Design of Structured Surfaces for Directional Mobility of Droplets

This paper deals with the directional mobility of droplets on structured surfaces. Structured surfaces were micro-patterned with rectangular lines and spaces of varying pitch and height in the sub-millimeter range. The material used was polydimethylsiloxane, which is hydrophobic and wettable by oil. First, we studied the effect of the structural design on the sliding angle of pure water or oil through experiments. For pure water droplets, we found that a wider pitch enhanced the directionality. On the other hand, oil droplets spread along the groove because of their low surface tension and strong capillary force. The directionality of the sliding angle of oil droplets was larger than that of pure water, especially when the groove was narrower and deeper. Second, we poured a large amount of liquid on the structure and evaluated the removal rate on the tilted surface. We found that a parallel structure enhanced the liquid mobility for both pure water and oil

Deficiency of the RIβ subunit of protein kinase A causes body tremor and impaired fear conditioning memory in rats

The RIβ subunit of cAMP-dependent protein kinase (PKA), encoded by Prkar1b, is a neuronal isoform of the type I regulatory subunit of PKA. Mice lacking the RIβ subunit exhibit normal long-term potentiation (LTP) in the Schaffer collateral pathway of the hippocampus and normal behavior in the open-field and fear conditioning tests. Here, we combined genetic, electrophysiological, and behavioral approaches to demonstrate that the RIβ subunit was involved in body tremor, LTP in the Schaffer collateral pathway, and fear conditioning memory in rats. Genetic analysis of WTC-furue, a mutant strain with spontaneous tremors, revealed a deletion in the Prkar1b gene of the WTC-furue genome. Prkar1b-deficient rats created by the CRISPR/Cas9 system exhibited body tremor. Hippocampal slices from mutant rats showed deficient LTP in the Schaffer collateral–CA1 synapse. Mutant rats also exhibited decreased freezing time following contextual and cued fear conditioning, as well as increased exploratory behavior in the open field. These findings indicate the roles of the RIβ subunit in tremor pathogenesis and contextual and cued fear memory, and suggest that the hippocampal and amygdala roles of this subunit differ between mice and rats and that rats are therefore beneficial for exploring RIβ function

Successful Long-Term Preservation of Rat Sperm by Freeze-Drying

Background: Freeze-drying sperm has been developed as a new preservation method where liquid nitrogen is no longer necessary. An advantage of freeze-drying sperm is that it can be stored at 4uC and transported at room temperature. Although the successful freeze-drying of sperm has been reported in a number of animals, the possibility of long-term preservation using this method has not yet been studied. Methodology/Principal Findings: Offspring were obtained from oocytes fertilized with rat epididymal sperm freeze-dried using a solution containing 10 mM Tris and 1 mM EDTA adjusted to pH 8.0. Tolerance of testicular sperm to freeze-drying was increased by pre-treatment with diamide. Offspring with normal fertility were obtained from oocytes fertilized with freeze-dried epididymal sperm stored at 4uC for 5 years. Conclusions and Significance: Sperm with –SS – cross-linking in the thiol-disulfide of their protamine were highly tolerant to freeze-drying, and the fertility of freeze-dried sperm was maintained for 5 years without deterioration. This is the first report to demonstrate the successful freeze-drying of sperm using a new and simple method for long-term preservation

マイクロプレート オ モチイタ オス シバヤギ ケッショウ チュウ 5αジヒドロテストステロン ノ コウソ メンエキ ソクテイ

雄シバヤギ血漿中5α-ジヒドロテストステロン(5α-DHT)に関する酵素免疫測定法(EIA)について検討した。抗血清は抗5α-DHT-11α-Succinate-BSAを,酵素標識ホルモンには5α-DHT-11α-Succinate-peroxidase(5α-DHT-HRP)を用いた。抗血清は100,000倍に希釈・使用が可能であった。また,抗血清にはテストステロンが30%交叉反応するため,Bond Elut CN-Uを用いたカラムクロマトグラフィーによる精製を行い,被検血漿中の5α-DHTとテストステロンを分離・測定した。その結果,酢酸エチル : ベンゼン=2 : 98の展開液を流したところ,1.00-4.25mlの範囲に5α-DHTが溶出された。添加回収試験において添加量0.1-1.0pgの各濃度での回収率は,平均100.45%±2.13となった。再現性試験における頸静脈血および精巣静脈血の測定内変動係数(n=6)は各々6.38%および5.94%となり,測定間変動係数は8.36%ならびに11.53%となった。以上の結果から,雄シバヤギの血漿中5α-DHT濃度を,本法を用いて測定することが可能であることを明らかにした。Enzymeimmunoassay (EIA) for 5α-dihydrotestosterone (5α-DHT) in male Shiba-goat blood plasma was examined. The antiserum used 5α-DHT-11α-succinate-peroxidase (5α-DHT-HRP) for the enzyme labeling hormone in respect of 5α-DHT-11α-succinate-BSA. The use was possible for the antiserum at 100,000 times. The 5α-DHT in plasma was purified and separated from the testosterone by column chromatography using Bond Elut CN-U, since antiserum for 5α-DHT had cross reaction (30%) to the testosterone. The 5α-DHT was washed away by the development liquid of ethyl acetate: benzene=2 : 98 and was collected in the range between 1.00 and 4.25ml. Recovery rates of 5α-DHT each concentration of the addition 0.1～1ng to Shiba-goat plasma were 100.45%±2.13 of the averages. Inter-assay coefficient of variation (C.V.) became respectively 6.38% and 5.94% in jugular and testicular vein blood,while for intra-assay, they became 8.36% and 11.53%. It was possible to analyse 5α-DHT concentration in blood plasma of the male Shiba-goat from the above result using this method

マイクロプレート オ モチイタ オス シバヤギ ケッショウ チュウ テストステロン ノ コウソ メンエキ ソクテイ ホウ ノ ケントウ

Testosterone-3(E)-carboxymethyloxime-BSAを抗原として作成した抗体,ならびに酵素標識ホルモンとしてTestosterone-3(E)-carboxymethyloxime-peroxidaseを使用し,雄シバヤギにおける血漿中テストステロン濃度の酵素免疫測定法(EIA)を検討した。本実験では高い測定感度が得られる二抗体法を用い,作成した抗血清は,350,000倍に希釈しても使用可能な高い力価を有していた。血漿に10～100pgのテストステロンを添加した添加回収試験では,回収率が平均102.8±2.8%となった。測定内変動係数(N=6)は雄シバヤギ頸静脈血漿で5.08%,精索静脈血漿では7.32%,測定間変動係数(N=6)は,頸静脈血漿で5.74%,精索静脈血漿で6.13%であった。以上の結果から,本方法によって雄シバヤギ血漿中テストステロンの測定が可能となった。Enzymeimmunoassay (EIA) of testosterone was examined in which an individual antibody, and testosterone-peroxidase-conjugate were used for that, was measured in male Shiba-goat plasma. An anti-testosterone-3(E)-carboxymethyloxime-BSA antibody was used as an anti-serum, and testosterone-3(E)-carboxymethyloxime-peroxidase was used as a steroid-enzyme conjugate. The anti-serum was diluted by using 2nd antibody method which could get high measurement sensitivity of 350,000 times. Recovery rates of testosterone for each concentration with the addition of 10～100pg to Shiba-goat plasma were 102.8±2.8% of the averages. Inter-assay coefficient of variation (C.V.) for testosterone level from jugular and testicular vein blood plasma samples were 5.08% and 7.32% respectively, as for intra-assay, they became 5.74% and 6.13%. These results suggest that the EIA method is extremely suitable to measure testosterone concentration in blood plasma of the male Shiba-goat

Simple genome editing of rodent intact embryos by electroporation

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-Associated (Cas) system is a powerful tool for genome editing in animals. Recently, new technology has been developed to genetically modify animals without using highly skilled techniques, such as pronuclear microinjection of endonucleases. Technique for animal knockout system by electroporation (TAKE) method is a simple and effective technology that produces knockout rats by introducing endonuclease mRNAs into intact embryos using electroporation. Using TAKE method and CRISPR/Cas system, the present study successfully produced knockout and knock-in mice and rats. The mice and rats derived from embryos electroporated with Cas9 mRNA, gRNA and single-stranded oligodeoxynucleotide (ssODN) comprised the edited targeted gene as a knockout (67% of mice and 88% of rats) or knock-in (both 33%). The TAKE method could be widely used as a powerful tool to produce genetically modified animals by genome editing

Simple knockout by electroporation of engineered endonucleases into intact rat embryos.

超簡単！遺伝子改変動物作製法の開発 -エレクトロポレーション（電気穿孔）法による哺乳類受精卵への ZFN、TALEN、CRISPR-Casの導入に成功-. 京都大学プレスリリース. 2014-10-03.Engineered endonucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, provide a powerful approach for genome editing in animals. However, the microinjection of endonucleases into embryos requires a high skill level, is time consuming, and may cause damage to embryos. Here, we demonstrate that the electroporation of endonuclease mRNAs into intact embryos can induce editing at targeted loci and efficiently produce knockout rats. It is noteworthy that the electroporation of ZFNs resulted in an embryonic survival rate (91%) and a genome-editing rate (73%) that were more than 2-fold higher than the corresponding rates from conventional microinjection. Electroporation technology provides a simple and effective method to produce knockout animals

In vitro maturation of immature rat oocytes under simple culture conditions and subsequent developmental ability

Rat oocytes can be produced artificially by superovulation. Because some strains show low sensitivity to superovulation treatment, in vitro maturation is an alternative method to produce numerous matured oocytes. Furthermore, establishment of an in vitro maturation system with simple culture conditions is cost effective and leads to easy handling of oocytes. This study examined developmental ability of rat germinal vesicle (GV) oocytes maturing in vitro under simple culture conditions. Significantly different numbers of ovulated oocytes reached the second metaphase of meiosis (MII) among Jcl:Wistar (17.0), F344/Stm (31.0), and BN/SsNSlc (2.2) rats in whom superovulation was induced by pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin. However, similar numbers of GV oocytes were obtained from ovaries of PMSG-injected Wistar (27.7), F344 (34.7), and BN (24.7) rats. These GV oocytes were cultured in vitro in HTF, αMEM, and a 1:1 HTF + αMEM or TYH + αMEM mixture. High proportions of Wistar and F344 oocytes that matured to MII in αMEM were parthenogenetically activated by strontium chloride treatment (78% and 74%, respectively). Additionally, 10% of matured oocytes of both strains developed into offspring after intracytoplasmic sperm injection and embryo transfer to foster mothers. Although BN oocytes cultured in αMEM could be parthenogenetically activated and developed into offspring, the success rate was lower than that for Wistar and F344 oocytes. This study demonstrated that numerous GV oocytes were produced in rat ovaries by PMSG injection. This simple in vitro maturation system of immature oocytes could be further developed to maintain valuable rat strains experiencing reproductive difficulties