The hematopoietic and endothelial lineages derive from mesoderm and are thought to develop through the maturation of a common progenitor, the hemangioblast. To investigate the developmental processes that regulate mesoderm induction and specification to the hemangioblast, we generated an embryonic stem cell line with the green fluorescent protein (GFP) targeted to the mesodermal gene, brachyury. After the in vitro differentiation of these embryonic stem cells to embryoid bodies, developing mesodermal progenitors could be separated from those with neuroectoderm potential based on GFP expression. Co-expression of GFP with the receptor tyrosine kinase Flk1 revealed the emergence of three distinct cell populations, GFP -Flk1 -, GFP + Flk1 -and GFP + Flk1 + cells, which represent a developmental progression ranging from pre-mesoderm to prehemangioblast mesoderm to the hemangioblast.
The considerable potential of Cre recombinase as a tool for in vivo fate-mapping studies depends on the availability of reliable reporter mice. By targeting a tandem-dimer red fluorescent protein (tdRFP) with advanced spectral and biological properties into the ubiquitously expressed ROSA26 locus of C57BL/6-ES cells, we have generated a novel inbred Cre-reporter mouse with several unique characteristics. We directly demonstrate the usefulness of our reporter strain in inter-crosses with a "universal Cre-deleter" strain and with mice expressing Cre recombinase in a T lineage-specific manner. Cytofluorometric and histological analyses illustrate: (i) non-toxicity and extraordinary brightness of the fluorescent reporter, allowing quantitative detection and purification of labeled cells with highest accuracy, (ii) reliable Cre-mediated activation of tdRFP from an antisense orientation relative to ROSA26 transcription, effectively excluding "leaky" reporter expression, (iii) absence of gene expression variegation effects, (iv) quantitative detection of tdRFP-expressing cells even in paraformaldehyde-fixed tissue sections, and (v) full compatibility with GFP/YFP-based fluorescent markers in multicolor experiments. Taken together, the data show that our C57BL/6-inbred reporter mice are ideally suited for sophisticated lineage-tracing experiments requiring sensitive and quantitative detection/purification of live Cre-expressing cells and their progeny. IntroductionCre recombinase is a small, bacteriophage P1-derived integrase that catalyses defined DNA recombination events between specific target sites, termed loxP (locus of crossover [x] in P1) [1]. LoxP sites are composed of two 13-bp inverted repeats and an asymmetric 8-bp spacer sequence, which endows individual loxP elements with a defined orientation [2]. The result of Cremediated recombination between two loxP sites de- pends on their specific orientation relative to one another: a DNA sequence flanked by two directly repeated loxP elements is cut out as a circular molecule, an effectively irreversible reaction due to loss of the excised product. In contrast, DNA flanked by two oppositely oriented sites will be inverted, a fully reversible process. Importantly, both activity and specificity of Cre recombinase are retained in eukaryotic cells [3], permitting the use of Cre as a genetic engineering tool in any cellular context. In combination with gene targeting techniques, the Cre/loxP recombination system has revolutionized the genetic analysis of mice [4,5]. Cre-mediated removal of loxP-flanked (floxed) selection cassettes, which are invariably introduced during the process of gene targeting into the mouse genome, and which may have unpredictable effects on neighboring genes, is already a routine manipulation. Also, conditional deletion of floxed genomic DNA sequences has become a rather standard approach for assessing tissue-, cell-type-or developmental stage-specific gene functions, provided appropriate Cre-transgenic mouse strains are available. Less common ...
In T-cell precursors, the T-cell-receptor beta chain is expressed before the T-cell-receptor alpha chain and is sufficient to advance T-cell development in the absence of T-cell receptor alpha chains. In immature T cells, the T-cell-receptor beta protein can form disulphide-linked heterodimers with the pre-T-cell-receptor alpha chain and associate with signal-transducing CD3 molecules. The recently cloned pre-T-cell-receptor alpha gene encodes a transmembrane protein that is expressed in immature but not mature T cells. Here we show that alpha beta, but not gamma delta, cell development is severely hampered in pre-T-cell-receptor alpha-gene-deficient mice, which establishes a crucial role for the pre-T-cell receptor in early thymocyte development.
Proteasomes degrade endogenous proteins. Two subunits, LMP-2 and LMP-7, are encoded in a region of the major histocompatibility complex (MHC) that is critical for class I-restricted antigen presentation. Mice with a targeted deletion of the gene encoding LMP-7 have reduced levels of MHC class I cell-surface expression and present the endogenous antigen HY inefficiently; addition of peptides to splenocytes deficient in LMP-7 restores wild-type class I expression levels. This demonstrates the involvement of LMP-7 in the MHC class I presentation pathway and suggests that LMP-7 functions as an integral part of the peptide supply machinery.
Proteasomes are the main proteases responsible for cytosolic protein degradation and the production of major histocompatibility complex class I ligands. Incorporation of the interferon γ–inducible subunits low molecular weight protein (LMP)-2, LMP-7, and multicatalytic endopeptidase complex–like (MECL)-1 leads to the formation of immunoproteasomes which have been associated with more efficient class I antigen processing. Although differences in cleavage specificities of constitutive and immunoproteasomes have been observed frequently, cleavage motifs have not been described previously.We now report that cells expressing immunoproteasomes display a different peptide repertoire changing the overall cytotoxic T cell–specificity as indicated by the observation that LMP-7−/− mice react against cells of LMP-7 wild-type mice. Moreover, using the 436 amino acid protein enolase-1 as an unmodified model substrate in combination with a quantitative approach, we analyzed a large collection of peptides generated by either set of proteasomes. Inspection of the amino acids flanking proteasomal cleavage sites allowed the description of two different cleavage motifs. These motifs finally explain recent findings describing differential processing of epitopes by constitutive and immunoproteasomes and are important to the understanding of peripheral T cell tolerization/activation as well as for effective vaccine development.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.