TALEN-mediated editing of the mouse Y chromosome

The functional study of Y chromosome genes has been hindered by a lack of mouse models with specific Y chromosome mutations. We used transcription activator-like effector nuclease (TALEN)-mediated gene editing in mouse embryonic stem cells (mESCs) to produce mice with targeted gene disruptions and insertions in two Y-linked genes—Sry and Uty. TALEN-mediated gene editing is a useful tool for dissecting the biology of the Y chromosome.National Institutes of Health (U.S.) (US NIH grant R01-HG000257)National Institutes of Health (U.S.) (US NIH grant R01-CA084198)National Institutes of Health (U.S.) (US NIH grant R37-HD045022)Croucher Foundation (Scholarship)Howard Hughes Medical Institute (Investigator

Effect of a CpG-ODN on the innate immune system of the horse: an in-vivo trial

Oligodeoxynucleotides containing cytosine-phosphate-guanosine motifs (CpG-ODN) represent a class of agonists of Toll-like Receptor 9 (TLR9). TLR9 activation induces the secretion of cytokines and the maturation of immune cells, thus initiating both innate and adaptive immune responses. Therefore, CpG-ODN has been investigated in different species as a potential immune-modulator targeting infectious, allergic and neoplastic diseases. It has been administered by nebulisation to RAO-affected horses with promising results. Nonetheless, there is no in-vivo study on the effect of CpG administered systemically to the horse. Therefore, we tested the effect of CpG, given by intramuscular injection, on the equine immune response. Eight horses were used for this study. Five mg/horse were injected to 4 horses at D0 and D7; the other horses received a placebo (PBS). Blood was collected 2 days prior to each injection, then regularly up to D21. A clinical exam was realised daily. Laboratory analyses included haematology, ELISA tests for IFN-alpha, IFN-gamma, TNF-alpha and IL-10 and cytometry analyses for MCHII and CD86 expressions on B-lymphocytes. A cross-over of the 2 groups was realised after 2 months of washout. CpG was well tolerated. Significant transient eosinopenia, monocytosis and leukopenia were observed after CpG injection, while ELISA and cytometry analyses did not reveal any significant modification. This trial represents the first in-vivo study where CpG is administered systemically to healthy horses. Further studies are needed to adjust the dose, the formulation and the sampling schedule and to fully investigate this molecule as potentiel modulator of the equine immune system

Molecular Definition of an Allelic Series of Mutations Disrupting the Myostatin Function and Causing Double-Muscling in Cattle

We have determined the entire myostatin coding sequence for 32 double-muscled cattle sampled from ten European cattle breeds. Seven DNA sequence polymorphisms were identified, of which five would be predicted to disrupt the function of the protein, one is a conservative amino acid substitution, and one a silent DNA sequence variant. Four additional DNA sequence polymorphisms were identified in myostatin intronic sequences. In all but two breeds, all double-muscled animals were either homozygous or compound heterozygotes for one of the five loss-of-function mutations. The absence of obvious loss-of-function mutations in the coding sequence of the two remaining breeds points either towards additional mutations in unexplored segments of the gene, or towards locus heterogeneity of double-muscling

Airway Macrophages Encompass Transcriptionally and Functionally Distinct Subsets Altered by Smoking

peer reviewedAlveolar macrophages (AMs) are functionally important innate cells involved in lung homeostasis and immunity and whose diversity in health and disease is a subject of intense investigations. Yet, it remains unclear to what extent conditions like smoking or chronic obstructive pulmonary disease (COPD) trigger changes in the AM compartment. Here, we aimed to explore heterogeneity of human AMs isolated from healthy nonsmokers, smokers without COPD, and smokers with COPD by analyzing BAL fluid cells by flow cytometry and bulk and single-cell RNA sequencing. We found that subpopulations of BAL fluid CD2061 macrophages could be distinguished based on their degree of autofluorescence in each subject analyzed. CD2061 autofluorescenthigh AMs were identified as classical, self-proliferative AM, whereas autofluorescentlow AMs were expressing both monocyte and classical AM-related genes, supportive of a monocytic origin. Of note, monocyte-derived autofluorescentlow AMs exhibited a functionally distinct immunoregulatory profile, including the ability to secrete the immunosuppressive cytokine IL-10. Interestingly, single-cell RNA-sequencing analyses showed that transcriptionally distinct clusters of classical and monocyte-derived AM were uniquely enriched in smokers with and without COPD as compared with healthy nonsmokers. Of note, such smoking-associated clusters exhibited gene signatures enriched in detoxification, oxidative stress, and proinflammatory responses. Our study independently confirms previous reports supporting that monocyte-derived macrophages coexist with classical AM in the airways of healthy subjects and patients with COPD and identifies smoking-associated changes in the AM compartment that may favor COPD initiation or progression. © 2022 by the American Thoracic Society

High-Resolution, Human-Bovine Comparative Mapping Based on a Closed Yac Contig Spanning the Bovine Mh Locus

A closed YAC contig spanning the mh locus was assembled by STS content mapping with seven microsatellite markers, eight genes or EST, and nine STS corresponding to YAC ends. The contig comprises 27 YACs, has an average depth of 4.3 YACs, and spans an estimated 1.2 Mb. A linkage map was constructed based on five of the microsatellite markers anchored to the contig and shown to span 7 cM, yielding a ratio of 160 kb/1 cM for the corresponding chromosome region. Comparative mapping data indicate that the constructed contig spans an evolutionary breakpoint connecting two chromosome segments that are syntenic but not adjacent in the human. Consolidation of human gene order by means of whole genome radiation hybrids and its comparison with the bovine order as inferred from the contig confirm conservation of gene order within segments

Polymorphic miRNA-target interactions : A Novel Source of Phenotypic Variation

Studying the muscular hypertrophy of Texel sheep by forward genetics, we have identified an A-to-G transition in the 3'UTRof the GDF8 gene that reveals an illegitimate target site for microRNAs miR-1 and miR-206 that are highly expressed in skeletal muscle. This causes the down-regulation of this muscle-specific chalone and hence contributes to the muscular hypertrophyof Texel sheep. We demonstrate that polymorphisms which alter the content of putative miRNA target sites are commonin human and mice, and provide evidence that both conserved and nonconserved target sites are selectively constrained. Wespeculate that these polymorphisms might be important mediators of phenotypic variation including disease. To facilitatestudies along those lines, we have constructed a database (www.patrocles.org) listing putative polymorphic microRNA–targetinteractions