Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations

Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B₄ (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types.     

Phase Relations and Volume Changes of Hafnia under High Pressure and High Temperature

Using multi-anvil high-pressure devices and synchrotron radiation, X-ray in situ observations of HfO₂ under high pressure and high temperature have been performed to investigate its phase relations and compression behavior. An orthorhombic phase (orthoI) is stable from 4 to 14.5 GPa below 1250°–1400°C and transforms to a tetragonal phase, which is one of the high-temperature forms of HfO₂, above these temperatures. Another orthorhombic phase (orthoII) with a cotunnite-type structure appears above 14.5 GPa. OrthoII is stable up to 1800°C at 21 GPa. OrthoII is quenchable to ambient conditions. The orthoI-to-orthoII transition is accompanied by ∼8 vol% decrease. The bulk moduli of orthoI and orthoII at room temperature are 220 and 312 GPa, respectively. This low compressibility of orthoII indicates that it is a potential candidate for very hard materials.     

Dehydrogenative polycondensation of telechelically bis(dimethylsilyl)functionalized oligo(dimethylsiloxane)s or aromatics and 1,3,3,5,5,7,7,7-octamethyltetrasiloxane

Telechelically bis(hydrodimethylsilyl)-functionalized oligo(dimethylsiloxane)s or aromatics were found to give polymers constituting of alternating disilylene and oligodimethylsiloxane or 4,4′-diylaromatics as repeating units by dehydrogenative coupling reaction in the presence of palladium dibenzylideneacetone complex. The same catalyst gave polysilylenes with trisiloxane pendant from 1,1-dihydro-1,3,3,5,5,7,7,7-octamethyltetrasiloxane. Received: 18 October 1996/Accepted: 31 October 1996     

Dependence of Lanthanide-Ion Binding Performance on HDEHP Concentration in HDEHP Impregnation to Porous Sheet

An octadecylamino-group-introduced polymer chain grafted onto a porous sheet was impregnated with bis(2-ethylhexyl)hydrogen phosphate (HDEHP). A mixture of HDEHP and ethanol of various HDEHP concentrations was used for the impregnation. The porous sheet into which a C₁₈H₃₇NH group was introduced was immersed in HDEHP/ethanol solution before ethanol evaporation. The liquid permeability of a cartridge charged with the HDEHP-impregnated porous sheet in disk form prepared in 50 (v/v)% HDEHP/ethanol solution was 96% that of the starting-porous-disk-packed cartridge. The equilibrium binding capacity of the HDEHP-impregnated porous disk for yttrium ions was 0.32 mol per kg of the disk. In addition, the HDEHP-impregnated-porous-disc-packed cartridge was found to be applicable to the preconcentration of trace amounts of lanthanides in a multielement solution prior to their measurement by inductively coupled plasma mass spectrometry.     

Relationship Between the B-Ring Hydroxylation Pattern of Condensed Tannins and their Protein-Precipitating Capacity

A series of condensed tannin derivatives with non-, mono-, di- and tri-hydroxylated B-rings were synthesized starting from phloroglucinol and benzaldehyde derivatives. The protein-precipitating capacity of these condensed tannin derivatives showed 1) A condensed tannin with only a 4′-hydroxylated B-ring has almost the same protein-precipitating capacity as that of a condensed tannin with 3′,4′-dihydroxylated or 3′,4′,5′-trihydroxylated B-rings. 2) The complexing ability of phenolic hydroxyl groups in the B-ring are effective in the order of p- > m- > o-positions.     

Core-Shell Type Polymer Microspheres Prepared from Diblock Copolymer Films

In the first part of this article, the preparation method for core-shell type polymer microspheres from diblock copolymer films is reviewed. The core-shell type polymer microspheres were obtained from the crosslinking reaction in spherical microdomains formed by diblock copolymer film. In the second section, the solution and solid properties of core-shell type microspheres are reviewed. The micelle of core-shell type microsphere moved like pseudolatex. Its solubility depended strongly on that of shell segments. Finally, the miscibility behaviors of binary core-shell type microsphere blends are reported briefly.     

Star-Block Copolymers: Organized Polymerization of Diblock Macromonomers

In the first part of this article, the method for preparation of heteroarm star (A _n B _n star-block) copolymers from diblock macromonomers possessing central functional groups is reviewed. These diblock macromonomers formed a microphase-separated structure in the solid state. The central functional groups at the position of the block junction were located regularly at the domain interface. The microgelation of diblock copolymer films formed A _n B _n star-block copolymers by organization effects. The second section reviews the methods for preparation of (AB) _n star-block copolymers from diblock macromonomers possessing a terminal vinylbenzyl group. The microgelation in micelles between diblock macromonomers and linking agent also formed (AB) _n star-block copolymers. Finally, the phase stability criteria of these star-block copolymers are reported briefly.     

Diffusional behavior of multi-arm star polymers by H pulsed field gradient spin-echo NMR method

The diffusional behavior of multi-arm star-shaped p(tBMA) was investigated in a concentration range from dilute to semidilute region with ¹H pulsed field gradient spin-echo NMR (PFGSE-NMR). An 142-arm star-shaped p(tBMA) showed two diffusional mode, which reflected the coexistence of liquid ordering phase and liquid phase near the ordering transition. On the other hand, for star-shaped p(tBMA) with 55 arms showed a single diffusional relaxation in all concentration ranges during observation time. In the semidilute region, the relationship between the diffusion coefficient against the polymer concentration was affected strongly not only the arm number but also liquid ordering structure.     

Effects of Annealing on Dielectric Loss and Microstructure of Aluminum Nitride Ceramics

The effect of annealing on tan δ and microstructures of aluminum nitride (AlN) ceramics were explored. Yttria was added as a sintering additive to AlN powders, and the powders were pressureless-sintered at 1900°C for 2 h in a nitrogen flow atmosphere. In succession to sintering, AlN samples were annealed at 720, 970 and 1210°C for 2 and 4 h. Very low tan δ values between 2.6 and 6.0 × 10⁻⁴ at 28 GHz were obtained when the AlN samples were annealed for 4 h at all the annealing temperatures.     

Sintering Kinetics at Constant Rates of Heating: Effect of Al2O3 on the Initial Sintering Stage of Fine Zirconia Powder

The sinterabilities of fine zirconia powders including 5 mass% Y₂O₃ were investigated, with emphasis on the effect of Al₂O₃ at the initial sintering stage. The shrinkage of powder compact was measured under constant rates of heating (CRH). The powder compact including a small amount of Al₂O₃ increased the densification rate with elevating temperature. The activation energies at the initial stage of sintering were determined by analyzing the densification curves. The activation energy of powder compact including Al₂O₃ was lower than that of a powder compact without Al₂O₃. The diffusion mechanisms at the initial sintering stage were determined using the new analytical equation applied for CRH techniques. This analysis exhibited that Al₂O₃ included in a powder compact changed the diffusion mechanism from grain boundary to volume diffusions (VD). Therefore, it is concluded that the effect of Al₂O₃ enhanced the densification rate because of decrease in the activation energy of VD at the initial sintering stage.