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. PITT was achieved by pre-installing so called landing pad sequences (such as heterotypic LoxP sites or attP sites) to create seed mice and then injecting Cre recombinase or PhiC31 integrase mRNAs along with a compatible donor plasmid into zygotes derived from the seed mice. PITT and its subsequent version, improved PITT (i-PITT), overcome disadvantages of conventional Tg mice such as lack of consistent and reliable expression of the cassettes among different Tg mouse lines, and the PITT approach is superior in terms of cost and labor. One of the limitations of PITT, particularly using Cre-mRNA, is that the approach cannot be used for insertion of conditional expression cassettes using Cre-LoxP site-specific recombination. This is because the LoxP sites in the donor plasmids intended for achieving conditional expression of the transgene will interfere with the PITT recombination reaction with LoxP sites in the landing pad. RESULTS: To enable the i-PITT method to insert a conditional expression cassette, we modified the approach by simultaneously using PhiC31o and FLPo mRNAs. We demonstrate the strategy by creating a model containing a conditional expression cassette at the Rosa26 locus with an efficiency of 13.7%. We also demonstrate that inclusion of FLPo mRNA excludes the insertion of vector backbones in the founder mice. CONCLUSIONS: Simultaneous use of PhiC31 and FLP in i-PITT approach allows insertion of donor plasmids containing Cre-loxP-based conditional expression cassettes.

Cross-contamination of CRISPR guides and other unrelated nucleotide sequences among commercial oligonucleotides.

Custom oligonucleotides (oligos) are widely used reagents in biomedical research. Some common applications of oligos include polymerase chain reaction (PCR), sequencing, hybridization, microarray, and library construction. The reliability of oligos in such applications depends on their purity and specificity. Here, we report that commercially available oligos are frequently contaminated with nonspecific sequences (i.e. other unrelated oligonucleotides). Most of the oligos that we designed to amplify clustered regularly interspersed palindromic repeats (CRISPR) guide sequences contained nonspecific CRISPR guides. These contaminants were detected in research-grade oligos procured from eight commercial oligo-suppliers located in three different geographic regions of the world. Deep sequencing of some of the oligos revealed a variety of contaminants. Given the wide range of applications of oligos, the impact of oligo cross-contamination varies greatly depending on the field and the experimental method. Incorporating appropriate control experiments in research design can help ensure that the quality of oligo reagents meets the intended purpose. This can also minimize risk depending on the purposes for which the oligos are used.

Embrittlement Fracture Behavior and Mechanical Properties in Heat-Affected Zone of Welded Maraging Steel.

In welded maraging steels, mechanical properties, particularly ductility and toughness, are often compromised in the heat-affected zone (HAZ). This study focuses on 300-grade maraging steel bars, solution annealed at 1123 K for 1.5 h (5.4 ks) and welded using gas tungsten arc welding, followed by a post-weld heat treatment at 753 K for 13.33 h (48 ks). In situ observations during three-point bending tests on HAZ samples featuring coarsened prior austenite grain sizes were conducted to examine damage behavior and the crack path near the crack tip. The main crack initiated at the peak applied load during the bending test and, upon further loading, exhibited significant deflection and extension accompanied by numerous microcracks and localized crack branching. Distinctive damage features, such as transgranular cracking across block regions, intense intergranular cracking along packet boundaries with a pronounced shear component, and crowding of microcracks ahead of the crack tip, were observed in the HAZ sample during the in situ test. The interaction between the main crack tip and microcracks and its influence on the local crack propagation driving force was discussed using fracture mechanics. Experimental results, including tensile fracture surface observations and in situ images, along with analysis of the stress anti-shielding effect by microcracks, suggest that the HAZ sample exhibits embrittlement fracture behavior with lower ductility and toughness compared to the base metal sample.

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). Such experiments are performed by highly skilled technicians (especially for MI). We recently developed a novel genome editing method, called "GONAD (Genome-editing via Oviductal Nucleic Acids Delivery)," which can completely eliminate the ex vivo handling of embryos. We also made improvements to the GONAD method, termed "improved-GONAD (i-GONAD)." The i-GONAD method involves injection of CRISPR reagents into the oviduct of an anesthetized pregnant female using a mouthpiece-controlled glass micropipette under a dissecting microscope, followed by EP of the entire oviduct allowing the CRISPR reagents to enter into the zygotes present inside the oviduct, in situ. After the i-GONAD procedure, the mouse recovered from anesthesia is allowed to continue the pregnancy to full term to deliver its pups. The i-GONAD method does not require pseudopregnant female animals for embryo transfer, unlike the methods relying on ex vivo handling of zygotes. Therefore, the i-GONAD method can reduce the number of animals used, compared to the traditional methods. In this chapter, we describe some newer technical tips about the i-GONAD method. Additionally, even though the detailed protocols of GONAD and i-GONAD have been published elsewhere (Gurumurthy et al., Curr Protoc Hum Genet 88:15.8.1-15.8.12, 2016 Nat Protoc 14:2452-2482, 2019), we provide all the protocol steps of i-GONAD in this chapter so that the reader can find most of the information, needed for performing i-GONAD experiments, in one place.

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). Although the seminal work demonstrates the SEND process in cultured cells, it is unknown whether this phenomenon occurs in vivo . Here, we demonstrate the SEND process in living organisms using specially designed genetically engineered mouse models. Our proof of principle study lays a foundation for the SEND-VLP system to potentially be used as a gene therapy tool to deliver therapeutically important mRNAs to tissues.

CRISPR-KRISPR: a method to identify on-target and random insertion of donor DNAs and their characterization in knock-in mice.

CRISPR tools can generate knockout and knock-in animal models easily, but the models can contain off-target genomic lesions or random insertions of donor DNAs. Simpler methods to identify off-target lesions and random insertions, using tail or earpiece DNA, are unavailable. We develop CRISPR-KRISPR (CRISPR-Knock-ins and Random Inserts Searching PRotocol), a method to identify both off-target lesions and random insertions. CRISPR-KRISPR uses as little as 3.4 µg of genomic DNA; thus, it can be easily incorporated as an additional step to genotype founder animals for further breeding.

Application of multi-directionally forged high-strength titanium to dental implants in beagle dogs.

Pure titanium is widely used as a material in dental implants. However, it possesses inferior mechanical strength. This study aimed to elucidate the efficacy of acid treated multi-directionally forged (MDF) pure titanium in vivo. We verified the temporal changes until osseointegration in beagle dogs. Using two types of experimental materials (conventional pure titanium or MDF pure titanium), new bone formation was assessed using morphological examinations, and the bone-to-implant contact (BIC) value was evaluated at each time point (14, 30, and 90 days after the operation). As such, new bone formation was observed around the acid-etched MDF group, in which the BIC value was highest, followed by that in the acid-etched pure titanium group. MDF pure titanium implants showed early promotion of new bone formation compared to conventional titanium implants. The new acid-treated MDF made of pure titanium could be applied to humans in the future to prove its practicality.

Fitness accuracy and retentive forces of milled titanium clasp.

Since cast titanium prostheses have many drawbacks, multi-directionally forged titanium grade 2 (MDF) was developed, and the application of the milling process was proposed for improving the titanium clasp. This in vitro study evaluated milled titanium clasps, including MDF titanium. Milling clasps were manufactured with commercially pure (CP) titanium grade 2 (CP 2), grade 4 (CP 4), Ti-6-Al-4V, and MDF. As a control, a CP 2 cast titanium clasp was fabricated in the conventional manner. No porosities and catastrophic failures were observed in the four milled titanium clasps. Fitness accuracy and retentive forces of milled CP 2 and CP 4 tended to be worse, and the milled MDF showed the higher retentive forces (12.45 N) than did cast and milled CP 2 clasps (9.32 N and 4.42 N). Milled titanium clasps can be recommended for longer-term clinical use as compared to cast clasps.

Cyclin D1 Binding Protein 1 Responds to DNA Damage through the ATM-CHK2 Pathway.

Cyclin D1 binding protein 1 (CCNDBP1) is considered a tumor suppressor, and when expressed in tumor cells, CCNDBP1 can contribute to the viability of cancer cells by rescuing these cells from chemotherapy-induced DNA damage. Therefore, this study focused on investigating the function of CCNDBP1, which is directly related to the survival of cancer cells by escaping DNA damage and chemoresistance. Hepatocellular carcinoma (HCC) cells and tissues obtained from Ccndbp1 knockout mice were used for the in vitro and in vivo examination of the molecular mechanisms of CCNDBP1 associated with the recovery of cells from DNA damage. Subsequently, gene and protein expression changes associated with the upregulation, downregulation, and irradiation of CCNDBP1 were assessed. The overexpression of CCNDBP1 in HCC cells stimulated cell growth and showed resistance to X-ray-induced DNA damage. Gene expression analysis of CCNDBP1-overexpressed cells and Ccndbp1 knockout mice revealed that Ccndbp1 activated the Atm-Chk2 pathway through the inhibition of Ezh2 expression, accounting for resistance to DNA damage. Our study demonstrated that by inhibiting EZH2, CCNDBP1 contributed to the activation of the ATM-CHK2 pathway to alleviate DNA damage, leading to chemoresistance.

Novel reporter mouse models useful for evaluating in vivo 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 in vivo 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. Here, we developed and validated reporter mouse models containing a single copy of disrupted EGFP (ΔEGFP) via frameshift mutations. We tested several delivery methods for validation of the reporters, and we demonstrated their utility to assess both non-homologous end-joining (NHEJ) and via homology-directed repair (HDR) processes in embryos and in somatic tissues. With the use of the reporters, we also show that hydrodynamic delivery of ribonucleoprotein (RNP) with Streptococcus pyogenes (Sp)Cas9 protein mixed with synthetic guide RNA (gRNA) elicits better genome-editing efficiencies than the plasmid vector-based system in mouse liver. The reporters can also be used for assessing HDR efficiencies of the Acidaminococcus sp. (As)Cas12a nuclease. The results suggest that the ΔEGFP mouse models serve as valuable tools for evaluation of in vivo genome editing.

Application of multi-directional forged titanium for prosthetic crown fabrication by CAD/CAM.

Titanium are often used as dental materials, pure titanium present low strength and titanium alloy is reported poor biocompatibility, respectively. To overcome the problem, we fabricated high-strength multi-directional forged (MDF) titanium with improved mechanical properties without changing the chemical composition and evaluated its applicability in prosthetic crowns. Cutting tests: the average absolute value of the difference before and after cutting was calculated as the uncut amount. Surface evaluations: MDF titanium, pure titanium, and the Ti-6Al-4V alloy were the surface properties (the surface roughness, the contact angles, glossiness) of the samples were evaluated. The fitness test used digital data. These demonstrated that the good workability of high-strength MDF titanium. The surface-roughness and contact-angle properties of MDF titanium and pure titanium were similar. The fitness test showed no significant differences between MDF titanium and pure titanium crowns. These results suggest that MDF titanium is promising for fabricating prosthetic crowns in dental applications.

Diatretol, an α, α'-dioxo-diketopiperazine, is a potent in vitro and in vivo antimalarial.

A fungal metabolite, diatretol, has shown to be a promising antimalarial agent. Diatretol displayed potent in vitro antiparasitic activity against the Plasmodium falciparum K1 strain, with an IC50 value of 378 ng ml-1, as well as in vivo efficacy in a Plasmodium berghei-infected mice model, with ca. 50% inhibition at 30 mg/kg (p.o.).

Alopecia areata susceptibility variant in MHC region impacts expressions of genes contributing to hair keratinization and is involved in hair loss.

BACKGROUND: Alopecia areata (AA) is considered a highly heritable, T-cell-mediated autoimmune disease of the hair follicle. However, no convincing susceptibility gene has yet been pinpointed in the major histocompatibility complex (MHC), a genome region known to be associated with AA as compared to other regions. METHODS: We engineered mice carrying AA risk allele identified by haplotype sequencing for the MHC region using allele-specific genome editing with the CRISPR/Cas9 system. Finally, we performed functional evaluations in the mice and AA patients with and without the risk allele. FINDINGS: We identified a variant (rs142986308, p.Arg587Trp) in the coiled-coil alpha-helical rod protein 1 (CCHCR1) gene as the only non-synonymous variant in the AA risk haplotype. Furthermore, mice engineered to carry the risk allele displayed a hair loss phenotype. Transcriptomics further identified CCHCR1 as a novel component interacting with hair cortex keratin in hair shafts. Both, these alopecic mice and AA patients with the risk allele displayed morphologically impaired hair and comparable differential expression of hair-related genes, including hair keratin and keratin-associated proteins (KRTAPs). INTERPRETATION: Our results implicate CCHCR1 with the risk allele in a previously unidentified subtype of AA based on aberrant keratinization in addition to autoimmune events. FUNDING: This work was supported by JSPS KAKENHI (JP16K10177) and the NIHR UCLH Biomedical Research center (BRC84/CN/SB/5984).

Effect of Diphtheria Toxin-Based Gene Therapy for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a major global malignancy, responsible for >90% of primary liver cancers. Currently available therapeutic options have poor performances due to the highly heterogeneous nature of the tumor cells; recurrence is highly probable, and some patients develop resistances to the therapies. Accordingly, the development of a novel therapy is essential. We assessed gene therapy for HCC using a diphtheria toxin fragment A (DTA) gene-expressing plasmid, utilizing a non-viral hydrodynamics-based procedure. The antitumor effect of DTA expression in HCC cell lines (and alpha-fetoprotein (AFP) promoter selectivity) is assessed in vitro by examining HCC cell growth. Moreover, the effect and safety of the AFP promoter-selective DTA expression was examined in vivo using an HCC mice model established by the hydrodynamic gene delivery of the yes-associated protein (YAP)-expressing plasmid. The protein synthesis in DTA transfected cells is inhibited by the disappearance of tdTomato and GFP expression co-transfected upon the delivery of the DTA plasmid; the HCC cell growth is inhibited by the expression of DTA in HCC cells in an AFP promoter-selective manner. A significant inhibition of HCC occurrence and the suppression of the tumor marker of AFP and des-gamma-carboxy prothrombin can be seen in mice groups treated with hydrodynamic gene delivery of DTA, both 0 and 2 months after the YAP gene delivery. These results suggest that DTA gene therapy is effective for HCC.

Thy1 promoter activity in the Rosa26 locus in mice: lessons from Dre-rox conditional expression system.

The pronuclear injection (PI)-based targeted transgenesis (PITT) method allows the generation of targeted transgenic (Tg) mice wherein a single copy of a transgene is integrated into the Rosa26 locus following PI. The Rosa26 locus allows unbiased ubiquitous expression of integrated transgenes; however, it remains little known whether tissue-specific promoters retain their functional properties when placed at the Rosa26 locus. We evaluated tissue-specific activity and reproducibility of exogenous tissue-specific promoters targeted to the Rosa26 locus by generating Thy1-Dre/Dre reporter mice using PITT and assessed spatial expression patterns of the transgenes. The Thy1 promoter targeted to the Rosa26 locus appeared active in virtually all Purkinje cells in the cerebellum and hippocampus. However, mosaic expression of the transgene under the Thy1 promoter was observed in many other organs. This phenomenon was consistent in all the Tg lines generated by PITT, indicating a high degree of reproducibility for this experiment.

In vitro evaluation of a removable partial denture framework using multi-directionally forged titanium.

PURPOSE: This study evaluated the availability of multi-directionally forged (MDF) titanium (Ti) as a component of removable partial dentures (RPDs). MDF-Ti remarkably improved the mechanical properties of RPDs due to its ultrafine-grained structure. MATERIALS AND METHODS: The wear resistance, plaque adhesion, and machinability of MDF-Ti were tested. As controls, commercially pure (CP) titanium was used for wear, plaque adhesion, and machinability tests. For wear resistance, the volume losses of the titanium teeth before and after wear tests were evaluated. Plaque adhesion was evaluated by the assay of Streptococcus mutans. In the machinability test, samples were cut and ground by a steel fissure bur and carborundum (SiC) point. An unpaired t-test was employed for the analysis of the significant differences between MDF-Ti and the control in the results for each test. RESULTS: Wear resistance and plaque adherence of MDF-Ti similar to those of CP-Ti (P>.05) were indicated. MDF-Ti exhibited significantly larger volume loss than CP-Ti in all conditions except 100/30,000 g/rpm in machinability tests (P<.05). CONCLUSION: Although the wear resistance and plaque adherence of MDF-Ti were comparable to those of controls, MDF-Ti showed better machinability than did CP-Ti. MDF-Ti could be used as a framework material for RPDs.

Electrochemical property and corrosion behavior of multi-directionally forged titanium in fluoride solution.

Multi-directional forging (MDFing) can improve the various properties of metals and alloys due to the evolution of an ultrafine-grained structure. In the present study, electrochemical properties and corrosion behaviors in a fluoride solution of MDFed pure titanium (MDF-Ti) were evaluated by comparing with conventional coarse-grained pure titanium (Ti). The Eopen value of MDF-Ti was significantly higher than that of Ti. However, similar potentiodynamic polarization profiles were obtained for Ti and MDF-Ti. Immersion in NaF solution caused no severe corrosion to Ti or MDF-Ti. However, immersion in acidulate phosphate fluoride solution (APF) revealed that MDF-Ti had better corrosion resistance than Ti at shorter time immersion periods and was more susceptible to corrosion for longer immersion. Significantly less release of titanium was observed for MDF-Ti in shorter immersion periods in APF. In conclusion, MDF-Ti showed similar electrochemical behaviors to Ti and less susceptible to corrosion in shorter time APF immersion.

Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD.

Methods to create genetically engineered mice involve three major steps: harvesting embryos from one set of females, microinjection of reagents into embryos ex vivo and their surgical transfer to another set of females. Although tedious, these methods have been used for more than three decades to create mouse models. We recently developed a method named GONAD (genome editing via oviductal nucleic acids delivery), which bypasses these steps. GONAD involves injection of CRISPR components (Cas9 mRNA and guide RNA (gRNA)) into the oviducts of pregnant females 1.5 d post conception, followed by in vivo electroporation to deliver the components into the zygotes in situ. Using GONAD, we demonstrated that target genes can be disrupted and analyzed at different stages of mouse embryonic development. Subsequently, we developed improved GONAD (i-GONAD) by delivering CRISPR ribonucleoproteins (RNPs; Cas9 protein or Cpf1 protein and gRNA) into day-0.7 pregnant mice, which made it suitable for routine generation of knockout and large-deletion mouse models. i-GONAD can also generate knock-in models containing up to 1-kb inserts when single-stranded DNA (ssDNA) repair templates are supplied. i-GONAD offers other advantages: it does not require vasectomized males and pseudo-pregnant females, the females used for i-GONAD are not sacrificed and can be used for other experiments, it can be easily adopted in laboratories lacking sophisticated microinjection equipment, and can be implemented by researchers skilled in small-animal surgery but lacking embryo-handling skills. Here, we provide a step-by-step protocol for establishing the i-GONAD method. The protocol takes ∼6 weeks to generate the founder mice.

Observation of Morphology Changes of Fine Eutectic Si Phase in Al-10%Si Cast Alloy during Heat Treatment by Synchrotron Radiation Nanotomography.

A series of three-dimensional morphology changes of fine eutectic Si-particles during heat treatment have been investigated in Self-modified and Sr-modified Al-10%Si cast alloys by means of synchrotron radiation nanotomography utilizing a Fresnel zone plate and a Zernike phase plate in this study. The coral-like shape particles observed in Sr-modified cast alloy fragmented at branch and neck during heat treatment at 773 K. The fragmentation occurred up to 900 s. After that, the fragmented particles grew and spheroidized by Ostwald ripening. On the other hand, rod-like shaped eutectic Si-particles observed in self-modified cast alloy were larger in size compared with the particle size in Sr-modified cast alloy. Separation of eutectic Si-particles in Self-modified cast alloy occurred up to approximately 900 s, which was similar tendency to that in Sr-modified cast alloy. However, it was found that the morphology change behavior was very complex in rod-like shape Si-particles. The three-dimensional morphology changes of fine eutectic Si-particles in both cast alloys, specifically fragmentation and spheroidizing, can be connected to changes in mechanical properties.

i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases.

We present a robust method called improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) that delivers CRISPR ribonucleoproteins to E0.7 embryos via in situ electroporation. The method generates mouse models containing single-base changes, kilobase-sized deletions, and knock-ins. The efficiency of i-GONAD is comparable to that of traditional microinjection methods, which rely on ex vivo handling of zygotes and require recipient animals for embryo transfer. In contrast, i-GONAD avoids these technically difficult steps, and it can be performed at any laboratory with simple equipment and technical expertise. Further, i-GONAD-treated females retain reproductive function, suggesting future use of the method for germline gene therapy.