Phenotypic and functional characterization of a mouse model of targeted Pig-a deletion in hematopoietic cells

Abstract

The online version of this paper has a supplementary appendix. BackgroundSomatic mutation in the X-linked phosphatidylinositol glycan class A gene (PIG-A) causes glycosyl phosphatidylinositol anchor deficiency in human patients with paroxysmal nocturnal hemoglobinuria. Design and MethodsWe produced an animal model of paroxysmal nocturnal hemoglobinuria by conditional Pig-a gene inactivation (Pig-a -/-) in hematopoietic cells; mice carrying two lox sites flanking exon 6 of the Pig-a gene were bred with mice carrying the transgene Cre-recombinase under the human c-fes promoter. We characterized the phenotypic and functional properties of glycosyl phosphatidylinositol-deficient and glycosyl phosphatidylinositol-normal hematopoietic cells from these Pig-a -/-mice using gene expression microarray, flow cytometry, bone marrow transplantation, spectratyping, and immunoblotting. ResultsIn comparison to glycosyl phosphatidylinositol-normal bone marrow cells, glycosyl phosphatidylinositol-deficient bone marrow cells from the same Pig-a -/-animals showed upregulation of the expression of immune function genes and contained a significantly higher proportion of CD8 T cells. Both characteristics were maintained when glycosyl phosphatidylinositol-deficient cells were transplanted into lethally-irradiated recipients. Glycosyl phosphatidylinositol-deficient T cells were inactive, showed pronounced Vβ5.1/5.2 skewing, had fewer γ-interferon-producing cells after lectin stimulation, and contained fewer CD4+ regulatory T cells. However, the levels of T-cell receptor signaling proteins from glycosyl phosphatidylinositol-deficient cells were normal relative to glycosyl phosphatidylinositol-normal cells from wild type animals, and cells were capable of inducing target cell apoptosis in vitro. ConclusionsDeletion of the Pig-a gene in hematopoietic cells does not cause frank marrow failure but leads to the appearance of clonally-restricted, inactive yet functionally competent CD8 T cells.Key words: Pig-a deletion, paroxysmal nocturnal hemoglobinuria, glycosyl phosphatidylinositol, T-cell mediated immunity.Citation: Visconte V, Raghavachari N, Liu D, Keyvanfar K, Desierto MJ, Chen J, and Young NS. Phenotypic and functional characterization of a mouse model of targeted Pig-a deletion in hematopoietic cells. Haematologica. 2010; 95:214-223. doi:10.3324/haematol.2009 This is an open-access paper. Phenotypic and functional characterization of a mouse model of targeted Pig-a deletion in hematopoietic cells

Inhibition of the DNA damage response phosphatase PPM1D reprograms neutrophils to enhance anti-tumor immune responses

“…Upon whole-body or tissue-selective exposure to cre recombinase, recombination between LoxP sites flanking exon 3 produces the knockout allele, Ppm1d Tm1d(KOMP)Wtsi , referred to as Ppm1d KO3 . The human FES promoter is highly active in hematopoietic progenitors and myeloid cells; mice expressing the Fes-cre transgene are useful for studying gene deletion in the hematopoietic system 44 , 45 . Through successive intercrosses of Fes-cre mice with Ppm1d fl/fl mice, we produced Ppm1d fl/fl ;Tg(Fes-cre) mice, referred to herein as Ppm1d Fes-cre mice, which lack expression of Wip1 in hematopoietic cells (Supplementary Fig.…”

CNS Glycosylphosphatidylinositol Deficiency Results in Delayed White Matter Development, Ataxia, and Premature Death in a Novel Mouse Model

“…This deletion resulted in cleft lip and cleft palate as well as hypoplasia of the craniofacial skeleton highlighting a critical role for GPI biosynthesis in neural crest cell survival [14]. Others have used this approach to reveal an important role for GPI biosynthesis in skin, limb, blood and craniofacial development [15–17]. However, germline knockout of Piga using CMV-Cre resulted in embryonic lethality with neural tube defect, edema, and cleft lip/palate precluding the analysis of any postnatal neurological phenotype [18].…”

CRISPR/Cas9 PIG-A gene editing in nonhuman primate model demonstrates no intrinsic clonal expansion of PNH HSPCs

“…4,9,10 However, definitive mechanisms remain unclear. There have been attempts to model PNH by using conditional knockout strategies in mice, 11,12 but these do not recapitulate the hemolytic phenotype or clonal dominance.…”