Transduction of human interleukin-9 receptor gene into human cord blood erythroid progenitors increases the number of erythropoietin-dependent erythroid colonies.
We constructed a retroviral vector, pLhIL-9RSN, containing CDNA encoding the human interleukin-9 receptor (IL-9R) along with a neomycin phosphotransferase gene (Neo). In order to study the biological effects of the IL-9R, high titer (1-5 x 10(5) CFU/ml) viral supernatant, generated from the packaging cell lines, ecotropic GPE86 and amphotropic PA317, was used to transduce the IL-9R gene into sorted populations of CD34++ CD33-cells from human cord blood which are highly enriched for erythroid progenitor cells (BFU-E). Colony formation by BFU-E transduced with the IL-9R gene and grown without selection in G418 and in the presence of erythropoietin (Epo) and interleukin (IL)-9 was significantly increased up to three-fold and the size of the erythroid colonies was significantly increased 50-100% compared to colony formation by mock virus transduced cells. Moreover, colony formation by IL-9R-transduced cells was more sensitive to stimulation with lower doses of IL-9 and Epo. Individual colonies formed with or without selection in G418 were evaluated. Proviral integration and mRNA expression were respectively assessed by polymerase chain reaction (PCR) and reverse transcriptase (RT) PCR analysis and were apparent in 93% and 84% of the G418-resistant colonies and 52% and 48% of the colonies grown in the absence of G418. Our study demonstrates that a functional human IL-9R gene can be efficiently transduced into human cord blood hematopoietic progenitors using retroviral vectors with increased cytokine-dependent erythroid colony formation.
Xiao
M
M
Department of Medicine (Hematology/Oncology), Indiana University School of Medicine, Indianapolis, 46202-5121, USA.
Yang
Y C
YC
Yang
L
L
Chang
M S
MS
Cornetta
K
K
Broxymeyer
H E
HE
Lu
L
L
D002455:Cell Division / Q000187:drug effects; D002999:Clone Cells / Q000166:cytology / Q000187:drug effects; D003114:Colony-Forming Units Assay; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000187:drug effects*; D004921:Erythropoietin / Q000494:pharmacology*; D005312:Fetal Blood / Q000166:cytology*; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D016906:Interleukin-9 / Q000494:pharmacology; D019868:Kanamycin Kinase; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics; D011533:Proviruses / Q000502:physiology; D018123:Receptors, Interleukin / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D053651:Receptors, Interleukin-9; D011994:Recombinant Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D012641:Selection, Genetic; D014162:Transfection; D016662:Virus Integration
C508045:IL9R protein, human; D016906:Interleukin-9; D018123:Receptors, Interleukin; D053651:Receptors, Interleukin-9; D011994:Recombinant Proteins; D004921:Erythropoietin; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase