Generation of PDX‐1 mutant porcine blastocysts by introducing CRISPR/Cas9‐system into porcine zygotes via electroporation     

Fuminori Tanihara  Maki Hirata  Nhien T. Nguyen  Quynh A. Le  Takayuki Hirano  Tatsuya Takemoto  Michiko Nakai  Dai‐ichiro Fuchimoto  Takeshige Otoi 《Animal Science Journal》2019,90(1):55-61

Recently, we established the GEEP (“gene editing by electroporation of Cas9 protein”) method, in which the CRISPR/Cas9 system, consisting of a Cas9 protein and single guide RNA (sgRNA), is introduced into pig zygotes by electroporation and thus induces highly efficient targeted gene disruption. In this study, we examined the effects of sgRNA on the blastocyst formation of porcine embryos and evaluated their genome‐editing efficiency. To produce an animal model for diabetes, we targeted PDX‐1 (pancreas duodenum homeobox 1), a gene that is crucial for pancreas development during the fetal period and whose monoallelic disruption impairs insulin secretion. First, Cas9 protein with different sgRNAs that targeted distinct sites in the PDX‐1 exon 1 was introduced into in vitro‐fertilized zygotes by the GEEP method. Of the six sgRNAs tested, three sgRNAs (sgRNA1, 2, and 3) successfully modified PDX‐1 gene. The blastocyst formation rate of zygotes edited with sgRNA3 was significantly (p < 0.05) lower than that of control zygotes without the electroporation treatment. Our study indicates that the GEEP method can be successfully used to generate PDX‐1 mutant blastocysts, but the development and the efficiency of editing the genome of zygotes may be affected by the sgRNA used for CRISPR/Cas9 system.  相似文献   

Effects of duration of electric pulse on in vitro development of cloned cat embryos with human artificial chromosome vector          下载免费PDF全文

LTK Do  M Wittayarat  T Terazono  Y Sato  M Taniguchi  F Tanihara  T Takemoto  Y Kazuki  K Kazuki  M Oshimura  T Otoi 《Reproduction in domestic animals》2016,51(6):1039-1043

The current applications for cat cloning include production of models for the study of human and animal diseases. This study was conducted to investigate the optimal fusion protocol on in vitro development of transgenic cloned cat embryos by comparing duration of electric pulse. Cat fibroblast cells containing a human artificial chromosome (HAC) vector were used as genetically modified nuclear donor cells. Couplets were fused and activated simultaneously with a single DC pulse of 3.0 kV/cm for either 30 or 60 μs. Low rates of fusion and embryo development to the blastocyst stage were observed in the reconstructed HAC‐transchromosomic embryos, when the duration of fusion was prolonged to 60 μs. In contrast, the prolongation of electric pulse duration improved the embryo development and quality in the reconstructed control embryos without HAC vector. Our results suggested that the optimal parameters of electric pulses for fusion in cat somatic cell nuclear transfer vary among the types used for donor cells.  相似文献   

Correlated responses of respiratory disease and immune capacity traits of Landrace pigs selected for Mycoplasmal pneumonia of swine (MPS) lesion          下载免费PDF全文

Toshihiro Okamura  Kouki Maeda  Wataru Onodera  Hiroshi Kadowaki  Chihiro Kojima‐Shibata  Eisaku Suzuki  Hirohide Uenishi  Masahiro Satoh  Keiichi Suzuki 《Animal Science Journal》2016,87(9):1099-1105

Five generations of Landrace pigs selected for average daily gain, backfat thickness, Mycoplasmal pneumonia of swine (MPS) lesion score, and plasma cortisol levels, was executed to decrease the MPS lesion score. Genetic parameters and correlated genetic responses for respiratory disease and peripheral blood immune traits were estimated in 1395 Landrace pigs. We estimated the negative genetic correlation of MPS lesion score with phagocytic activity (PA) at 7 weeks of age (‐0.67). The breeding values of PA at 7 weeks of age and 105 kg body weight and the correlated selection response of the ratio of granular leukocytes to lymphocytes at 105 kg body weight were significantly increased, and sheep red blood cell‐specific antibody production (AP) was significantly decreased in a selection‐dependent manner. Increasing of natural immunological indicators (e.g. PA) and decreasing of humoral immunological indicator (e.g. AP) were observed due to genetically decreasing MPS lesion score.  相似文献   

Equilibrium vitrification of mouse embryos at various developmental stages using low concentrations of cryoprotectants     

Juan QIU  Kazutsugu MATSUKAWA  Chihiro KOSHIMOTO  Keisuke EDASHIGE 《The Journal of reproduction and development》2021,67(2):109

We previously developed a new vitrification method (equilibrium vitrification) by which two-cell mouse embryos can be vitrified in liquid nitrogen in a highly dehydrated/concentrated state using low concentrations of cryoprotectants. In the present study, we examined whether this method is effective for mouse embryos at multiple developmental stages. Four-cell embryos, eight-cell embryos, morulae, and blastocysts were vitrified with EDFS10/10a, 10% (v/v) ethylene glycol and 10% (v/v) DMSO in FSa solution. The FSa solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 0.5 M sucrose. The state of dehydration/concentration was assessed by examining the survival of vitrified embryos after storage at –80°C. When four-cell embryos and eight-cell embryos were vitrified with EDFS10/10a in liquid nitrogen and then stored at –80°C, the survival rate was high, even after 28 days, with relatively high developmental ability. On the other hand, the survival of morulae and blastocysts vitrified in liquid nitrogen and stored at –80°C for four days was low. Therefore, morulae and blastocysts cannot be vitrified in a highly dehydrated/concentrated state using the same method as with two-cell embryos. However, when blastocysts were shrunken artificially before vitrification, survival was high after storage at –80°C for four days with high developmental ability. In conclusion, the equilibrium vitrification method using low concentrations of cryoprotectants, which is effective for two-cell mouse embryos, is also useful for embryos at multiple stages. This method enables the convenient transportation of vitrified embryos using dry ice.  相似文献   

Maturation in larch: age-related changes in xylem development in the long-shoot foliage and the main stem     

Takemoto Y  Greenwood MS 《Tree physiology》1993,13(3):253-262

As the meristems of eastern larch (Larix laricina (Du Roi) K. Koch) mature, they produce long-shoot foliage with progressively higher chlorophyll content and net photosynthesis and progressively lower specific leaf area (Hutchison et al. 1990). Despite the apparent increase in foliar vigor, both shoot height and diameter growth rates declined during maturation. These changes were associated with changes in xylem morphology of the vascular tissue of both the long-shoot foliage and the main stem. Although the number of xylem cells remained constant in a cross section of the foliage, their diameters increased with age. With increasing maturation, the same number of xylem cells were produced per radial file in the annual rings of the main stem, but their radial diameters decreased, which accounted for the decrease in diameter growth. There was no change in tracheid length during maturation. We conclude that the decrease in growth rate with increased maturation is not due to a decrease in physiological vigor of the foliage, but is a function of reduced sink strength of the growing shoots.  相似文献   

Identification of herds with cattle persistently infected with bovine viral diarrhea virus by virological evaluation of three calves     

Seki Y  Seimiya YM  Yaegashi G  Sato C 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2006,68(3):255-258

For the identification of herds with cattle persistently infected (PI) with bovine viral diarrhea virus, 1,272 animals from 20 herds were subjected to serum neutralizing (SN) test using the Nose strain and virus isolation. Eighteen PI cattle were detected from 5 herds. On the phylogenetic tree based on the nucleotide sequences of the 5' untranslated region, the isolates from the PI cattle were classified into genotypes-1a or -1b. Of 3 unvaccinated calves aged 6 to 12 months selected from each herd, the probabilities of obtaining 2 or more non-PI cattle with SN antibody titers of 64 or more (P(SN)), one or more PI cattle (P(VI)), and either of the conditions (P(Total)) were calculated using the hypergeometric probability model. P(Total) for the 5 herds with PI cattle was 1.000. P(SN) for 3 herds with many PI cattle within the selected age group was as low as 0.500 or less, and P(VI) was as high as 0.886 or more. P(SN) in the 2 other herds with few PI cattle was 1.000, and P(VI) was as low as 0.375 or less. P(Total) in 13 of 15 herds without PI cattle was 0.000, and was 0.714 or 0.774 for the 2 other herds. These results suggest that herds with PI cattle can be predicted with high accuracy when both SN test and virus isolation are performed on only 3 unvaccinated calves aged 6 to 12 months selected from a herd.  相似文献   

Henneguya corruscans n. sp. (Myxozoa,Myxosporea, Myxobolidae), a parasite of Pseudoplatystoma corruscans (Osteichthyes,Pimelodidae) from the Paraná River,Brazil: A morphological and morphometric study     

J.C. Eiras  R.M. Takemoto  G.C. Pavanelli 《Veterinary parasitology》2009

This paper describes the parasite Henneguya corruscans n. sp. which infects the gills of Pseudoplatystoma corruscans Spix and Agassiz, 1829 found in the Paraná River, Brazil. The parasites belong to the interlamellar-epithelial type as defined by Molnár (2002) [Molnár, K., 2002. Site preference of fish myxosporeans in the gills. Dis. Aquat. Org. 48, 197–207]. The spores examined had thin, smooth walls with symmetric valves; the total length of the spores was 27.6 (25–29) μm. The spore body was ellipsoidal in frontal view and biconvex in lateral view and they measured 14.3 (13–15) μm long by 5 μm wide and 4 μm in thickness. The polar capsules were small and elongated, equally sized, with a rounded posterior extremity and tapering anteriorly, and they corresponded more or less the half the length of the spore body; they were 6.8 (6–7) μm long by 2 μm wide, and the polar filament formed 5–6 coils obliquely to the axis of the polar capsule. The tail was 13.7 (12–15) μm long and bifurcated shortly after the end of the spore body. The importance of the infection for the farming of P. corruscans is discussed.  相似文献   

Superficial necrolytic dermatitis associated with extrapancreatic glucagonoma in a dog     

Takuya Mizuno  Hiroko Hiraoka  Chihiro Yoshioka  Yoshiho Takeda  Yuki Matsukane†  Natsumi Shimoyama‡  Masahiro Morimoto‡  Toshiharu Hayashi‡  Masaru Okuda 《Veterinary dermatology》2009,20(1):72-79

An 11-year-old Shih Tzu presented with crusting and erythema, mainly on the abdomen and the root of the tail. Based on histopathological findings, blood examinations and necropsy findings, the condition was diagnosed as superficial necrolytic dermatitis associated with a glucagon-secreting extrapancreatic neuroendocrine tumour. Gross necropsy revealed tumour invasion into the spleen, liver, adrenal glands and mesenteric lymph nodes. Immunohistochemical analysis of the neoplastic cells revealed that the tumour was a glucagonoma, consistent with earlier findings of persistent glucagonaemia and hypoaminoacidaemia.  相似文献   

Evaluation of loop-mediated isothermal amplification (LAMP), PCR and parasitological tests for detection of Trypanosoma evansi in experimentally infected pigs   总被引：6，自引：0，他引：6  

Thekisoe OM  Inoue N  Kuboki N  Tuntasuvan D  Bunnoy W  Borisutsuwan S  Igarashi I  Sugimoto C 《Veterinary parasitology》2005,130(3-4):327-330

Six surra negative piglets (6-week-old) were infected with Trypanosoma evansi and two uninfected piglets were used as negative controls. Detection performances of various diagnostic tests (LAMP, PCR and parasitological tests) were compared by analysing blood samples collected weekly over a period of 11 weeks. With a two by two analysis without a gold standard, all methods were 100% specific. MI had the highest sensitivity of 65%, while LAMP, PCR, MHCT and TBS had sensitivities of 45, 33, 38 and 24%, respectively. However, when the analysis was done using MI as a gold standard, the sensitivity of MHCT was the highest at 53% followed by LAMP, PCR and TBS at 49, 44 and 35%, respectively. All methods gave high specificity above 60%. This study validates LAMP as an alternative method for the diagnosis of surra.  相似文献   

Expression of a gene encoding Trypanosoma congolense putative Abc1 family protein is developmentally regulated   总被引：1，自引：0，他引：1  

Baticados WN  Witola WH  Inoue N  Kim JY  Kuboki N  Xuan X  Yokoyama N  Sugimoto C 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2005,67(2):157-164

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