Stable integration of retrovirally transduced genes into human umbilical cord blood high-proliferative potential colony-forming cells (HPP-CFC) as assessed after multiple HPP-CFC colony replatings in vitro.
We previously demonstrated stable integration of a transduced thymidine kinase (TK)-neo gene into immature and replatable stem and progenitor cells, as assessed by the presence of the gene in second-generation colonies. To evaluate whether this integration was still present in third- and fourth-generation colonies, nonadherent low-density T-lymphocyte-depleted (NALT-) cells from human umbilical cord blood were prestimulated with recombinant human (rhu) erythropoietin (Epo), steel factor (SLF), interleukin-3 (IL-3), granulocyte-macrophage (GM) colony-stimulating factor (CSF), and granulocyte (G)-CSF. Prestimulated NALT- cells were incubated with retroviral-containing supernatant obtained from TK-neo vector-producing cells, washed, and assayed for colony formation in the presence of Epo, SLF, IL-3, GM-CSF, and G-CSF -/+ G418. The results confirmed that the TK-neo gene could be efficiently introduced into hematopoietic progenitor cells without stromal cells as a source of virus. As previously reported, proviral integration was detected in primary G418R-colonies, and in second-generation replated colonies derived from G418R granulocyte erythroid macrophage megakaryocyte colony-forming units and high-proliferative potential colony-forming cells (HPP-CFCs). Moreover, we now document that proviral integration was apparent in cells from colonies derived from third- and fourth-generation replated HPP-CFC, suggesting a high degree of stable integration of the transduced gene.
Lu
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L
Department of Medicine (Hematology/Oncology), University School of Medicine, Indianapolis, Indiana 46202-5121, USA.
Xiao
M
M
Clapp
D W
DW
Li
Z H
ZH
Broxmeyer
H E
HE
D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D003238:Connective Tissue / Q000502:physiology; D046508:Culture Techniques / Q000379:methods; D004274:DNA, Recombinant / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D005312:Fetal Blood / Q000166:cytology*; D005813:Genes, Synthetic; D005822:Genetic Vectors / Q000235:genetics*; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000821:virology*; D006801:Humans; D019868:Kanamycin Kinase; D009355:Neomycin / Q000494:pharmacology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics; D011533:Proviruses / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics* / Q000502:physiology; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000235:genetics; D016662:Virus Integration
D004274:DNA, Recombinant; D004279:DNA, Viral; D016298:Hematopoietic Cell Growth Factors; D011993:Recombinant Fusion Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D013937:Thymidine Kinase; D019868:Kanamycin Kinase; D009355:Neomycin