Monitoring the effect of gene silencing by RNA interference in human CD34+ cells injected into newborn RAG2-/- γc-/- mice: functional inactivation of p53 in developing T cells

Abstract

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 opti- IntroductionThe tumor suppressor p53 plays an important role in regulating the cell cycle and apoptosis in response to DNA damage caused by irradiation or exposure to genotoxic mediators. In addition, p53 can mediate several cellular responses, including cell cycle arrest, senescence, differentiation, and apoptosis, depending on the cell type and the microenvironment. 1 Although mutations occur in the gene encoding p53 in human cancers, including tumors of hematopoietic origin, its function in normal human hematopoietic development remains largely unexplored.Recently, we obtained evidence that p53 plays a role in regulating the replicative lifespan of mature human T cells in vitro through the suppression of human telomerase reverse transcriptase (hTERT) (R.G., E.W., R. Beijersbergen, and H.S., manuscript in preparation). Telomeres are DNA repeats at the distal ends of the chromosomes that protect against chromosome end-to-end fusion. 2 Telomeres are shortened at each cell division, and cells with critically short telomeres undergo cell cycle arrest and become senescent. [3][4][5] hTERT, which prevents telomere shortening, is transiently up-regulated in T cells on stimulation through the T-cell receptor (TCR), 4,5 and expression of a dominant-negative mutant of hTERT significantly decreased the lifespan of CD4 ϩ and CD8 ϩ T cells, 5 indicating that hTERT plays a regulatory role in the lifespan of human T cells. Recently, we observed that the down-regulation of p53 by RNA interference (RNAi) extends the lifespan of mature human T cells and neutralizes the inhibition by dominant-negative hTERT, indicating that p53 regulates hTERT expression in primary human T cells. Given the function of p53 in mature human T cells, we asked whether the p53 loss would affect the homeostatic proliferation of T cells. Mice deficient in p53 did not show obvious defects in T-cell homeostasis. However, T cells from inbred mouse strains have longer telomeres than T cells from humans, resulting in a delay in the onset of replicative senescence and in an extended lifespan of these murine T cells. It is, therefore, of interest to test the effect of p53 inactivation on human T-cell homeostasis.In addition, we examined the role of p53 in thymic T-cell development because studies in the mouse have revealed that p53 is induced after the initiation of TCR rearrangement 6 and that it plays an important role in early T-cell development, specifically in pre-T-cell receptor signaling. 7 Results obtained in mice cannot always be extrapolated to humans, and the role of p53 in human T-cell developm...

Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo

“…[13][14][15][16] Along with others, we have recently established BALB/c Rag-2 À/À IL-2Rg c À/À newborn mice as a new xenograft model for human hematopoietic stem cell (HSC) transplantation. 17,18 In contrast with other immunodeficient mouse strains humanized for the immune system (for example, C.B-17 SCID mice or NOD/SCID mice), 14 the resulting HIS (BALB-Rag/g) mice show all major human myeloid and lymphoid cellular compartments, giving access to in vivo and ex vivo experimentation on human immune cells. [14][15][16] Dox-dependent gene expression strategies have been applied to humanized NOD/SCID mice, 19 but the more robust human reconstitution obtained in the HIS (BALBRag/g) mice renders this model more attractive for prospective study of gene function in vivo during human hematopoiesis.…”

Autonomous and extrinsic regulation of thymopoiesis inhuman immune system (HIS) mice

“…Mice were maintained in isolators with autoclaved food and water. Mice with a HIS were generated as previously described [1][2][3]40]. Briefly, newborn (3-5-day-old) mice received sub-lethal (3.3 Gy) total body irradiation from a Cs source, and were injected intra-hepatic (i.h.)…”

“…The recent advances in the development of HIS mice provide a new model to experimentally dissect human lymphocyte biology that may eventually allow us to bridge the knowledge gap between murine and human in vivo studies of immune responses. One welldescribed HIS mouse model involves engraftment of newborn Balb/c Rag2 À/À g c À/À mice with human fetal liver or cord blood haematopoietic stem cells (HSCs), which seed the murine BM and thymus and in turn differentiate into an almost complete spectrum of mature human myeloid and lymphoid cell subsets [1][2][3]. Human B-cell development predominates in this model, with T cells encompassing o10% of the peripheral lymphocyte pool [1][2][3].…”

“…One welldescribed HIS mouse model involves engraftment of newborn Balb/c Rag2 À/À g c À/À mice with human fetal liver or cord blood haematopoietic stem cells (HSCs), which seed the murine BM and thymus and in turn differentiate into an almost complete spectrum of mature human myeloid and lymphoid cell subsets [1][2][3]. Human B-cell development predominates in this model, with T cells encompassing o10% of the peripheral lymphocyte pool [1][2][3]. The reasons for the reduced T-cell homeostasis is not known, but could involve central and/or peripheral mechanisms.…”

“…turnover rate yet fail to accumulate [1][2][3][4][5]. On the other hand, interaction of haematopoietic precursors with murine thymic epithelial cell (TEC) ligands or soluble factors during thymopoiesis and subsequent selection of T cells on murine major histocompatibility complexes (MHC) may generate suboptimal signals for T-cell maintenance.…”