![]() Transgenic mouse lines using a Sca-1 promoter driving LacZ or green fluorescent protein expression as well as an enhanced green fluorescent protein (eGFP) knock in have thus proved instrumental in facilitating more detailed expression studies. Redundancy among Ly6 genes prevents Northern analysis from being useful and requires exceptionally stringent reverse transcription- or quantitative-polymerase chain reaction conditions, whereas various Sca-1 antibodies are less than ideal for Western analysis and immunostaining. With the exception of flow cytometry (fluorescence-activated cell sorting), traditional methods for detecting this Sca-1 expression have posed many challenges. Thus, Sca-1 is used routinely in combination with negative selection against mature markers for enrichment of stem and progenitor cells. Outside the hematopoietic system, Sca-1 is similarly expressed by a mixture of stem, progenitor, and differentiated cell types in a wide variety of tissues and organs. Sca-1 expression is further upregulated in activated lymphocytes and in the presence of interferon-α/β and interferon-γ, whereas tumor necrosis factor-α or anti-Fas antibody downregulate Sca-1. Immature thymocytes turn off Sca-1 expression but then re-express it on mature single-positive medullary thymocytes and peripheral T cells. As HSCs commit to lymphoid progenitors, Sca-1 expression decreases and prothymocytes that seed the thymic cortex upregulate expression of both Sca-1 and Sca-2, another Ly6 gene. As HSCs differentiate into common myeloid progenitors, Sca-1 expression is downregulated, becoming upregulated on a proportion of spleen colony-forming unit (CFU-S) and culture colony-forming unit (CFU-C) progenitors. Sca-1 expression is regulated in a complex fashion in hematopoietic ontology. This allele-specific expression appears to be due to post-transcriptional regulation or variable protein expression levels, as is suggested by studies in Sca-1 transgenic reporter mice. Essentially all HSCs express Sca-1 in Ly6.2 strains, such as C57Bl/6, although only 25% of HSCs express Sca-1 in Ly6.1 strains, such as BALB/c. Not surprisingly, embryonic and fetal Sca-1 expression is consistent with the emergence and expansion of definitive HSCs, being first observed at E9 in the ventral dorsal aorta, with continued expression in the endothelial layer of the E11 ventral dorsal aorta and in the fetal liver. Sca-1 is the most common marker used to enrich adult murine hematopoietic stem cells (HSCs) and can be used to isolate a nearly pure HSC population when used in conjunction with additional markers. ![]() Like other GPI-APs, Ly6 proteins are thus physically located in an ideal position from which to regulate or coactivate cell signaling via receptor-ligand binding or other protein-protein interactions however, the exact molecular mechanism by which these proteins act remains unclear. Consisting of saturated sphingolipids and cholesterol, lipid rafts play critical roles in cell signaling by excluding or concentrating key signaling molecules, such as Src family kinases, as well as regulating receptor recycling and degradation. These cysteine-rich GPI-APs contain two or three protruding fingers, based upon the structure of Ly6 superfamily member cobra toxin proteins, and are localized to lipid rafts of the plasma membrane. The murine Ly6 gene family encodes at least 18 highly homologous, cross-hybridizing genes closely linked on mouse chromosome 15, many of which demonstrate greater than 80% sequence similarity with Sca-1. Originally identified as an antigen upregulated on activated lymphocytes more than 30 years ago, Sca-1, or lymphocyte activation protein-6A (Ly-6A), is encoded by two strain-specific alleles. Stem cell antigen-1 (Sca-1) is an 18-kDa mouse glycosyl phosphatidylinositol-anchored cell surface protein (GPI-AP) of the Ly6 gene family. ![]() ![]() Stem cell antigen-1, Lymphocyte activation protein-6A, Stem cell, Self-renewal, Glycosyl phosphatidylinositol-anchored protein, Lipid rafts I ntroduction
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