ABSTRACT
Recent advances in genetic analyses have significantly refined human type 1 diabetes (T1D) associated loci. The goal of such effort is to identify the causal genes and have a complete understanding of the molecular pathways that independently or interactively influence cellular processes leading to the destruction of insulin producing pancreatic ß cells. UBASH3A has been suggested as the underlying gene for a human T1D associated region on chromosome 21. To further evaluate the role of UBASH3A in T1D, we targeted Ubash3a in NOD mice using zinc-finger nuclease mediated mutagenesis. In both 10-week-old females and males, significantly more advanced insulitis was observed in UBASH3A-deficient than in wild-type NOD mice. Consistently, UBASH3A-deficient NOD mice developed accelerated T1D in both sexes, which was associated with increased accumulation of ß-cell autoreactive T cells in the spleen and pancreatic lymph node. Adoptive transfer of splenic T cells into NOD.Rag1-/- mice demonstrated that UBASH3A deficiency in T cells was sufficient to promote T1D development. Our results provide strong evidence to further support a role of UBASH3A in T1D. In addition to T1D, UBASH3A deficiency also promoted salivary gland inflammation in females, demonstrating its broad impact on autoimmunity.
Subject(s)
Diabetes Mellitus, Type 1/genetics , Genetic Predisposition to Disease/genetics , Receptors, Antigen, T-Cell/deficiency , Receptors, Antigen, T-Cell/genetics , Sialadenitis/metabolism , Adoptive Transfer , Animals , Autoimmunity , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 1/metabolism , Disease Models, Animal , Female , Insulin-Secreting Cells/metabolism , Male , Mice , Mice, Inbred NOD , Mice, Knockout , Mutagenesis/drug effects , Zinc Finger Nucleases/pharmacologyABSTRACT
Studies in NOD mice have provided important insight into the genetics and pathogenesis of type 1 diabetes (T1D). Our goal was to further explore novel methods of genetic manipulation in this mouse model. We tested the feasibility of using zinc-finger nucleases (ZFNs) to knock out a gene directly in a pure NOD background, bypassing the need of embryonic stem cells. We report here the successful application of ZFN pairs to specifically and efficiently knock out Tnfrsf9 (encoding CD137/4-1BB) directly in the NOD mouse by embryo microinjection. Histology and T1D incidence studies indicated that CD137 was dispensable for the development of insulitis but played a role to promote progression to overt diabetes in NOD mice. We also demonstrated that CD137-deficient T-cells were less diabetogenic than their wild-type counterpart when adoptively transferred into NOD.Rag1(-/-) recipients, even when CD25(+) cells were predepleted. In vitro assays suggested that CD137 deficiency had a limited effect on the suppressive function of CD4(+)CD25(+) regulatory T-cells (Tregs). Therefore, CD137 deficiency predominately affected effector T-cells rather than Tregs. Our study demonstrates the ability to generate gene-targeted knockouts in a pure NOD background by using ZFNs without potential confounding factors introduced by contaminating genetic materials obtained from other strains.
Subject(s)
Diabetes Mellitus, Type 1/genetics , Gene Knockout Techniques/methods , T-Lymphocytes, Regulatory/immunology , Zinc Fingers/genetics , Animals , Diabetes Mellitus, Type 1/immunology , Mice , Mice, Inbred NOD , Tumor Necrosis Factor Receptor Superfamily, Member 9/genetics , Tumor Necrosis Factor Receptor Superfamily, Member 9/immunology , Zinc Fingers/immunologyABSTRACT
BACKGROUND: Respiratory Syncytial Virus (RSV) is the leading cause of lower respiratory-tract infections in infants and young children worldwide. Despite this, only six complete genome sequences of original strains have been previously published, the most recent of which dates back 35 and 26 years for RSV group A and group B respectively. METHODOLOGY/PRINCIPAL FINDINGS: We present a semi-automated sequencing method allowing for the sequencing of four RSV whole genomes simultaneously. We were able to sequence the complete coding sequences of 13 RSV A and 4 RSV B strains from Milwaukee collected from 1998-2010. Another 12 RSV A and 5 RSV B strains sequenced in this study cover the majority of the genome. All RSV A and RSV B sequences were analyzed by neighbor-joining, maximum parsimony and Bayesian phylogeny methods. Genetic diversity was high among RSV A viruses in Milwaukee including the circulation of multiple genotypes (GA1, GA2, GA5, GA7) with GA2 persisting throughout the 13 years of the study. However, RSV B genomes showed little variation with all belonging to the BA genotype. For RSV A, the same evolutionary patterns and clades were seen consistently across the whole genome including all intergenic, coding, and non-coding regions sequences. CONCLUSIONS/SIGNIFICANCE: The sequencing strategy presented in this work allows for RSV A and B genomes to be sequenced simultaneously in two working days and with a low cost. We have significantly increased the amount of genomic data that is available for both RSV A and B, providing the basic molecular characteristics of RSV strains circulating in Milwaukee over the last 13 years. This information can be used for comparative analysis with strains circulating in other communities around the world which should also help with the development of new strategies for control of RSV, specifically vaccine development and improvement of RSV diagnostics.
