Retroviral transfer of genes into erythroid progenitors derived from human peripheral blood.
Human erythroblasts are a logical target for studies of expression of transferred globin genes because high-level expression is a prerequisite for gene therapy of hemoglobinopathies. Early erythroid progenitors (erythroid burst-forming units, BFU-E) are readily available from human peripheral blood and can be cultured to produce erythroblasts. However, conditions for efficient transfer into these normal progenitors have not been previously described. Here we demonstrate efficient transfer of the neomycin resistance gene into human peripheral blood BFU-E using the retrovirus vector, N2. We show that liquid culture of mononuclear cells from peripheral blood for 18-24 h prior to retroviral infection leads to increased transfer efficiency of N2 as determined by G418 resistance, and we are able to detect viral DNA by polymerase chain reaction (PCR) analysis. In addition, a second retrovirus, beta(gamma)-SVX, prepared with a human beta-globin gene containing a gamma-globin second exon to facilitate transcript detection and the 3'-enhancer sequence, was also used to determine whether similar results could be obtained when more than one gene is transferred. Using the beta(gamma)-SVX virus, increased transfer efficiency into BFU-E was similarly found after liquid culture for up to 4 days. Expression of the transferred globin gene was also detected by PCR analysis of cDNA made from erythroblast RNA. The human peripheral blood BFU-E system described should allow determination of sequences required for high-level expression of transferred globin and other erythroid genes.
Department of Anatomy, University of Pennsylvania School of Medicine, Philadelphia.
C J
CJ
Stoeckert
N C
NC
Nicolaides
K M
KM
Haines
S
S
Surrey
E
E
Bayever
D001483:Base Sequence; D004279:DNA, Viral / Q000032:analysis; D004351:Drug Resistance / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000235:genetics; D015672:Erythroid Precursor Cells / Q000378:metabolism; D005914:Globins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009355:Neomycin; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection
D004279:DNA, Viral; D012313:RNA; D005914:Globins; D009355:Neomycin