University of Tsukuba

About: University of Tsukuba is a education organization based out in Tsukuba, Ibaraki, Japan. It is known for research contribution in the topics: Population & Gene. The organization has 36352 authors who have published 79483 publications receiving 1934752 citations. The organization is also known as: Tsukuba daigaku & Tsukuba University.

A genome-editing strategy to treat β-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition

Abstract: Disorders resulting from mutations in the hemoglobin subunit beta gene (HBB; which encodes β-globin), mainly sickle cell disease (SCD) and β-thalassemia, become symptomatic postnatally as fetal γ-globin expression from two paralogous genes, hemoglobin subunit gamma 1 (HBG1) and HBG2, decreases and adult β-globin expression increases, thereby shifting red blood cell (RBC) hemoglobin from the fetal (referred to as HbF or α2γ2) to adult (referred to as HbA or α2β2) form. These disorders are alleviated when postnatal expression of fetal γ-globin is maintained. For example, in hereditary persistence of fetal hemoglobin (HPFH), a benign genetic condition, mutations attenuate γ-globin-to-β-globin switching, causing high-level HbF expression throughout life. Co-inheritance of HPFH with β-thalassemia- or SCD-associated gene mutations alleviates their clinical manifestations. Here we performed CRISPR-Cas9-mediated genome editing of human blood progenitors to mutate a 13-nt sequence that is present in the promoters of the HBG1 and HBG2 genes, thereby recapitulating a naturally occurring HPFH-associated mutation. Edited progenitors produced RBCs with increased HbF levels that were sufficient to inhibit the pathological hypoxia-induced RBC morphology found in SCD. Our findings identify a potential DNA target for genome-editing-mediated therapy of β-hemoglobinopathies.

PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors

Abstract: We introduce a PCR-based procedure for generating a gene disruption construct. This method depends on DNA fragment fusion by the PCR technique and requires only two steps of PCR to obtain a sufficient amount of the gene disruption construct for one transformation experiment. The first step involves three separate PCR syntheses of a selectable marker cassette and the 5'- and 3'-regions of a target gene. Of the four primers used in amplification of the 5'- and 3'-regions of the target gene, two primers placed proximal to the site of the marker cassette are designed to have sequence tags complementary to the 5'- or 3'-side of the marker cassette. The two primers used in PCR synthesis of the marker cassette are complementary to the tagged primers. By fusion PCR, the 5' and 3' PCR products are linked to the marker cassette via the regions of tagged primers that overlap. A sufficient amount of the disruption construct can be directly amplified with the outermost primers. This method is simple, rapid and relatively inexpensive. In addition, there is the freedom of attaching long flanking regions to any selectable marker cassette.

Subventricular Zone-Derived Neural Progenitor Cells Migrate Along a Blood Vessel Scaffold Toward the Post-stroke Striatum

Abstract: The subventricular zone (SVZ) of the adult brain contains neural stem cells that have the capacity to regenerate new neurons after various insults. Brain ischemia causes damage to brain tissue and induces neural regeneration together with angiogenesis. We previously reported that, after ischemic injury in mice, SVZ-derived neural progenitor cells (NPCs) migrate into the striatum, and these NPCs are frequently associated with blood vessels in the regenerating brain tissue. Here we studied the role of blood vessels during the neural regeneration in more detail. BrdU administration experiments revealed that newly generated NPCs were associated with both newly formed and pre-existing blood vessels in the ischemic striatum, suggesting that the angiogenic environment is not essential for the neuron-blood vessel interaction. To observe migrating NPCs and blood vessels simultaneously in damaged brain tissue, we performed live imaging of cultured brain slices after ischemic injury. In this system, we virally labeled SVZ-derived NPCs in Flk1-EGFP knock-in mice in which the blood vessels are labeled with EGFP. Our results provide direct evidence that SVZ-derived NPCs migrate along blood vessels from the SVZ toward the ischemic region of the striatum. The leading process of the migrating NPCs was closely associated with blood vessels, suggesting that this interaction provides directional guidance to the NPCs. These findings suggest that blood vessels play an important role as a scaffold for NPCs migration toward the damaged brain region.

Direct Visualization of the Reversible O2- /O- Redox Process in Li-Rich Cathode Materials.

Abstract: Conventional cathodes of Li-ion batteries mainly operate through an insertion-extraction process involving transition metal redox. These cathodes will not be able to meet the increasing requirements until lithium-rich layered oxides emerge with beyond-capacity performance. Nevertheless, in-depth understanding of the evolution of crystal and excess capacity delivered by Li-rich layered oxides is insufficient. Herein, various in situ technologies such as X-ray diffraction and Raman spectroscopy are employed for a typical material Li1.2 Ni0.2 Mn0.6 O2 , directly visualizing O- O- (peroxo oxygen dimers) bonding mostly along the c-axis and demonstrating the reversible O2- /O- redox process. Additionally, the formation of the peroxo OO bond is calculated via density functional theory, and the corresponding OO bond length of ≈1.3 A matches well with the in situ Raman results. These findings enrich the oxygen chemistry in layered oxides and open opportunities to design high-performance positive electrodes for lithium-ion batteries.