Rie Ohga

Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish

The CRISPR/Cas9 system provides a powerful tool for genome editing in various model organisms, including zebrafish. The establishment of targeted gene-disrupted zebrafish (knockouts) is readily achieved by CRISPR/Cas9-mediated genome modification. Recently, exogenous DNA integration into the zebrafish genome via homology-independent DNA repair was reported, but this integration contained various mutations at the junctions of genomic and integrated DNA. Thus, precise genome modification into targeted genomic loci remains to be achieved. Here, we describe efficient, precise CRISPR/Cas9-mediated integration using a donor vector harbouring short homologous sequences (10–40 bp) flanking the genomic target locus. We succeeded in integrating with high efficiency an exogenous mCherry or eGFP gene into targeted genes (tyrosinase and krtt1c19e) in frame. We found the precise in-frame integration of exogenous DNA without backbone vector sequences when Cas9 cleavage sites were introduced at both sides of the left homology arm, the eGFP sequence and the right homology arm. Furthermore, we confirmed that this precise genome modification was heritable. This simple method enables precise targeted gene knock-in in zebrafish.

Efficient Multiple Genome Modifications Induced by the crRNAs, tracrRNA and Cas9 Protein Complex in Zebrafish

The type II clustered regularly interspaced short palindromic repeats (CRISPR) associated with Cas9 endonuclease (CRISPR/Cas9) has become a powerful genetic tool for understanding the function of a gene of interest. In zebrafish, the injection of Cas9 mRNA and guide-RNA (gRNA), which are prepared using an in vitro transcription system, efficiently induce DNA double-strand breaks (DSBs) at the targeted genomic locus. Because gRNA was originally constructed by fusing two short RNAs CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), we examined the effect of synthetic crRNAs and tracrRNA with Cas9 mRNA or Cas9 protein on the genome editing activity. We previously reported that the disruption of tyrosinase (tyr) by tyr-gRNA/Cas9 mRNA causes a retinal pigment defect, whereas the disruption of spns2 by spns2-gRNA1/Cas9 mRNA leads to a cardiac progenitor migration defect in zebrafish. Here, we found that the injection of spns2-crRNA1, tyr-crRNA and tracrRNA with Cas9 mRNA or Cas9 protein simultaneously caused a migration defect in cardiac progenitors and a pigment defect in retinal epithelial cells. A time course analysis demonstrated that the injection of crRNAs and tracrRNA with Cas9 protein rapidly induced genome modifications compared with the injection of crRNAs and tracrRNA with Cas9 mRNA. We further show that the crRNA-tracrRNA-Cas9 protein complex is functional for the visualization of endogenous gene expression; therefore, this is a very powerful, ready-to-use system in zebrafish.

Effect of resin use in the post-embedding procedure on immunoelectron microscopy of membranous antigens, with special reference to sensitivity.

To investigate quantitatively the effect of resins on the sensitivity of immunoelectron microscopy of membranous antigen, ultra-thin sections of bovine epithelial tissue embedded in five different kinds of resins [JB-4 (JB4), LR Gold (LRG), Lowicryl K4M (K4M), Quetol 812 (Q812), and Spurrs (Spurr) resin] were labeled specifically with anti-desmosomal glycoprotein I(DGI) antibody followed by protein A-gold (PAG) conjugates. When we compared the labeling intensity expressed as the number of PAG particles per 500-nm length of the desmosomal region along the membrane, three hydrophilic resins (JB4, LRG, and K4M) showed much greater levels of labeling intensity than did epoxy resins (Q812 and Spurr), which had a negative value. The three hydrophilic resins showed only minor differences in their levels of labeling intensity. The intensity obtained with JB4, which was the highest of the three, was further increased by pretreatment of the ultra-thin sections with methyl methacrylate monomer (MM) for 5 min. On the basis of these results, wide applicability of this new technique for membranous antigens, which have been difficult to detect positively by any previously employed techniques, is suggested.

Functional visualization and disruption of targeted genes using CRISPR/Cas9-mediated eGFP reporter integration in zebrafish.

The CRISPR/Cas9 complex, which is composed of a guide RNA (gRNA) and the Cas9 nuclease, is useful for carrying out genome modifications in various organisms. Recently, the CRISPR/Cas9-mediated locus-specific integration of a reporter, which contains the Mbait sequence targeted using Mbait-gRNA, the hsp70 promoter and the eGFP gene, has allowed the visualization of the target gene expression. However, it has not been ascertained whether the reporter integrations at both targeted alleles cause loss-of-function phenotypes in zebrafish. In this study, we have inserted the Mbait-hs-eGFP reporter into the pax2a gene because the disruption of pax2a causes the loss of the midbrain-hindbrain boundary (MHB) in zebrafish. In the heterozygous Tg[pax2a-hs:eGFP] embryos, MHB formed normally and the eGFP expression recapitulated the endogenous pax2a expression, including the MHB. We observed the loss of the MHB in homozygous Tg[pax2a-hs:eGFP] embryos. Furthermore, we succeeded in integrating the Mbait-hs-eGFP reporter into an uncharacterized gene epdr1. The eGFP expression in heterozygous Tg[epdr1-hs:eGFP] embryos overlapped the epdr1 expression, whereas the distribution of eGFP-positive cells was disorganized in the MHB of homozygous Tg[epdr1-hs:eGFP] embryos. We propose that the locus-specific integration of the Mbait-hs-eGFP reporter is a powerful method to investigate both gene expression profiles and loss-of-function phenotypes.

Sangivamycin induces apoptosis by suppressing Erk signaling in primary effusion lymphoma cells

Sangivamycin, a structural analog of adenosine and antibiotic exhibiting antitumor and antivirus activities, inhibits protein kinase C and the synthesis of both DNA and RNA. Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposis sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients and HIV-infected homosexual males. PEL cells are derived from post-germinal center B cells, and are infected with KSHV. Herein, we asked if sangivamycin might be useful to treat PEL. We found that sangivamycin killed PEL cells, and we explored the underlying mechanism. Sangivamycin treatment drastically decreased the viability of PEL cell lines compared to KSHV-uninfected B lymphoma cell lines. Sangivamycin induced the apoptosis of PEL cells by activating caspase-7 and -9. Further, sangivamycin suppressed the phosphorylation of Erk1/2 and Akt, thus inhibiting activation of the proteins. Inhibitors of Akt and MEK suppressed the proliferation of PEL cells compared to KSHV-uninfected cells. It is known that activation of Erk and Akt signaling inhibits apoptosis and promotes proliferation in PEL cells. Our data therefore suggest that sangivamycin induces apoptosis by inhibiting Erk and Akt signaling in such cells. We next investigated whether sangivamycin, in combination with an HSP90 inhibitor geldanamycin (GA) or valproate (valproic acid), potentiated the cytotoxic effects of the latter drugs on PEL cells. Compared to treatment with GA or valproate alone, the addition of sangivamycin enhanced cytotoxic activity. Our data thus indicate that sangivamycin may find clinical utility as a novel anti-cancer agent targeting PEL.

Maternal and Zygotic Sphingosine Kinase 2 Are Indispensable for Cardiac Development in Zebrafish.

Background: Developmental functions of Sphk1 and Sphk2 remain unclear in vertebrates. Results: Maternal-zygotic sphk2 zebrafish mutant exhibited cardia bifida, whereas maternal-zygotic sphk1, maternal sphk2, and zygotic sphk2 mutants did not. Conclusion: Maternal and zygotic Sphk2 cooperatively regulate cardiac development. Significance: The contribution of maternally supplied lipid mediators presents as a critical requirement for maternal-zygotic Sphk2 during cardiac development. Sphingosine 1-phosphate (S1P) is synthesized from sphingosine by sphingosine kinases (SPHK1 and SPHK2) in invertebrates and vertebrates, whereas specific receptors for S1P (S1PRs) selectively appear in vertebrates, suggesting that S1P acquires novel functions in vertebrates. Because the developmental functions of SPHK1 and SPHK2 remain obscure in vertebrates, we generated sphk1 or sphk2 gene-disrupted zebrafish by introducing premature stop codons in their coding regions using transcription activator-like effector nucleases. Both zygotic sphk1 and sphk2 zebrafish mutants exhibited no obvious developmental defects and grew to adults. The maternal-zygotic sphk2 mutant (MZsphk2), but not the maternal-zygotic sphk1 mutant and maternal sphk2 mutant, had a defect in the cardiac progenitor migration and a concomitant decrease in S1P level, leading to a two-heart phenotype (cardia bifida). Cardia bifida in MZsphk2, which was rescued by injecting sphk2 mRNA, was a phenotype identical to that of zygotic mutants of the S1P transporter spns2 and S1P receptor s1pr2, indicating that the Sphk2-Spns2-S1pr2 axis regulates the cardiac progenitor migration in zebrafish. The contribution of maternally supplied lipid mediators during vertebrate organogenesis presents as a requirement for maternal-zygotic Sphk2.

The Ubiquitin System and Kaposi's Sarcoma-Associated Herpesvirus.

Ubiquitination is a post-translational modification in which one or more ubiquitin molecules are covalently linked to lysine residues of target proteins. The ubiquitin system plays a key role in the regulation of protein degradation, which contributes to cell signaling, vesicular trafficking, apoptosis, and immune regulation. Bacterial and viral pathogens exploit the cellular ubiquitin system by encoding their own proteins to serve their survival and replication in infected cells. Recent studies have revealed that Kaposi’s sarcoma-associated herpesvirus (KSHV) manipulates the ubiquitin system of infected cells to facilitate cell proliferation, anti-apoptosis, and evasion from immunity. This review summarizes recent developments in our understanding of the molecular mechanisms used by KSHV to interact with the cellular ubiquitin machinery.

Comprehensive analysis of sphingosine-1-phosphate receptor mutants during zebrafish embryogenesis

The lipid mediator sphingosine‐1‐phosphate (S1P) regulates various physiological and pathological phenomena such as angiogenesis and oncogenesis. Secreted S1P associates with the G‐protein‐coupled S1P receptors (S1PRs), leading to the activation of downstream signaling molecules. In mammals, five S1prs have been identified and the genetic disruption of a single S1pr1 gene causes vascular defects. In zebrafish, seven s1prs have been isolated. We found that individual s1prs showed unique expression patterns with some overlapping expression domains during early embryogenesis. We generated all s1pr single‐mutant zebrafish by introducing premature stop codons in their coding regions using transcription activator‐like effector nucleases and analyzed their phenotypes during early embryogenesis. Zygotic s1pr1, s1pr3a, s1pr3b, s1pr4, s1pr5a and s1pr5b mutants showed no developmental defects and grew into adults, whereas zygotic s1pr2 mutant showed embryonic lethality with a cardiac defect, showing quite distinct embryonic phenotypes for individual S1pr mutants between zebrafish and mouse. We further generated maternal‐zygotic s1pr1, s1pr3a, s1pr3b, s1pr4, s1pr5a and s1pr5b mutants and found that these maternal‐zygotic mutants also showed no obvious developmental defects, presumably suggesting the redundant functions of the S1P receptor‐mediated signaling in zebrafish.

Spatiotemporal expression of the cocaine- and amphetamine-regulated transcript-like (cart-like) gene during zebrafish embryogenesis

The cocaine- and amphetamine-regulated transcript (CART) genes are involved in the neural regulation of energy homeostasis; however, their developmental expressions and functions are not fully understood in vertebrates. We have identified a novel zebrafish cart-like gene that encodes a protein of 105 amino acids possessing sequence similarity to zebrafish and mammalian CART proteins. RT-PCR analysis revealed that the cart-like transcripts were maternally supplied and gradually decreased during the cleavage, blastula and gastrula stages; then, transcripts subsequently reaccumulated at the segmentation, pharyngula and hatching stages. Based on a whole-mount in situ hybridization analysis using an antisense cart-like RNA probe, we found that the cart-like transcript was predominantly expressed in both the Rohon-Beard neurons and trigeminal ganglia, suggesting the involvement of the cart-like gene in zebrafish neural development.

Developmental expression of the slurp-like1/ly2.3/ly97.3 and slurp-like2/ly2.2/ly97.2 genes during zebrafish early embryogenesis

Mammalian SLURP1 and SLURP2 belong to the Ly-6/uPAR superfamily and are involved in maintaining the physiological integrity of keratinocytes. However, the developmental expression and functions of other Ly-6/uPAR family genes in vertebrates are still obscure. We have isolated novel Ly-6/uPAR family genes slurp-like1 (ly2.3/ly97.3) and slurp-like2 (ly2.2/ly97.2) in zebrafish. Both the Slurp-like1 and Slurp-like2 proteins contain the typical signal sequence and carboxy-terminal CCXXXXCN (X: an arbitrary amino acid) consensus sequence of the Ly-6/uPAR family but lack a transmembrane domain and a GPI-anchoring signal sequence, suggesting that both proteins may function as secretory proteins. Whole-mount in situ hybridization analysis revealed that slurp-like1 was predominantly expressed in the floor plate of the neural tube and in the hypochord of the notochord at 24 h post-fertilization (hpf) and detected in the liver and intestinal bulb at 72 hpf, while slurp-like2 was expressed in the midbrain and hindbrain at 24 hpf and detected in the liver and pancreas at 72 hpf. Differential expression profiles of the slurp-like1 and slurp-like2 genes suggest the distinct physiological involvement of these genes in zebrafish early embryogenesis.