Lentiviral gene transfer into peripheral blood-derived CD34+ NOD/SCID-repopulating cells.
This study reports a lentiviral gene transfer protocol for efficient transduction of adult human peripheral blood (PB)-derived CD34+ NOD/SCID-repopulating cells (SRCs) using vesicular stomatitis virus-G protein (VSV-G)-pseudotyped lentiviruses encoding for enhanced green fluorescence protein (eGFP). Lentiviral stocks were concentrated by anion exchange chromatography, and transduction was performed under serum-free conditions at a multiplicity of infection (MOI) between 3 and 50. Similar transduction efficiencies were achieved in the presence and absence of cytokines. Transduction of PB-derived CD34+ cells at a MOI of 3 resulted in gene transfer efficiencies into SRCs of 9.2% and 12.0% in the absence and presence of cytokines, respectively. Using improved lentiviral vectors, transduction frequency varied between 42.0% (MOI 10) and 36.0% (MOI 50) with multilineage transgene expression within SRC-derived myeloid and lymphoid cells. The protocol described can be adapted for clinical application of lentiviral gene transfer into PB-derived CD34+ cells from adult patients.
Scherr
Michaela
M
Hannover Medical School, Department Hematology and Oncology, Hannover, Germany. m.scherr@t-online.de
Battmer
Karin
K
Blömer
Ulrike
U
Schiedlmeier
Bernd
B
Ganser
Arnold
A
Grez
Manuel
M
Eder
Matthias
M
D000328:Adult; D000818:Animals; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D016895:Culture Media, Serum-Free; D016207:Cytokines / Q000494:pharmacology; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics* / Q000302:isolation & purification; D006085:Graft Survival; D049452:Green Fluorescent Proteins; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000378:metabolism / Q000821:virology*; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000302:isolation & purification; D008164:Luminescent Proteins / Q000235:genetics; D008562:Membrane Glycoproteins; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D014162:Transfection; D014721:Vesicular stomatitis Indiana virus / Q000502:physiology; D014759:Viral Envelope Proteins / Q000502:physiology; D019065:Virus Assembly
D016895:Culture Media, Serum-Free; D016207:Cytokines; C028391:G protein, vesicular stomatitis virus; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D049452:Green Fluorescent Proteins