FES-Cre targets phosphatidylinositol glycan class A (PIGA) inactivation to hematopoietic stem cells in the bone marrow.
A somatic mutation in the X-linked phosphatidylinositol glycan class A (PIGA) gene causes the loss of glycosyl phosphatidylinositol (GPI)-linked proteins on blood cells from patients with paroxysmal nocturnal hemoglobinuria. Because all blood cell lineages may be affected it is thought that the mutation occurs in a hematopoietic stem cell. In transgenic mice, germline transmission of an inactive Piga gene is embryonic lethal. To inactivate the murine Piga gene in early hematopoiesis we therefore chose conditional gene inactivation using the Cre/loxP system. We expressed Cre recombinase under the transcription regulatory sequences of the human c-fes gene. FES-Cre inactivated PIGA in hematopoietic cells of mice carrying a floxed Piga allele (LF mice). PIGA(-) cells were found in all hematopoietic lineages of definitive but not primitive hematopoiesis. Their proportions were low in newborn mice but subsequently increased continuously to produce for the first time mice that have almost exclusively PIGA(-) blood cells. The loss of GPI-linked proteins occurred mainly in c-kit(+)CD34(+)Lin(-) progenitor cells before the CFU-GEMM stage. Using bone marrow reconstitution experiments with purified PIGA(-) cells we demonstrate that LF mice have long-term bone marrow repopulating cells that lack GPI-linked proteins, indicating that recombination of the floxed Piga allele occurs in the hematopoietic stem cell.
Keller
P
P
Division of Hematology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
Payne
J L
JL
Tremml
G
G
Greer
P A
PA
Gaboli
M
M
Pandolfi
P P
PP
Bessler
M
M
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000502:physiology; D003114:Colony-Forming Units Assay; D005260:Female; D005313:Fetal Death; D018507:Gene Expression Regulation, Developmental; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000502:physiology*; D006457:Hemoglobinuria, Paroxysmal / Q000235:genetics; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051578:Proto-Oncogene Proteins c-fes; D011519:Proto-Oncogenes; D011995:Recombination, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D014764:Viral Proteins; C080086:phosphatidylinositol glycan-class A protein; D011505:Protein-Tyrosine Kinases; C496645:FES protein, human; C496646:Fes protein, mouse; D051578:Proto-Oncogene Proteins c-fes; C045073:Cre recombinase; D019426:Integrases