%}) on BSA coated plates, and 337/ 2,010 (16.7{\%}) on stromal support. Two transductions at 24 hour intervals proved to be optimal. To verify the high extents of transduction on CH-296, individual, well-isolated G418 colonies were plucked and the genomic DNA was extracted and studied. PCR for the neo gene, carried by me LN vector, confirmed that each colony that had attained a size of at least 100 cells was derived from a progenitor cell that carried an integrated provins DNA from each colony was then subjected to clonal integration analysis by inverse PCR (Noita ft at., Proc- Nail. Acad. Sei. USA, Vol. 93, in press 1996). Unique proviral integrants were found for each colony, demonstrating dut the observed results were not due to several G4l g-resistant colonies rescuing or contaminating other clones in the same plate. Retroviral - mediated transduction on plates coated with the CH-296 fragment of fibronectin represents a major improvement over previously established human hematopoietic progenitor transduction protocols.",
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T1 - High efficiency transduction of human CD34' progenitors on fibronectin CH-296 verified by clonal integration analysis

AU - Dao, M. A.

AU - Hashino, K.

AU - Kato, L.

AU - Kohn, D. B.

AU - Williams, P. A.

AU - Nolta, Jan

PY - 1996

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N2 - The recombinant fibronectin fragment CH-296 has been previously demonstrated to co-localize retroviral vector particles and hematopoietic progenitors, leading to efficient transduction of human hematopoietic progenitors (Hanenberg et al.. Expl. Hem. 23:19a. 1995). We further studied retroviral-mediated gene transfer into human CD34+ progenitors on CH-296 by clonal integration analysis of individual transduced colonies. CD34+ cells (>95% pure) from normal donors were transduced by the high liter retroviral vector. LN, packaged in the GALV pseudotype cell line PG13 (Miller ft al, J. Virol. 65: 2220, 1994). Cells were transduced on plates coated with CH-296. irradiated allogenek stroma, or BSA. Vector supernatant, prepared al 32°C, was added to the plates at 24 hour intervals for 1, 2, or 3 days, in the presence of IL-3, IL-6, and SCF. Following transduction, methylcellulose-based colony-forming assays were plated +/-G418, and the remainder of the cells from each condition were transplanted into immune deficient mice to allow subsequent analysis of the transduction extent of more primitive hematopoietic progenitors. Transduction on the fibronectin domain was significantly higher than that obtained using the other conditions. A total of 515/ 1,112 G418 CFU (46.3%) were obtained from cells transduced on CH-296, as opposed to 56/ 1,128 (4.9%) on BSA coated plates, and 337/ 2,010 (16.7%) on stromal support. Two transductions at 24 hour intervals proved to be optimal. To verify the high extents of transduction on CH-296, individual, well-isolated G418 colonies were plucked and the genomic DNA was extracted and studied. PCR for the neo gene, carried by me LN vector, confirmed that each colony that had attained a size of at least 100 cells was derived from a progenitor cell that carried an integrated provins DNA from each colony was then subjected to clonal integration analysis by inverse PCR (Noita ft at., Proc- Nail. Acad. Sei. USA, Vol. 93, in press 1996). Unique proviral integrants were found for each colony, demonstrating dut the observed results were not due to several G4l g-resistant colonies rescuing or contaminating other clones in the same plate. Retroviral - mediated transduction on plates coated with the CH-296 fragment of fibronectin represents a major improvement over previously established human hematopoietic progenitor transduction protocols.

AB - The recombinant fibronectin fragment CH-296 has been previously demonstrated to co-localize retroviral vector particles and hematopoietic progenitors, leading to efficient transduction of human hematopoietic progenitors (Hanenberg et al.. Expl. Hem. 23:19a. 1995). We further studied retroviral-mediated gene transfer into human CD34+ progenitors on CH-296 by clonal integration analysis of individual transduced colonies. CD34+ cells (>95% pure) from normal donors were transduced by the high liter retroviral vector. LN, packaged in the GALV pseudotype cell line PG13 (Miller ft al, J. Virol. 65: 2220, 1994). Cells were transduced on plates coated with CH-296. irradiated allogenek stroma, or BSA. Vector supernatant, prepared al 32°C, was added to the plates at 24 hour intervals for 1, 2, or 3 days, in the presence of IL-3, IL-6, and SCF. Following transduction, methylcellulose-based colony-forming assays were plated +/-G418, and the remainder of the cells from each condition were transplanted into immune deficient mice to allow subsequent analysis of the transduction extent of more primitive hematopoietic progenitors. Transduction on the fibronectin domain was significantly higher than that obtained using the other conditions. A total of 515/ 1,112 G418 CFU (46.3%) were obtained from cells transduced on CH-296, as opposed to 56/ 1,128 (4.9%) on BSA coated plates, and 337/ 2,010 (16.7%) on stromal support. Two transductions at 24 hour intervals proved to be optimal. To verify the high extents of transduction on CH-296, individual, well-isolated G418 colonies were plucked and the genomic DNA was extracted and studied. PCR for the neo gene, carried by me LN vector, confirmed that each colony that had attained a size of at least 100 cells was derived from a progenitor cell that carried an integrated provins DNA from each colony was then subjected to clonal integration analysis by inverse PCR (Noita ft at., Proc- Nail. Acad. Sei. USA, Vol. 93, in press 1996). Unique proviral integrants were found for each colony, demonstrating dut the observed results were not due to several G4l g-resistant colonies rescuing or contaminating other clones in the same plate. Retroviral - mediated transduction on plates coated with the CH-296 fragment of fibronectin represents a major improvement over previously established human hematopoietic progenitor transduction protocols.

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