Cryopreserved cord blood myeloid progenitor cells can serve as targets for retroviral-mediated gene transduction and gene-transduced progenitors can be cryopreserved and recovered.
To determine future possibilities for gene transfer, we evaluated whether myeloid progenitor cells from human umbilical cord blood (CB) could be frozen, thawed in viable form and transduced with a Neomycin resistance (NeoR) gene using retroviral vectors, and if fresh progenitor cells transduced with a Neo gene could be cryopreserved and recovered. Fresh and thawed cryopreserved nonadherent low-density T-lymphocyte depleted (NALT-) CB cells were assayed before and after gene transduction for colony formation in the presence of multiple growth factors in the absence and presence of G418. The results demonstrate that the NeoR gene could be introduced into thawed cryopreserved myeloid progenitor cells at an efficiency similar to that of fresh cells and that fresh cells transduced with the NeoR gene could be frozen in a cryopreserved state and recovered after thawing. Proviral integration, as assessed by PCR/Southern Analysis, confirmed the G418R colony data. Proviral integration was detected not only in primary G418R-colonies, but also in replated colonies in secondary dishes derived from G418R-multipotential progenitor cells (CFU-GEMM) suggesting stable integration of the transduced gene into early subsets of replatable progenitors. This information may be of use clinically.
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003114:Colony-Forming Units Assay; D015925:Cryopreservation; D046508:Culture Techniques / Q000379:methods; D017931:DNA Primers; D005260:Female; D005312:Fetal Blood / Q000166:cytology*; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D007231:Infant, Newborn; D019868:Kanamycin Kinase; D008212:Lymphocyte Depletion; D051379:Mice; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D012190:Retroviridae / Q000235:genetics*; D013601:T-Lymphocytes; D014161:Transduction, Genetic; D014162:Transfection; D016662:Virus Integration
D017931:DNA Primers; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase