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Naruse, Kenji,Quan, Yan Shi,Park, Tae Young,Kim, Beak Chul,Park, Chang Sik,Jin, Dong Il 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10
Abnormal fusion is thought to be related to the incorrect reprogramming of nucleus following transfer into the oocytes. For this reason, the purpose of current experiments was to investigate the development of porcine nuclear transfer embryos reconstructed by using treated donor cells with streptolysin O (SLO). SLO-treated group (200 ng/ml, 50 min) significantly increased (P<0.05) the percentage of fusion rates and blastocyst developmental rates compared with control. Exposure to SLO-treated donor cells with the matured cytoplasm for 40 min significantly increased (P<0.05) the rates of fusion and cleavage and blastocysts formation of nuclear transfer (NT) embryos. In conclusion, treatment to fetal fibroblast cells with Streptolysin O (SLO) and exposed to the matured cytoplasm could lead to improvements of the nuclear transfer methods.
Naruse, K.,Quan, Y.S.,Kim, B.C.,Lee, J.H.,Park, C.S.,Jin, D.I. 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10
To investigate the effects of cycloheximide exposure before electrical activation of in vitro-matured porcine oocytes on the subsequent development of parthenogenetic embryos, cumulus-free mature oocytes were exposed to NCSU-23 medium containing cycloheximide (10μg/mL) for 0, 5, 10, 20, 30 and 60 min, activated by electrical pulse treatment (1.5 kV/cm, 100μs) and then cultured in PZM-3 for 7 days. To evaluate the effects of cycloheximide on the activation of nuclear transfer embryos, reconstructed embryos were electrically activated by two DC pulses (1.2 kV/cm, 30μs) before or after exposure to cycloheximide. The reconstructed embryos were allocated into four groups: electrical pulse treatment alone (Ele); exposure to cycloheximide for 10 min followed by electrical activation (CHX + Ele); electrical activation followed by exposure to cycloheximide for 6 h (Ele + CHX); exposure to cycloheximide for 10 min, followed by electrical activation and a further exposure to cycloheximide for 6 h (CHX + Ele + CHX). The activated reconstructed embryos were cultured in PZM-3 for 6 days. Oocytes treated with 10 min exposure to cycloheximide followed by electrical activation had a significantly higher percentage of blastocyst formation compared to control oocytes and oocytes exposed for≥30 min. In the reconstructed embryos, the blastocyst development rates of embryos exposed to cycloheximide (CHX + Ele, Ele + CHX and CHX + Ele + CHX) were significantly higher than those of the control group (Ele). Among the cycloheximide-treated groups, the CHX + Ele group had increased development rate and total blastocyst cell number, though these values were not significantly different from those observed in the other cycloheximide-treated groups. To evaluate the quality of NT embryos treated with cycloheximide, apoptosis in blastocysts was analyzed by TUNEL assay. The 10 min exposure to cycloheximide prior to electrical activation significantly reduced cell death compared with longer exposure to cycloheximide after electrical fusion. In conclusion, brief exposure to cycloheximide prior to electrical activation may increase the subsequent blastocyst development rates in porcine parthenogenetic and reconstructed embryos.
Naruse, Kenji,Kim, Hong Rye,Shin, Young Min,Chang, Suk Min,Lee, Hye Ran,Park, Chang Sik,Jin, Dong Il 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10
To investigate the effects of water-soluble vitamin supplementation for IVM/IVC of porcine oocytes and evaluate maturation and developmental capacity in vitro, porcine cumulus oocyte complexes (COCs) was matured in NCSU-23-based medium with water-soluble vitamins for 44 h and then cultured in PZM-3 for 7 days following activation. The COCs were allocated into five treatment groups and matured in various concentrations of MEM vitamins (control, 0.05, 0.1,0.2,0.4, and 1×). Metaphase II plates of the cumulus-free oocytes were observed following Hoechest 33258 staining. The COCs were allocated into four treatment groups, matured in various concentrations of MEM vitamins (control, 0.05, 0.1, 0.2, and O.4×) and cultured in PZM-3 following activation. Also, COCS were matured without MEM vitamins and cultured in PZM-3 with various concentrations (control, 0.1,0.4, 1.0, and 2.0×) of MEM vitamins. Furthermore, 2 × 2 factorial (IVM/IVC) experiments were performed in IVM medium with or without 0.05 × MEM vitamins and IVC medium with or without 0.4× MEM vitamins to examine the in vitro development of parthenogenetic embryos. Maturation rates of COCs treated with MEM vitamins did not differ significantly among groups. However, compared to the control group, oocytes matured with the addition of 0.05×MEM vitamins developed to blastocysts at a higher percentage (P < 0.05) following activation and culture in PZM-3 without MEM vitamins. Total cell number of blastocysts was significantly higher in the 0.05×group. Addition of O.4× MEM vitamins decreased (P < 0.05) cleavage and blastocyst developmental rates compared with 0.05× MEM vitamins-treated group. In contrast, addition of vitamins to PZM-3 medium for in vitro culture of activated porcine oocytes did not affect development. In conclusion, addition of a low concentration of MEM vitamins to IVM medium for porcine oocytes enhanced subsequent development and improved embryo quality.
NARUSE, Kenji,YOO, Seung Kwon,KIM, Sun Myoung,CHOI, Yun Jaie,LEE, Hong Mie,JIN, Dong Il Japan Society for Bioscience, Biotechnology, and A 2006 Bioscience, Biotechnology, and Biochemistry Vol.70 No.1
<P>To investigate the ability of 1.8 kb or 3.1 kb bovine β-casein promoter sequences for the expression regulation of transgene <I>in vivo</I>, transgenic mice were produced with human type II collagen gene fused to 1.8 kb and 3.1 kb of bovine β-casein promoter by DNA microinjection. Five and three transgenic founder mice were produced using transgene constructs with 1.8 kb and 3.1 kb of bovine β-casein promoters respectively. Founder mice were outbred with the wild type to produce F<SUB>1</SUB> and F<SUB>2</SUB> progenies. Total RNAs were extracted from four tissues (mammary gland, liver, kidney, and muscle) of female F<SUB>1</SUB> transgenic mice of each transgenic line following parturition. RT-PCR and Northern blot analysis revealed that the expression level of transgene was variable among the transgenic lines, but transgenic mice containing 1.8 kb of promoter sequences exhibited more leaky expression of transgene in other tissues compared to those with 3.1 kb promoter. Moreover, Western blot analysis of transgenic mouse milk showed that human type II collagen proteins secreted into the milk of lactating transgenic mice contained 1.8 kb and 3.1 kb of bovine β-casein promoter. These results suggest that promoter sequences of 3.1 kb bovine β-casein gene can be used for induction of mammary gland-specific expression of transgenes in transgenic animals.</P>
Naruse Kenji,Kim Hong-Rye,Shin Young-Min,Chang Suk-Min,Lee Hye-Ran,Tarte Vaishali,Quan Yan-Shi,Kim Beak-Chul,Park Tae-Young,Choi Su-Min,Park Chang-Sik,Jin Dong-Il 한국동물생명공학회(구 한국동물번식학회) 2006 Reproductive & developmental biology Vol.30 No.1
Electrical treatment has been widely used for porcine oocytes activation. However, developmental rates following electrical activation of porcine oocytes is relatively inefficient compared to other domestic animals. To investigate the effects of porcine oocytes on combined activation by both chemical and electrical treatment, in-vitro matured oocytes were activated by combined cycloheximide and electrical pulses treatment. Cumulus-free oocytes were exposed with NCSU-23 medium containing cycloheximide (10μgml) for 0, 5, 10, 20, 30 min and then activated by electrical pulse treatment and cultured in PZM-3 for 8 days. Also effects of exposure to 6.25μM calcium ionophore for 2 min for cumulus-free oocytes were tested. The percentage of blastocyst formation in 10 min exposure to 10μgml cycloheximide and electrical pulse treatment was significantly increased (P<0.05) than in the control group. And exposure to 6.25μM calcium ionophore for 2 min with 10μgml cycloheximide for 10min and electrical pulse treatment significantly increased (P<0.05) the percentage of blastocyst developmental rates than the control group. In conclusion, activation by combined cycloheximide and electrical stimulation treatment promoted the subsequent development of porcine oocytes and improved the subsequence blastocyst development