This genetic stock was maintained in positive individually ventilated racks by sib mating NOD- scid IL2Rγ null mice in a barrier mouse room. The NOD- scid IL2Rγ null females and NOD- scid IL2Rγ null/Y males were intercrossed. NOD- scid IL2Rγ null female and NOD- scid IL2Rγ null/ Y male offspring were identified by real time quantitative PCR ( 28) and confirmed by flow cytometric validation of the absence of IL-2Rγ-chain expression on peripheral blood myeloid cells, as described below, using PE-conjugated rat anti-mouse IL2Rγ common chain mAb, clone TUGm2 (BD Biosciences). Heterozygosity for the IL2Rγ null allele was determined by PCR for the neomycin resistance gene (neo) ().Īfter backcrossing the IL2Rγ null allele for 8 generations onto the NOD- scid background, NOD.Cg- Prkdc scid IL2Rγ Tm1Wjl/Sz (abbreviated as NOD- scid IL2Rγ +/ null) females were crossed with NOD- scid IL2Rγ null/ Y males. Homozygosity for scid was determined by flow cytometric analysis of blood for the absence of CD3 + T cells and Ig + B cells ( 10, 15) and confirmed by PCR analyses of tail DNA ( 27). After two backcross generations, females homozygous for scid and heterozygous for the disrupted IL2Rγ gene were identified by flow cytometry and PCR. (NOD × B6) F 1 +/ scid IL2Rγ null hemizygous males were backcrossed with NOD- scid females. The NOD.Cg- Prkdc scid IL2rg tmWjl/Sz (abbreviated as NOD- scid IL2 Rγ null) mouse genetic stock was developed by first crossing NOD- scid females with B6.129S4- IL2Rγ tm1Wjl/J males. B6.129S4- IL2rg tmWjl/ J mice (abbreviated as B6- IL2Rγ null) were obtained from the Animal Resources colony at The Jackson Laboratory. NOD/LtJ (abbreviated as NOD), C57BL/6J, NOD.CB17- Prkdc scid (abbreviated as NOD- scid), and B6.129S7- Rag1 tm1Mom/J (abbreviated as B6- Rag1 null) mice were raised in our research colony at The Jackson Laboratory under specific pathogen-free conditions as previously described ( 10, 18). Although improved human HSC engraftment was observed in each of these newer immunodeficient NOD models, human HSC failed to differentiate into mature human lymphoid and myeloid cells. NOD- Rag1 null Prf1 null mice have an increased life span as compared with NOD- scid mice, and they support relatively high levels of human hematolymphoid engraftment ( 19). Alternative immunodeficient NOD mouse models that have been developed include NOD mice bearing a targeted mutation at the recombination activation gene 1 (NOD- Rag1 null) ( 18) and NOD- Rag1 null mice bearing a targeted mutation at the perforin ( Prf1) locus (NOD- Rag1 null Prf1 null) ( 19). However, long term studies in NOD- scid B2m null mice have been constrained by a markedly shortened life span due to accelerated thymic lymphomagenesis ( 15). These mice are severely deficient in NK cell activity and support higher levels of human HSC engraftment than do NOD- scid mice ( 15, 16, 17). To begin to address these limitations, we developed NOD- scid mice homozygous for a targeted mutation in the β 2-microglobulin structural gene (NOD- scid B2m null mice). However, HSC function in NOD- scid mice is limited by remaining NK cell activity and a relatively short life span due to the early occurrence of thymic lymphomas. Thus, NOD- scid IL2Rγ null mice engrafted with human mobilized blood stem cells provide a new in vivo long-lived model of robust multilineage human HSC engraftment. De novo human T cell development in NOD- scid IL2Rγ null mice was validated by 1) high levels of TCR excision circles, 2) complex TCRβ repertoire diversity, and 3) proliferative responses to PHA and streptococcal superantigen, streptococcal pyrogenic exotoxin. Coadministration of human Fc-IL7 fusion protein results in high percentages of human CD4 +CD8 + thymocytes as well human CD4 +CD8 − and CD4 −CD8 + peripheral blood and splenic T cells. Spleens from engrafted NOD- scid IL2Rγ null mice contain human Ig + B cells and lower numbers of human CD3 + T cells. These human cells include B cells, NK cells, myeloid cells, plasmacytoid dendritic cells, and HSC. Engraftment of NOD- scid IL2Rγ null mice with human HSC generate 6-fold higher percentages of human CD45 + cells in host bone marrow than with similarly treated NOD- scid mice. NOD- scid IL2Rγ null mice are deficient in mature lymphocytes and NK cells, survive beyond 16 mo of age, and even after sublethal irradiation resist lymphoma development. We report the development and characterization of a new genetic stock of IL-2R common γ-chain deficient NOD/LtSz- scid (NOD- scid IL2Rγ null) mice and document their ability to support human mobilized blood HSC engraftment and multilineage differentiation. To overcome this limitation, small animal models of human HSC engraftment have been used. Ethical considerations constrain the in vivo study of human hemopoietic stem cells (HSC).
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