Project description:The screening of putative candidate genes downstream of IL7R-INS in vitro KSL/CLP and in vivo (depicted as B-neoplasm, Notch-T-ALL, or myeloid disorder) in comparison to WT. Five-condition experiment: WT-KSL vs. INS-KSL cells; WT-CLP vs.INS-CLP; WT/ICN vs. INS/ICN; WT-CMP vs. INS-CMP. Biological replicate: each 1, One replicate per array.
Project description:Here we report the expansion of the genetic code of Mus musculus with various unnatural amino acids including N?-acetyl-lysine. Stable integration of transgenes encoding an engineered N?-acetyl-lysyl-tRNA synthetase (AcKRS)/tRNAPyl pair into the mouse genome enables site-specific incorporation of unnatural amino acids into a target protein in response to the amber codon. We demonstrate temporal and spatial control of protein acetylation in various organs of the transgenic mouse using a recombinant green fluorescent protein (GFPuv) as a model protein. This strategy will provide a powerful tool for systematic in vivo study of cellular proteins in the most commonly used mammalian model organism for human physiology and disease.
Project description:Germinal centers (GCs) are clusters of activated B cells built on stromal cells known as follicular dendritic cells (FDCs). In the Peyer’s patches (PPs), GCs are chronically induced by bacteria and are the major sites for generation of gut IgA immune responses. Whether FDCs directly contribute to the IgA production in PP GCs is unknown. To investigate the role FDCs in gut immune system, we examined comprehensive gene profiles of FDCs purified from PPs or perypheral lymph nodes (pLNs) with or without immunization. We also tried to reconstitute the PP FDC signature in vitro by pulsed or continuous stimulation of pLN FDCs through TLRs, RARs or simultaneously through TLRs and RARs. The number of samples is as follows; ex vivo naïve pLN FDC=3, ex vivo immunized pLN FDC=2, ex vivo PP FDCs=3, in vitro without stimulation=2, in vitro with LPS stimulation for 5 hours=1, in vitro with LPS stimulation for 72h=1, in vitro with Pam2CSK4 (Pam) stimulation for 96h=2, in vitro with retinoic acid(RA) stimulation for 96h=2,in vitro with RA+Pam stimulation for 96h=2. In addition, ex vivo FDCs with or without immunizations were compared (3 samples), or in vitro cultured FDCs with or without various stimulations were compared (6 samples)
Project description:Lymph node (LN) development depends on prenatal interactions occurring between LN inducer and LN organizer cells. We have distinguished defects in LN formation due to failure in embryonic development (aly/aly) from defects in postnatal maturation (Il2rgamma(-/-)Rag2(-/-)). Both mutant strains form normal primordial LNs with differing fate. In aly/aly mice, the LN primordium dissipates irreversibly late in gestation; in contrast, Il2rgamma(-/-)Rag2(-/-) LN anlage persists for a week after birth but disperses subsequently, a process reversible by neonatal transfer of WT IL7r(+) TCR(+) T or natural killer (NK) cells, suggesting a role for IL7/IL7r interactions. Thus, we reveal a unique stage of postnatal LN development during which mature lymphocytes and IL7/IL7r interactions may play an important role.
Project description:A mutation in the IL7R? locus has been identified as a risk factor for multiple sclerosis (MS), a neurodegenerative autoimmune disease characterized by inflammation, demyelination, and axonal damage. IL7R? has well documented roles in lymphocyte development and homeostasis, but its involvement in disease is largely understudied. In this study, we use the experimental autoimmune encephalomyelitis (EAE) model of MS to show that a less severe form of the disease results when IL7R? expression is largely restricted to thymic tissue in IL7RTg(IL7R-/-) mice. Compared with wild-type (WT) mice, IL7RTg(IL7R-/-) mice exhibited reduced paralysis and myelin damage that correlated with dampened effector responses, namely decreased TNF production. Furthermore, treatment of diseased WT mice with neutralizing anti-IL7R? Ab also resulted in significant improvement of EAE. In addition, chimeric mice were generated by bone marrow transplant to limit expression of IL7R? to cells of either hematopoietic or nonhematopoietic origin. Mice lacking IL7R? only on hematopoietic cells develop severe EAE, suggesting that IL7R? expression in the nonhematopoietic compartment contributes to disease. Moreover, novel IL7R? expression was identified on astrocytes and oligodendrocytes endogenous to the CNS. Chimeric mice that lack IL7R? only on nonhematopoietic cells also develop severe EAE, which further supports the role of IL7R? in T cell effector function. Conversely, mice that lack IL7R? throughout both compartments are dramatically protected from disease. Taken together, these data indicate that multiple cell types use IL7R? signaling in the development of EAE, and inhibition of this pathway should be considered as a new therapeutic avenue for MS.
Project description:BACKGROUND: Due to their high level of genotypic and phenotypic variability, Mus spretus strains were introduced in laboratories to investigate the genetic determinism of complex phenotypes including quantitative trait loci. Mus spretus diverged from Mus musculus around 2.5 million years ago and exhibits on average a single nucleotide polymorphism (SNP) in every 100 base pairs when compared with any of the classical laboratory strains. A genoproteomic approach was used to assess polymorphism of the major milk proteins between SEG/Pas and C57BL/6J, two inbred strains of mice representative of Mus spretus and Mus musculus species, respectively. RESULTS: The milk protein concentration was dramatically reduced in the SEG/Pas strain by comparison with the C57BL/6J strain (34 ± 9 g/L vs. 125 ± 12 g/L, respectively). Nine major proteins were identified in both milks using RP-HPLC, bi-dimensional electrophoresis and MALDI-Tof mass spectrometry. Two caseins (? and ?s1) and the whey acidic protein (WAP), showed distinct chromatographic and electrophoresis behaviours. These differences were partly explained by the occurrence of amino acid substitutions and splicing variants revealed by cDNA sequencing. A total of 34 SNPs were identified in the coding and 3'untranslated regions of the SEG/Pas Csn1s1 (11), Csn2 (7) and Wap (8) genes. In addition, a 3 nucleotide deletion leading to the loss of a serine residue at position 93 was found in the SEG/Pas Wap gene. CONCLUSION: SNP frequencies found in three milk protein-encoding genes between Mus spretus and Mus musculus is twice the values previously reported at the whole genome level. However, the protein structure and post-translational modifications seem not to be affected by SNPs characterized in our study. Splicing mechanisms (cryptic splice site usage, exon skipping, error-prone junction sequence), already identified in casein genes from other species, likely explain the existence of multiple ?s1-casein isoforms both in SEG/Pas and C57BL/6J strains. Finally, we propose a possible mechanism by which the hallmark tandem duplication of a 18-nt exon (14 copies) may have occurred in the mouse genome.
Project description:The laboratory mouse is the workhorse of immunology, used as a model of mammalian immune function, but how well immune responses of laboratory mice reflect those of free-living animals is unknown. Here we comprehensively characterize serological, cellular and functional immune parameters of wild mice and compare them with laboratory mice, finding that wild mouse cellular immune systems are, comparatively, in a highly activated (primed) state. Associations between immune parameters and infection suggest that high level pathogen exposure drives this activation. Moreover, wild mice have a population of highly activated myeloid cells not present in laboratory mice. By contrast, in vitro cytokine responses to pathogen-associated ligands are generally lower in cells from wild mice, probably reflecting the importance of maintaining immune homeostasis in the face of intense antigenic challenge in the wild. These data provide a comprehensive basis for validating (or not) laboratory mice as a useful and relevant immunological model system.
Project description:Copy number variation is an important dimension of genetic diversity and has implications in development and disease. As an important model organism, the mouse is a prime candidate for copy number variant (CNV) characterization, but this has yet to be completed for a large sample size. Here we report CNV analysis of publicly available, high-density microarray data files for 351 mouse tail samples, including 290 mice that had not been characterized for CNVs previously.We found 9634 putative autosomal CNVs across the samples affecting 6.87% of the mouse reference genome. We find significant differences in the degree of CNV uniqueness (single sample occurrence) and the nature of CNV-gene overlap between wild-caught mice and classical laboratory strains. CNV-gene overlap was associated with lipid metabolism, pheromone response and olfaction compared to immunity, carbohydrate metabolism and amino-acid metabolism for wild-caught mice and classical laboratory strains, respectively. Using two subspecies of wild-caught Mus musculus, we identified putative CNVs unique to those subspecies and show this diversity is better captured by wild-derived laboratory strains than by the classical laboratory strains. A total of 9 genic copy number variable regions (CNVRs) were selected for experimental confirmation by droplet digital PCR (ddPCR).The analysis we present is a comprehensive, genome-wide analysis of CNVs in Mus musculus, which increases the number of known variants in the species and will accelerate the identification of novel variants in future studies.