Background Human leukocyte antigen (HLA)‐DP is much less studied than other HLA class II antigens, that is, HLA‐DR and HLA‐DQ, etc. However, the accumulating data have suggested the important roles of DP‐restricted responses in the context of cancer, allergy, and infectious disease. Lack of animal models expressing these genes as authentic cis‐haplotypes blocks our understanding for the role of HLA‐DP haplotypes in immunity. Methods To explore the potential cis‐acting control elements involved in the transcriptional regulation of the HLA‐DPA1/DPB1 gene, we performed the expression analysis using bacterial artificial chromosome (BAC)‐based transgenic humanized mice in the C57BL/6 background, which carried the entire HLA‐DP401 gene locus. We further developed a mouse model of Staphylococcus aureus pneumonia in HLA‐DP401 humanized transgenic mice, and performed the analysis on the expression pattern of HLA‐DP401 and immunological responses in the model. Results In this study, we screened and identified a BAC clone spanning the entire HLA‐DP gene locus. DNA from this clone was analyzed for integrity by pulsed‐field gel electrophoresis and then microinjected into fertilized mouse oocytes to produce transgenic founder animals. Nine sets of PCR primers for regional markers with an average distance of 15 kb between each primer were used to confirm the integrity of the transgene in the five transgenic lines carrying the HLA‐DPA1/DPB1 gene. Transgene copy numbers were determined by real‐time PCR analysis. HLA‐DP401 gene expression was analyzed at the mRNA and protein level. Although infection with S aureus Newman did not alter the percentage of immune cells in the spleen and thymus from the HLA‐DP401‐H2‐Aβ1 humanized mice. Increased expression of HLA‐DP401 was observed in the thymus of the humanized mice infected by S aureus. Conclusions We generated several BAC transgenic mice, and analyzed the expression of HLA‐DPA1/DPB1 in those mice. A model of S aureus‐induced pneumonia in the HLA‐DP401‐H2‐Aβ1−/− humanized mice was further developed, and S aureus infection upregulated the HLA‐DP401 expression in thymus of those humanized mice. These findings demonstrate the potential of those HLA‐DPA1/DPB1 transgenic humanized mice for developing animal models of infectious diseases and MHC‐associated immunological diseases.

Background: Myostatin (MSTN) encodes a negative regulator of skeletal muscle mass that might have applications for promoting muscle growth in livestock. In this study, we aimed to test whether targeted MSTN editing, mediated by transcription activator-like effector nucleases (TALENs), is a viable approach to create myostatin-modified goats (Capra hircus). Results: We obtained a pair of TALENs (MTAL-2) that could recognize and cut the targeted MSTN site in the goat genome. Fibroblasts from pedigreed goats were co-transfected with MTAL-2, and 272 monoclonal cell strains were confirmed to have mono-or bi-allelic mutations in MSTN. Ten cell strains with different genotypes were used as donor cells for somatic cell nuclear transfer, which produced three cloned kids (K179/MSTN −/− , K52-2/MSTN +/− , and K52-1/MSTN +/+ ).

T cells play a critical role in coronavirus diseases. How they do so in COVID-19 may be revealed by analyzing the epigenetic chromatin accessibility of cis- and trans-regulatory elements and creating transcriptomic immune profiles. We performed single-cell assay for transposase-accessible chromatin (scATAC) and single-cell RNA (scRNA) sequencing (seq) on the peripheral blood mononuclear cells (PBMCs) of severely ill/critical patients (SCPs) infected with COVID-19, moderate patients (MPs), and healthy volunteer controls (HCs). About 76,570 and 107,862 single cells were used, respectively, for analyzing the characteristics of chromatin accessibility and transcriptomic immune profiles by the application of scATAC-seq (nine cases) and scRNA-seq (15 cases). The scATAC-seq detected 28,535 different peaks in the three groups; among these peaks, 41.6 and 10.7% were located in the promoter and enhancer regions, respectively. Compared to HCs, among the peak-located genes in the total T cells and its subsets, CD4+ T and CD8+ T cells, from SCPs and MPs were enriched with inflammatory pathways, such as mitogen-activated protein kinase (MAPK) signaling pathway and tumor necrosis factor (TNF) signaling pathway. The motifs of TBX21 were less accessible in the CD4+ T cells of SCPs compared with those in MPs. Furthermore, the scRNA-seq showed that the proportion of T cells, especially the CD4+ T cells, was decreased in SCPs and MPs compared with those in HCs. Transcriptomic results revealed that histone-related genes, and inflammatory genes, such as NFKBIA, S100A9, and PIK3R1, were highly expressed in the total T cells, CD4+ T and CD8+ T cells, both in the cases of SCPs and MPs. In the CD4+ T cells, decreased T helper-1 (Th1) cells were observed in SCPs and MPs. In the CD8+T cells, activation markers, such as CD69 and HLA class II genes (HLA-DRA, HLA-DRB1, and HLA-DRB5), were significantly upregulated in SCPs. An integrated analysis of the data from scATAC-seq and scRNA-seq showed some consistency between the approaches. Cumulatively, we have generated a landscape of chromatin epigenetic status and transcriptomic immune profiles of T cells in patients with COVID-19. This has provided a deeper dissection of the characteristics of the T cells involved at a higher resolution than from previously obtained data merely by the scRNA-seq analysis. Our data led us to suggest that the T-cell inflammatory states accompanied with defective functions in the CD4+ T cells of SCPs may be the key factors for determining the pathogenesis of and recovery from COVID-19.

Background: There are remarkable genetic differences between animal major histocompatibility complex (MHC) systems and the human leukocyte antigen (HLA) system. HLA transgenic humanized mouse model systems offer a much better method to study the HLA-A-related principal mechanisms for vaccine development and HLA-Arestricted responses against infection in human. Methods: A recombinant gene encoding the chimeric HLA-A30 monochain was constructed. This HHD molecule contains the following: α1-α2 domains of HLA-A30, α3 and cytoplasmic domains of H-2D b , linked at its N-terminus to the C-terminus of human β2m by a 15-amino-acid peptide linker. The recombinant gene encoding the chimeric HLA-A30 monochain cassette was introduced into bacterial artificial chromosome (BAC) CH502-67J3 containing the HLA-A01 gene locus by Red-mediated homologous recombination. Modified BAC CH502-67J3 was microinjected into the pronuclei of wild-type mouse oocytes. This humanized mouse model was further used to assess the immune responses against influenza A virus (H1N1) pdm09 clinically isolated from human patients. Immune cell population, cytokine production, and histopathology in the lung were analyzed. Results: We describe a novel human β2m-HLA-A30 (α1α2)-H-2D b (α3 transmembrane cytoplasmic) (HHD) monochain transgenic mouse strain, which contains the intact HLA-A01 gene locus including 49 kb 5′-UTR and 74 kb 3′-UTR of HLA-A01*01. Five transgenic lines integrated into the large genomic region of HLA-A gene locus were obtained, and the robust expression of exogenous transgene was detected in various tissues from A30-18# and A30-19# lines encompassing the intact flanking sequences. Flow cytometry revealed that the introduction of a large genomic region in HLA-A gene locus can influence the immune cell constitution in humanized mice. Pdm09 infection caused a similar immune response among HLA-A30 Tg humanized mice and wild-type mice, and induced the rapid increase of cytokines, including IFNγ, TNFα, and IL-6, in both HLA-A30 humanized Tg mice and wild-type mice. The expression of This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Background Lingual epithelia in the tongue tip are among the most rapidly regenerating tissues, but the mechanism of cell genesis in this tissue is still unknown. Previous study has suggested the existence of multiple stem cell pools in lingual epithelia and papillae. Like K14+ and Sox2+ cells, NTPD ase2+ cells have characteristics of stem cells. Methods We employed a system using doxycycline to conditionally ablate NTPD ase2+ cells in lingual epithelia and papillae by regulated expression of the diphtheria toxin A ( DTA ) gene. Transgenic lines, which expressed the rt TA gene in NTPD ase2+ cells, were produced by pronuclear injection of zygotes from C57 BL /6 mice using the BAC clone RP 23‐47P18. The NTPD ase2‐rt TA transgenic mice were crossed with the TetO‐ DTA transgenic animals. The double transgenic mice were treated with doxycycline. Doxycycline (Dox) was diluted in 5% sucrose in water to a final concentration of 0.3‐0.5 mg/mL and supplied as drinking water. Results After 15 days of Dox induction, the expression of NTPD ase2, Sox2 and K14 was ablated from lingual epithelia. DTA expression in NTPD ase2+ cells did not inhibit the turnover of GNAT 3+ or PLC β2+ cells in taste buds, nor the expression of S100β beneath lingual epithelia and papillae. After 35 days ablation of NTPD ase2+ cells, the basic structure of lingual epithelia and papillae remained intact. However, the ratio of cell to total tissue area was decreased in lingual epithelia and circumvallate ( CV ) papillae. DTA expression also inhibited the regeneration of filiform papillae on the dorsal surface of the tongue tip. Conclusions These studies provide important insights into the understanding of dynamic equilibrium among the multiple stem cell populations present in the lingual epithelia and papillae.