Synaptotagmin-9 in mouse retina.

Synaptotagmin-9 (Syt9) is a Ca sensor mediating fast synaptic release expressed in various parts of the brain. The presence and role of Syt9 in retina is unknown. We found evidence for Syt9 expression throughout the retina and created mice to conditionally eliminate Syt9 in a cre-dependent manner.

Targeted insertion of conditional expression cassettes into the mouse genome using the modified i-PITT.

Background: Transgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called "pronuclear injection-based targeted transgenesis" (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci.

SMARCA4 is a haploinsufficient B cell lymphoma tumor suppressor that fine-tunes centrocyte cell fate decisions.

SMARCA4 encodes one of two mutually exclusive ATPase subunits in the BRG/BRM associated factor (BAF) complex that is recruited by transcription factors (TFs) to drive chromatin accessibility and transcriptional activation. SMARCA4 is among the most recurrently mutated genes in human cancer, including ∼30% of germinal center (GC)-derived Burkitt lymphomas. In mice, GC-specific Smarca4 haploinsufficiency cooperated with MYC over-expression to drive lymphomagenesis.

Transgenic Mice Expressing Functional TCRs Specific to Cardiac Myhc-α 334-352 on Both CD4 and CD8 T Cells Are Resistant to the Development of Myocarditis on C57BL/6 Genetic Background.

Myocarditis is a predominant cause of congestive heart failure and sudden death in children and young adolescents that can lead to dilated cardiomyopathy. Lymphocytic myocarditis mediated by T cells can result from the recognition of cardiac antigens that may involve CD4 or CD8 T cells or both. In this report, we describe the generation of T cell receptor (TCR) transgenic mice on a C57BL/6 genetic background specific to cardiac myosin heavy chain (Myhc)-α 334-352 and make the following observations: First, we verified that Myhc-α 334-352 was immunogenic in wild-type C57BL/6 mice and induced antigen-specific CD4 T cell responses despite being a poor binder of IA; however, the immunized animals developed only mild myocarditis.

SYNAPTOTAGMIN-9 IN MOUSE RETINA.

Synaptotagmin-9 (Syt9) is a Ca sensor mediating fast synaptic release expressed in various parts of the brain. The presence and role of Syt9 in retina is unknown. We found evidence for Syt9 expression throughout the retina and created mice to conditionally eliminate Syt9 in a cre-dependent manner.

Rescue of auditory function by a single administration of AAV-TMPRSS3 gene therapy in aged mice of human recessive deafness DFNB8.

Patients with mutations in the TMPRSS3 gene suffer from recessive deafness DFNB8/DFNB10. For these patients, cochlear implantation is the only treatment option. Poor cochlear implantation outcomes are seen in some patients.

Improved Genome Editing via Oviductal Nucleic Acids Delivery (i-GONAD): Protocol Steps and Additional Notes.

The clustered regularly interspaced short palindromic repeats (CRISPR) technology has made it possible to produce genome-edited (GE) animals more easily and rapidly than before. In most cases, GE mice are produced by microinjection (MI) or by in vitro electroporation (EP) of CRISPR reagents into fertilized eggs (zygotes). Both of these approaches require ex vivo handling of isolated embryos and their subsequent transfer into another set of mice (called recipient or pseudopregnant mice).

Rescue of Auditory Function by a Single Administration of AAV- Gene Therapy in Aged Mice of Human Recessive Deafness DFNB8.

Patients with mutations in the gene suffer from recessive deafness DFNB8/DFNB10 for whom cochlear implantation is the only treatment option. Poor cochlear implantation outcomes are seen in some patients. To develop biological treatment for TMPRSS3 patients, we generated a knock-in mouse model with a frequent human DFNB8 mutation.

Delivering mRNAs to mouse tissues using the SEND system.

mRNAs produced in a cell are almost always translated within the same cell. Some mRNAs are transported to other cells of the organism through processes involving membrane nanotubes or extracellular vesicles. A recent report describes a surprising new phenomenon of encapsulating mRNAs inside virus-like particles (VLPs) to deliver them to other cells in a process that was named SEND (Selective Endogenous eNcapsidation for cellular Delivery).

Muc4 loss mitigates epidermal growth factor receptor activity essential for PDAC tumorigenesis.

Mucin4 (MUC4) appears early during pancreatic intraepithelial neoplasia-1 (PanIN1), coinciding with the expression of epidermal growth factor receptor-1 (EGFR). The EGFR signaling is required for the onset of Kras-driven pancreatic ductal adenocarcinoma (PDAC); however, the players and mechanisms involved in sustained EGFR signaling in early PanIN lesions remain elusive. We generated a unique Esai-CRISPR-based Muc4 conditional knockout murine model to evaluate its effect on PDAC pathology.

Synaptotagmins 1 and 7 in vesicle release from rods of mouse retina.

Synaptotagmins are the primary Ca sensors for synaptic exocytosis. Previous work suggested synaptotagmin-1 (Syt1) mediates evoked vesicle release from cone photoreceptor cells in the vertebrate retina whereas release from rods may involve another sensor in addition to Syt1. We found immunohistochemical evidence for syntaptotagmin-7 (Syt7) in mouse rod terminals and so performed electroretinograms (ERG) and single-cell recordings using mice in which Syt1 and/or Syt7 were conditionally removed from rods and/or cones.

Knock-in mouse models for studying somatostatin and cholecystokinin expressing cells.

Background: Somatostatin (SST) and cholecystokinin (CCK) are peptide hormones that regulate the endocrine system, cell proliferation and neurotransmission.

New Method: We utilized the novel Easi-CRISPR system to generate two knock-in mouse strains with Cre recombinase in SST- and CCK-expressing cells and validated their utility in the developing and adult brain tissues.

Results: The full nomenclature for the newly generated strains are C57BL/6-Sst and C57BL/6-Cck.

Prototype mouse models for researching SEND-based mRNA delivery and gene therapy.

One of the major challenges of gene therapy-an approach to treat diseases caused by faulty genes-is a lack of technologies that deliver healthy gene copies to target tissues and cells. Some commonly used approaches include viral vectors or coating therapeutic nucleic acids with lipid-based nanoparticles to pass through cell membranes, but these technologies have had limited success. A revolutionary tool, the CRISPR-Cas gene-editing system, offers tremendous promise, but it too suffers from problems with delivery.

Ecdysoneless Overexpression Drives Mammary Tumorigenesis through Upregulation of C-MYC and Glucose Metabolism.

Unlabelled: Ecdysoneless (ECD) protein is essential for embryogenesis, cell-cycle progression, and cellular stress mitigation with an emerging role in mRNA biogenesis. We have previously shown that ECD protein as well as its mRNA are overexpressed in breast cancer and ECD overexpression predicts shorter survival in patients with breast cancer. However, the genetic evidence for an oncogenic role of ECD has not been established.

Structure of Long-Range Direct and Indirect Spinocerebellar Pathways as Well as Local Spinal Circuits Mediating Proprioception.

Proprioception, the sense of limb and body position, generates a map of the body that is essential for proper motor control, yet we know little about precisely how neurons in proprioceptive pathways are wired. Defining the anatomy of secondary neurons in the spinal cord that integrate and relay proprioceptive and potentially cutaneous information from the periphery to the cerebellum is fundamental to understanding how proprioceptive circuits function. Here, we define the unique anatomic trajectories of long-range direct and indirect spinocerebellar pathways as well as local intersegmental spinal circuits using genetic tools in both male and female mice.

Novel reporter mouse models useful for evaluating gene editing and for optimization of methods of delivering genome editing tools.

The clustered regularly interspersed palindromic repeats (CRISPR) system is a powerful genome-editing tool to modify genomes, virtually in any species. The CRISPR tool has now been utilized in many areas of medical research, including gene therapy. Although several proof-of-concept studies show the feasibility of gene therapy applications for correcting disease-causing mutations, and new and improved tools are constantly being developed, there are not many choices of suitable reporter models to evaluate genome editor tools and their delivery methods.

Biogenesis, physiological functions and potential applications of extracellular vesicles in substance use disorders.

Substance use disorder (SUD) is a growing health problem that affects several millions of people worldwide, resulting in negative socioeconomic impacts and increased health care costs. Emerging evidence suggests that extracellular vesicles (EVs) play a crucial role in SUD pathogenesis. EVs, including exosomes and microvesicles, are membrane-encapsulated particles that are released into the extracellular space by most types of cells.

A transgenic pig model expressing a CMV-ZsGreen1 reporter across an extensive array of tissues.

Since genetic engineering of pigs can benefit both biomedicine and agriculture, selecting a suitable gene promoter is critically important. The cytomegalovirus (CMV) promoter, which can robustly drive ubiquitous transgene expression, is commonly used at present, yet recent reports suggest tissue-specific activity in the pig. The objective of this study was to quantify ZsGreen1 protein (in lieu of CMV promoter activity) in tissues from pigs harboring a CMV-ZsGreen1 transgene with a single integration site.

Designing and generating a mouse model: frequently asked questions.

Genetically engineered mouse (GEM) models are commonly used in biomedical research. Generating GEMs involve complex set of experimental procedures requiring sophisticated equipment and highly skilled technical staff. Because of these reasons, most research institutes set up centralized core facilities where custom GEMs are created for research groups.