KOnezumi: a web application for automating gene disruption strategies to generate knockout mice

A Summary: Although gene editing using the CRISPR/Cas9 system enables the rapid generation of knockout mice, constructing an optimal gene disruption strategy is still labourious. Here, we propose KOnezumi, a simple and user-friendly web application, for use in automating the design of knockout strategies for multiple genes. Users only need to input gene symbols, and then KOnezumi returns target exons, gRNA candidates to delete the target exons, genotyping PCR primers, nucleotide sequences of the target exons and coding sequences of expected deletion products. KOnezumi enables users to easily and rapidly apply a rational strategy to accelerate the generation of KO mic

Molecular-size-distribution-dependent aggregation of humic substances by Na(I), Ag(I), Ca(II), and Eu(III)

Molecular-size-distribution (MSD)-dependent aggregation of Fluka and International Humic Substance Society humic acids (HAs) in the presence of Na(I), Ca(II), Ag(I), and Eu(III) were studied using high-performance size-exclusion chromatography. Larger molecular size fractions (>8000. Da) rich in aliphatic carbons were observed to aggregate in the presence of Na(I) and Ca(II), whereas both large and small molecular size fractions concurrently aggregated in the presence of Ag(I) and Eu(III). In view of the non-specific binding of Na(I) and Ca(II) with HAs, the electric-field screening effect is probably the main cause of HA aggregation by Na(I) and Ca(II), preserving the intrinsic hydrophobicity of the HAs. Reduced surface charges and the hydrophobic heterogeneities of different MSD fractions of HAs by direct binding of Ag(I) and Eu(III) are postulated to induce insignificant dependence of aggregation on the different MSD fractions. © 2013 Elsevier B.V

Optimizing Charge Switching in Membrane Lytic Peptides for Endosomal Release of Biomacromolecules.

Endocytic pathways are practical routes for the intracellular delivery of biomacromolecules. Along with this, effective strategies for endosomal cargo release into the cytosol are desired to achieve successful delivery. Focusing on compositional differences between the cell and endosomal membranes and the pH decrease within endosomes, we designed the lipid-sensitive and pH-responsive endosome-lytic peptide HAad. This peptide contains aminoadipic acid (Aad) residues, which serve as a safety catch for preferential permeabilization of endosomal membranes over cell membranes, and His-to-Ala substitutions enhance the endosomolytic activity. The ability of HAad to destabilize endosomal membranes was supported by model studies using large unilamellar vesicles (LUVs) and by increased intracellular delivery of biomacromolecules (including antibodies) into live cells. Cerebral ventricle injection of Cre recombinase with HAad led to Cre/loxP recombination in a mouse model, thus demonstrating potential applicability of HAad in vivo

Simple generation of hairless mice for in vivo imaging

The in vivo imaging of mice makes it possible to analyze disease progress non-invasively through reporter gene expression. As the removal of hair improves the accuracy of in vivo imaging, gene-modified mice with a reporter gene are often crossed with Hos:HR-1 mutant mice homozygous for the spontaneous Hrhr mutation that exhibit a hair loss phenotype. However, it is time consuming to produce mice carrying both the reporter gene and mutant Hrhr gene by mating. In addition, there is a risk that genetic background of the gene-modified mice would be altered by mating. To resolve these issues, we established a simple method to generate hairless mice maintaining the original genetic background by CRISPR technology. First, we constructed the pX330 vector, which targets exon 3 of Hr. This DNA vector (5 ng/µl) was microinjected into the pronuclei of C57BL/6J mice. Induced Hr gene mutations were found in many founders (76.1%) and these mutations were heritable. Next, we performed in vivo imaging using these gene-modified hairless mice. As expected, luminescent objects in their body were detected by in vivo imaging. This study clearly showed that hairless mice could be simply generated by the CRISPR/Cas9 system, and this method may be useful for in vivo imaging studies with various gene-modified mice

Disruption of entire Cables2 locus leads to embryonic lethality by diminished Rps21 gene expression and enhanced p53 pathway

In vivo function of CDK5 and Abl enzyme substrate 2 (Cables2), belonging to the Cables protein family, is unknown. Here, we found that targeted disruption of the entire Cables2 locus (Cables2d) caused growth retardation and enhanced apoptosis at the gastrulation stage and then induced embryonic lethality in mice. Comparative transcriptome analysis revealed disruption of Cables2, 50% down-regulation of Rps21 abutting on the Cables2 locus, and up-regulation of p53-target genes in Cables2d gastrulas. We further revealed the lethality phenotype in Rps21-deleted mice and unexpectedly, the exon 1-deleted Cables2 mice survived. Interestingly, chimeric mice derived from Cables2d ESCs carrying exogenous Cables2 and tetraploid wild-type embryo overcame gastrulation. These results suggest that the diminished expression of Rps21 and the completed lack of Cables2 expression are intricately involved in the embryonic lethality via the p53 pathway. This study sheds light on the importance of Cables2 locus in mouse embryonic development.Grant-in-Aid for Scientific Research(B), Japan Society for the Promotion of Science (JSPS KAKENHI 17H03568) Fumihiro Sugiyama Grant-in-Aid for Scientific Research(S), Japan Society for the Promotion of Science (JSPS KAKENHI 26221004) Satoru Takahashi Grant-in-Aid for Scientific Research(C), Japan Society for the Promotion of Science (JSPS KAKENHI 17K07130) Hiroyoshi Iseki Grant-in-Aid for Young Scientists (B), Japan Society for the Promotion of Science (JSPS KAKENHI 19K16020) Tra Thi Huong Dinh Grant-in-Aid for Scientific Research(A), Japan Society for the Promotion of Science (JSPS KAKENHI 20H00444) Fumihiro Sugiyama The Cooperative Research Project Program of Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA Center), University of Tsukuba, Japan (182107) Fumihiro Sugiyam

Ribosome binding protein GCN1 regulates the cell cycle and cell proliferation and is essential for the embryonic development of mice.

Amino acids exert many biological functions, serving as allosteric regulators and neurotransmitters, as constituents in proteins and as nutrients. GCN2-mediated phosphorylation of eukaryotic initiation factor 2 alpha (elF2α) restores homeostasis in response to amino acid starvation (AAS) through the inhibition of the general translation and upregulation of amino acid biosynthetic enzymes and transporters by activating the translation of Gcn4 and ATF4 in yeast and mammals, respectively. GCN1 is a GCN2-binding protein that possesses an RWD binding domain (RWDBD) in its C-terminus. In yeast, Gcn1 is essential for Gcn2 activation by AAS; however, the roles of GCN1 in mammals need to be established. Here, we revealed a novel role of GCN1 that does not depend on AAS by generating two Gcn1 mutant mouse lines: Gcn1-knockout mice (Gcn1 KO mice (Gcn1-/-)) and RWDBD-deleted mutant mice (Gcn1ΔRWDBD mice). Both mutant mice showed growth retardation, which was not observed in the Gcn2 KO mice, such that the Gcn1 KO mice died at the intermediate stage of embryonic development because of severe growth retardation, while the Gcn1ΔRWDBD embryos showed mild growth retardation and died soon after birth, most likely due to respiratory failure. Extension of pregnancy by 24 h through the administration of progesterone to the pregnant mothers rescued the expression of differentiation markers in the lungs and prevented lethality of the Gcn1ΔRWDBD pups, indicating that perinatal lethality of the Gcn1ΔRWDBD embryos was due to simple growth retardation. Similar to the yeast Gcn2/Gcn1 system, AAS- or UV irradiation-induced elF2α phosphorylation was diminished in the Gcn1ΔRWDBD mouse embryonic fibroblasts (MEFs), suggesting that GCN1 RWDBD is responsible for GCN2 activity. In addition, we found reduced cell proliferation and G2/M arrest accompanying a decrease in Cdk1 and Cyclin B1 in the Gcn1ΔRWDBD MEFs. Our results demonstrated, for the first time, that GCN1 is essential for both GCN2-dependent stress response and GCN2-independent cell cycle regulation

Generating Vegfr3 reporter transgenic mouse expressing membrane-tagged Venus for visualization of VEGFR3 expression in vascular and lymphatic endothelial cells

Vascular endothelial growth factor receptor 3 (Vegfr3) has been widely used as a marker for lymphatic and vascular endothelial cells during mouse embryonic development and in adult mouse, making it valuable for studying angiogenesis and lymphangiogenesis under normal and pathological conditions. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a membrane-localized fluorescent reporter protein, Gap43-Venus, under the control of the Vegfr3 regulatory sequence. Vegfr3-Gap43-Venus BAC Tg recapitulated endogenous Vegfr3 expression in vascular and lymphatic endothelial cells during embryonic development and tumor development. Thus, this Tg mouse line contributes a valuable model to study angiogenesis and lymphangiogenesis in physiological and pathological contexts

Generation and Characterization of a Cell Type-Specific, Inducible Cre-Driver Line to Study Olfactory Processing

In sensory systems of the brain, mechanisms exist to extract distinct features from stimuli to generate a variety of behavioral repertoires. These often correspond to different cell types at various stages in sensory processing. In the mammalian olfactory system, complex information processing starts in the olfactory bulb, whose output is conveyed by mitral cells (MCs) and tufted cells (TCs). Despite many differences between them, and despite the crucial position they occupy in the information hierarchy, Cre-driver lines that distinguish them do not yet exist. Here, we sought to identify genes that are differentially expressed between MCs and TCs of the mouse, with an ultimate goal to generate a cell type-specific Cre-driver line, starting from a transcriptome analysis using a large and publicly available single-cell RNA-seq dataset (Zeisel et al., 2018). Many genes were differentially expressed, but only a few showed consistent expressions in MCs and at the specificity required. After further validating these putative markers using ISH, two genes (i.e., Pkib and Lbdh2) remained as promising candidates. Using CRISPR/Cas9-mediated gene editing, we generated Cre-driver lines and analyzed the resulting recombination patterns. This indicated that our new inducible Cre-driver line, Lbhd2-CreERT2, can be used to genetically label MCs in a tamoxifen dose-dependent manner, both in male and female mice, as assessed by soma locations, projection patterns, and sensory-evoked responses in vivo. Hence, this is a promising tool for investigating cell type-specific contributions to olfactory processing and demonstrates the power of publicly accessible data in accelerating science

ZMYND10 functions in a chaperone relay during axonemal dynein assembly

Molecular chaperones promote the folding and macromolecular assembly of a diverse set of ‘client’ proteins. How ubiquitous chaperone machineries direct their activities towards specific sets of substrates is unclear. Through the use of mouse genetics, imaging and quantitative proteomics we uncover that ZMYND10 is a novel co-chaperone that confers specificity for the FKBP8-HSP90 chaperone complex towards axonemal dynein clients required for cilia motility. Loss of ZMYND10 perturbs the chaperoning of axonemal dynein heavy chains, triggering broader degradation of dynein motor subunits. We show that pharmacological inhibition of FKBP8 phenocopies dynein motor instability associated with the loss of ZMYND10 in airway cells and that human disease-causing variants of ZMYND10 disrupt its ability to act as an FKBP8-HSP90 co-chaperone. Our study indicates that primary ciliary dyskinesia (PCD), caused by mutations in dynein assembly factors disrupting cytoplasmic pre-assembly of axonemal dynein motors, should be considered a cell-type specific protein-misfolding disease