Monitoring the effect of gene silencing by RNA interference in human CD34+ cells injected into newborn RAG2-/- gammac-/- mice: functional inactivation of p53 in developing T cells.
Tumor suppressor p53 plays an important role in regulating cell cycle progression and apoptosis. Here we applied RNA interference to study the role of p53 in human hematopoietic development in vivo. An siRNA construct specifically targeting the human tumor-suppressor gene p53 was introduced into human CD34(+) progenitor cells by lentivirus-mediated gene transfer, which resulted in more than 95% knockdown of p53. We adapted the human-SCID mouse model to optimize the development of hematopoietic cells, particularly of T cells. This was achieved by the intraperitoneal injection of CD34(+) precursor cells into newborn Rag2(-/-) gammac(-/-) mice that lack T, B, and NK cells. Robust development of T cells was observed in these mice, with peripheral T-cell repopulation 8 weeks after injection of the precursor cells. Other lymphocyte and myeloid subsets also developed in these mice. Injecting p53 siRNA-transduced CD34(+) cells resulted in stable expression and down-modulation of p53 in the mature T-cell offspring. Inactivating p53 did not affect the development of CD34(+) cells into various mature leukocyte subsets, including T cells, but it conferred resistance to gamma-irradiation and other p53-dependent apoptotic stimuli to the T cells.
Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, Netherlands.
Ramon
R
Gimeno
Kees
K
Weijer
Arie
A
Voordouw
Christel H
CH
Uittenbogaart
Nicolas
N
Legrand
Nuno L
NL
Alves
Erwin
E
Wijnands
Bianca
B
Blom
Hergen
H
Spits
D000818:Animals; D000831:Animals, Newborn; D018952:Antigens, CD34 / Q000276:immunology*; D017209:Apoptosis; D055316:Cystatin C; D015891:Cystatins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D005434:Flow Cytometry; D005720:Gamma Rays; D020868:Gene Silencing; D005865:Gestational Age; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D006801:Humans; D008099:Liver / Q000166:cytology / Q000196:embryology; D051379:Mice; D018345:Mice, Knockout; D009687:Nuclear Proteins; D034622:RNA Interference; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D033581:Stem Cell Transplantation; D013601:T-Lymphocytes / Q000276:immunology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D018952:Antigens, CD34; C525030:CST3 protein, human; C525031:Cst3 protein, mouse; D055316:Cystatin C; D015891:Cystatins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C499311:RAG2 protein, human; C499308:Rag2 protein, mouse; D011948:Receptors, Antigen, T-Cell; D016159:Tumor Suppressor Protein p53; C064655:V(D)J recombination activating protein 2