| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
b Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo,Kashiwa City, Chiba 277-8562, Japan
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
developmental biology, early development, embryo
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The methods used for cloning procedures fall into two groups in terms of the cell cycle stage of the recipient cytoplasm. One method is transplanting the nuclei into metaphase II (MII)-arrested unfertilized oocytes. This approach leads to nuclear envelope breakdown (NEBD) and subsequent premature chromosome condensation (PCC) due to the high level of maturation/M-phase promoting factor (MPF) in the oocyte cytoplasm [9–12]. The other method is to use activated ova, which have been relieved from MII arrest and have resumed the cell cycle, as the recipient cytoplasm. In this case, NEBD and PCC of the donor nuclei do not occur because the activity of MPF has already declined [9, 13–15]. Earlier studies demonstrated that MII-arrested nonactivated oocytes are far more effective for supporting development of embryos reconstructed with differentiated nuclei than are activated oocytes [16, 17]. Thus, success in chromatin remodeling, from differentiated nuclei to totipotent ones, is dependent on the cell cycle stage of the recipient's cytoplasm. The ability to allow remodeling of chromatin apparently exists in MII-arrested nonactivated oocytes and disappears after activation.
The purpose of this study was to investigate the molecular/biochemical basis of chromatin remodeling that occurs in the somatic nuclei transferred into oocytes. We reconstructed the embryos by two different nuclear transfer protocols, transfer of nuclei into the oocytes before and after activation, to investigate the difference in the molecular/biochemical events involved in chromatin remodeling when the nuclei are successfully remodeled and when they are not. This comparison would provide us with an important key to understanding the mechanism of chromatin remodeling. The embryos reconstructed by two different protocols were compared with each other using molecular/biochemical criteria such as transcriptional activity, distribution of basal transcription factor, and DNase I sensitivity of the chromosomes.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Female B6D2F1 (C57BL/6 x DBA/2 hybrid; SLC, Shizuoka, Japan) mice 8–9 wk of age were superovulated with 7.5 IU eCG followed by 7.5 IU hCG 48 h later. Mice were killed by cervical dislocation. Cumulus-oocyte complexes were collected from ampullae of oviducts 14–17 h after hCG injection and placed in KSOM [18] containing 0.3 mg/ml bovine testicular hyaluronidase. After complete removal of cumulus cells, oocytes were transferred to fresh KSOM and incubated in a humidified atmosphere of 5% CO2/95% air at 37.5°C. Removed cumulus cells were dispersed on Hepes-KSOM containing 12% (w/v) polyvinylpyrolidone and kept at room temperature until used as nuclear donors.
Nuclear Transfer and Oocyte Activation Procedure
The oocytes were treated with 5 µg/ml cytochalasin B in Hepes-buffered KSOM for 5–10 min at room temperature in the micromanipulation chamber (made from the cover of a plastic dish, Falcon no. 1006; Becton Dickinson, Franklin Lakes, NJ) on the inverted microscope equipped with Hoffman or Nomarski optics. Cytochalasin B (500 µg/ml) in dimethylsulfoxide (DMSO) was diluted with Hepes-buffered KSOM immediately before use to final concentration of 5 µg/ml cytochalasin B and 1% (v/v) DMSO. The zona pellucida was cored by a Piezo impact-driven micromanipulator (Prime Tech, Ibaraki, Japan), and MII chromosomes were aspirated with an enucleation pipette (approximately 7 µm inner diameter) with a minimal volume of oocyte cytoplasm [2]. Enucleated oocytes were transferred into cytochalasin B-free KSOM and left for >1 h at 37.5°C before being used as recipients for nuclear transfer.
Two nuclear transfer protocols were employed. In the first protocol, the nucleus was transferred first and then the oocyte was activated (transfer before activation, TA) [2]. In the second protocol, the nucleus was transferred into an enucleated oocyte after the oocyte was activated (activation before transfer, AT). Somatic nuclei were removed from cumulus cells by gentle aspiration of the Piezo pulse-actuated injection pipette (approximately 5 µm inner diameter). In the TA protocol, each nucleus was then injected into an enucleated oocyte with weak Piezo pulse to minimize distortion of the oocyte cell membrane [2, 19]. After 1 h of culture, the reconstructed oocytes were activated by treatment with 10 mM SrCl2 in Ca2+-free KSOM containing 5 µg/ml cytochalasin B (1% DMSO) for 1 h, followed by a further 5 h of incubation in KSOM containing 5 µg/ml cytochalasin B (1% DMSO) [2, 19–21]. In the AT protocol, the enucleated oocytes were activated by treatment with 10 mM SrCl2 in Ca2+-free KSOM for 20 min and then transferred into KSOM. After incubation for 1 h, oocytes were subjected to nuclear transfer. The injection of the nucleus was conducted in the same manner as in the TA protocol. In this procedure, all oocytes were parthenogenetically activated by the treatment with 10 mM SrCl2 in Ca2+-free KSOM for 20 min [20, 21].
Parthenogenesis of Control Embryos
Unfertilized oocytes were activated by treatment with 10 mM SrCl2 in Ca2+-free KSOM containing 5 µg/ml cytochalasin B (1% DMSO) for 20 min, followed by an additional 5.5-h incubation in KSOM containing 5 µg/ml cytochalasin B (1% DMSO) to prevent extrusion of the polar bodies from the oocytes. The diploid parthenogenetic embryos thus obtained were used as the controls for the reconstructed embryos.
In Vitro Transcriptional Activity Assay
The in vitro transcriptional activity assay was conducted as described by Aoki et al. [22]. All treatments were performed at room temperature unless otherwise specified. Embryos were washed in a drop of physiological buffer (PB) that consisted of 100 mM potassium acetate, 30 mM KCl, 1 mM MgCl2, 10 mM Na2HPO4, 1 mM ATP supplemented with 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and 50 units/ml of RNase inhibitor (Promega, Madison, WI), and the plasma membrane was permeabilized by treating embryos for 1 min with 0.05% Triton X-100 in PB. Following this treatment, the embryos were briefly washed three times with PB and then transferred to 100 mM potassium acetate, 1 mM MnCl2, 50 mM (NH4)2SO4, 30 mM KCl, 10 mM Na2HPO4 containing 2 mM ATP, 0.4 mM each GTP, CTP, and BrUTP, and 1 mM MgCl2. After 15 min of incubation at 33°C, the embryos were washed briefly three times with PB, and the nuclear membrane was permeabilized by a 3-min treatment in PB containing 0.2% Triton X-100. The embryos were then washed in PB three times and fixed for 1 h with 3.7% paraformaldehyde in PB. The incorporated BrUTP was detected by indirect immunostaining with anti-BrdU antibodies. The embryos were washed five times in 15-µl drops of PBS containing 3 mg/ml BSA (PBS/3BSA) over a period of 15 min and then incubated for 45 min with PBS containing 2 µg/ml anti-BrdU monoclonal antibody (Boehringer Mannheim, Indianapolis, IN). The embryos were then washed four times with PBS/3BSA over the course of 15 min and subsequently incubated in PBS containing 0.5 µg/ml anti-mouse IgG antibody conjugated with Texas Red (Jackson ImmunoResearch, West Grove, PA) for 45 min. The samples were then washed with PBS/3BSA and mounted on glass slides in VectaShield antibleaching solution (Vector Laboratories, Burlingame, CA).
Immunocytochemistry of TATA Box Binding Protein
The TATA box binding protein (TBP) was detected by an anti-TBP monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), which recognizes the amino terminal domain of TFIID (TBP). Embryos were collected and fixed for over 1 h with 3.7% paraformaldehyde. Fixed embryos were washed three times with PBS containing 1 mg/ml BSA (PBS/1BSA) and permeabilized in 0.5% Triton-X/PBS for 10 min. Embryos were then washed briefly followed by incubation with 0.4 µg/ml anti-TBP antibody in PBS for 1 h at room temperature. The cells were washed in four drops of PBS/1BSA over 10 min and incubated with 0.5 µg/ml anti-rabbit IgG antibody conjugated with Texas Red in PBS for 45 min at room temperature. The samples were then washed again with PBS/1BSA before mounting on glass slides in VectaShield antibleaching solution.
In Situ DNase I Sensitivity Assay
All treatments were performed at room temperature unless otherwise specified. Embryos were collected and briefly washed in PBS containing 4 mg/ml polyvinylpyrolidone (PBS/PVP) before permeabilization in 0.2 % Triton X-100/PBS for 6 min. Cells were then washed in two drops of PBS/PVP and DNase I buffer (50 mM Tris-HCl, pH 7.8, 5 mM MgCl2, 10 mM 2-mercaptoethanol, 10 µg/ml BSA) and transferred to DNase I buffer containing 0.3 U/ml DNase I (Gibco BRL, Gaithersburg, MD). After a 15-min incubation at 37°C, embryos were incubated in stop solution (PBS containing 15 mM EDTA) for 10 min, washed three times with PBS/PVP, and fixed with 3.7% paraformaldehyde in PBS for 1 h.
A TUNEL reaction was conducted to introduce the fluorescent nucleotides into digested DNA. Fixed embryos were washed five times in PBS/PVP over a period of 15 min and then preincubated in 5 µl of TUNEL labeling mixture (Boehringer Mannheim). Following this treatment, the embryos were transferred to 50 µl of TUNEL labeling mixture containing 5 µl of TUNEL enzyme (Boehringer Mannheim). After 60 min of incubation at 37°C, the embryos were washed four times in PBS/PVP and mounted on glass slides in VectaShield antibleaching solution.
Quantification of Fluorescence Intensityby Laser-Scanning Confocal Microscopy
Fluorescence was detected using a Carl Zeiss 510 laser-scanning confocal microscope (Oberkochen, Germany), and signal was quantified using an NIH-Image (National Institutes of Health, Bethesda, MD) as follows. The average nucleus pixel value/unit area was subtracted from the average cytoplasmic pixel value/unit area and multiplied by nuclear dimensions to yield the relative values to compare BrUTP incorporation, the amount of TBP, and DNase I sensitivity. Dimensions instead of volume were used in this calculation procedure, because the embryos were pressed on glass slides so that the values of the dimensions precisely reflected the difference in volume. In every quantification procedure, the value of the control embryo (diploid parthenote) 12 h postactivation was arbitrarily set as 100%, and the fluorescence intensity observed in each sample was expressed relative to this value.
Statistical Analysis
The data were obtained from two or three independent experiments and analyzed by ANOVA.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The development to the morula/blastocyst stage was significantly different between the embryos prepared by the two different nuclear transfer protocols (Table 1). When the donor nuclei were transferred to the enucleated unfertilized oocytes (TA protocol), more than half of the reconstructed embryos developed to the morula/blastocyst stage at Day 4 (96 h after activation). However, when the nuclei were transferred to the enucleated activated oocytes (AT protocol), few (1.0%) of the resulting embryos developed to the morula/blastocyst stage; most of them (98.0%) did not cleave to two cells and fragmented at Day 2 (24 h postactivation) while they entered S phase at 10–11 h postactivation. Because the origin of the donor nuclei and the nuclear transfer method was essentially identical for these two protocols, the observed difference in development was considered due to inequality of the ability of recipient cytoplasm to support development. Because the chromatin structure of the transferred somatic nuclei should be remodeled to change its gene expression pattern for further development, the cytoplasm of MII-arrested unfertilized oocytes would be able to remodel the introduced somatic nuclei but that of the activated oocytes would not.
|
The diploid parthenogenetic embryos, activated by the same chemical method as used with the reconstructed embryos, were employed as the control cells in the following experiments. Almost all of these embryos developed to the morula/blastocyst stage.
Changes in the Transcriptional Activityof Reconstructed Embryos
The mouse zygote starts its intrinsic transcription at the late 1-cell stage after a transcriptionally silent period [22–25]. Therefore, we examined the changes in transcriptional activity of the transferred somatic nuclei during the 1-cell stage to investigate whether those nuclei start their intrinsic transcription normally (Fig. 1). The embryos were collected at 5 h and 12 h postactivation. In our culture system, the control diploid parthenogenetic embryos entered into the first mitosis 13 h after activation stimuli.
|
In the control embryos, transcriptional activity was not detected at 5 h postactivation but became detectable at 12 h. This pattern of changes in transcriptional activity was the same as that previously observed in fertilized embryos [22].
When the unfertilized oocytes were used as recipient cytoplast (TA protocol), the transcription in the reconstructed embryos was silenced after nuclear transfer, i.e., transcriptional activity was not detected at 5 h postactivation. At the late 1-cell stage (12 h postactivation), however, the fluorescence that reflects the BrUTP incorporation reappeared with nearly the same intensity as that of the control embryos. This finding is consistent with our previous observation that the transcriptional activity in the haploid nuclei of parthenogenetic embryos was almost the same as that in the diploid nuclei of normally fertilized embryos [22]. When the donor nuclei were transferred into enucleated activated oocytes (AT protocol), they remained transcriptionally active at 5 h postactivation, and BrUTP incorporation was not significant different from that of intact donor nuclei that had not been transferred (P > 0.05, ANOVA). The reconstructed embryos prepared by TA protocol showed a higher percentage of development than did AT embryos. These results suggest that remodeling of chromatin in the fully differentiated nuclei results in silencing and resumption of transcriptional activity after nuclear transfer. The difference in the duration of Sr2+ treatment (1 h and 20 min) probably did not contribute to the difference in the transcriptional activity between these two protocols; the transcriptional activity was not different between the parthenogenetically activated embryos treated with Sr2+ for 20 min and 1 h (data not shown).
Changes in the Accumulation of TBP in the Nucleusof the Reconstructed Embryos
TBP is a basal transcription factor required for general transcription. Several lines of evidence suggest that exchange of TBP between nucleus and cytoplasm reflects global transition of nuclear function and structure [26] and that the nuclear accumulation of TBP is involved in zygotic gene activation during the 1-cell stage [27]. Therefore, we examined the changes in the amount of nuclear TBP after nuclear transfer to explore the role of TBP in nuclear remodeling.
The nuclear transfer protocol affected temporal changes in the amount of TBP in the transferred somatic nuclei (Fig. 2). When TA protocol was employed, the transferred nuclei underwent NEBD and PCC. TBP was displaced from condensed chromatin before activation (Fig. 2a). Although TBP was not yet detected at 2 h postactivation, it appeared again in the nucleus at 5 h postactivation. However, such translocation of TBP was not observed in the embryos reconstructed by AT protocol, where somatic nuclei did not undergo NEBD or PCC. In these embryos, TBP was always detected in the nucleus at 2 and 5 h after nuclear transfer (Fig. 2b).
|
Nuclear TBP was quantified in the reconstructed embryos (Fig. 3). A similar pattern of change in TBP amount was observed for the control embryos and the reconstructed embryos prepared using the TA protocol. TBP was not detected at 2 h postactivation, was detected at 5 h, and then significantly increased at 12 h, at which time the amount was not significantly different between the control and the reconstructed embryos (P > 0.05, ANOVA). In the embryos prepared using the AT protocol, the nuclear TBP was almost the same as that in the intact donor nuclei at 2 h postactivation. It did not change until 5 h and then slightly increased at 12 h. However, the amount was significantly less than that of the control embryos (P < 0.001, ANOVA). The difference in the duration of Sr2+ treatment (1 h and 20 min) probably did not contribute to the difference in the accumulation of nuclear TBP between these two protocols; the amount of nuclear TBP was not different between the parthenogenetically activated embryos treated with Sr2+ for 20 min and 1 h (data not shown).
|
DNase I Sensitivity of Reconstructed Embryos
To directly investigate the changes in the chromatin structure in the transferred nuclei, an in situ DNase I sensitivity assay was conducted (Fig. 4). Cho et al. [28] showed that DNase I sensitivity was decreased from the early to the late 1-cell stage but that the magnitude of the decrease was smaller in the female nuclei than in the male nuclei. The control diploid parthenogenetic embryos showed changes in DNase I sensitivity similar to those in the fertilized embryos during the 1-cell stage (Fig. 5). Sensitivity was slightly but significantly decreased from 5 to 12 h postactivation (P < 0.05, ANOVA). In the reconstructed embryos derived using the TA protocol, DNase I sensitivity was increased after nuclear transfer. At 5 h postactivation, the sensitivity in the transferred nuclei was about 2-fold that of the donor somatic nuclei. After that, DNase I sensitivity was significantly decreased at 12 h (P < 0.001, ANOVA). However, in the embryos reconstructed using the AT protocol, DNase I sensitivity did not change significantly during the 1-cell stage (P > 0.05, ANOVA).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Silencing of transcription in the early 1-cell stage seems to be a phenomenon correlated with remodeling of the differentiated nucleus into a totipotent nucleus. The two highly differentiated cells, sperm and oocyte, should delete their program of gene expression pattern after fertilization to make totipotent zygotes. The mouse zygotic genome starts its intrinsic transcription at the late 1-cell stage after a transcriptionally silent period [22–25]. Our results showed that after transfer of somatic nuclei, the transcriptional activity was inactivated at the early 1-cell stage and then resumed at the late stage in the TA embryos. In addition, the basal transcription factor TBP was also displaced from the nucleus after transfer in the TA embryos. However, in the AT embryos, which showed significantly retarded development, the nucleus maintained its transcriptional activity after being transferred into the oocyte and TBP was not displaced from the nucleus after nuclear transfer. These results experimentally showed that a transcriptionally inert state is involved in successful chromatin remodeling. The transient displacement of TBP may, at least in part, contribute to this transcriptional inactivation.
Changes in DNase I sensitivity were different in TA and AT embryos, suggesting that DNase I is involved in chromatin remodeling. The accessibility of transcription factors to endogenous DNA is correlated with the sensitivity of genomic DNA to DNase attack. Hence, changes in DNase I sensitivity of the nucleus could be used to monitor the changes in chromatin structure. In TA and control embryos, DNase I sensitivity of the nucleus was significantly decreased from the early to the late 1-cell stage. This result was consistent with a previous finding that a transcriptionally repressive chromatin structure was established following S-phase in the 1-cell mouse embryos [29]. Furthermore, DNase I sensitivity increased about 2-fold at the early 1-cell stage of TA embryos when compared with intact donor nuclei, although there were no significant changes in AT embryos. Because successful development to morulae/blastocysts was observed in the TA embryos but not in AT embryos, changes in chromatin structure to the DNase I accessible state would be required for successful chromatin remodeling.
In several studies on successful production of cloned animals, exposure to the cytoplasm of nonactivated oocytes was crucial for the remodeling of the differentiated somatic nuclei [3, 16, 17, 30]. In the TA protocol, transferred somatic nuclei underwent NEBD and subsequent PCC due to active MPF in the cytoplasm of unfertilized oocytes. When activated oocytes with declined MPF activity were used as recipients (AT protocol), the membranes and chromosomes of somatic nuclei remained intact and decondensed, respectively, after transfer. Because both protocols were implemented using by the same microinjection method and with an identical source of donor nuclei, observed differences in developmental potential and behavior of transferred nuclei with these two protocols were attributable to inequality of the recipient cytoplasm's ability to induce chromatin remodeling. Similar results have been reported elsewhere; when somatic nuclei were introduced into activated mouse oocytes by cell fusion, the nucleus did not develop a pronucleus-like configuration. In these nuclei, transcriptional activity also remained active [14, 15]. These results suggest that the cytoplasm of activated mouse oocytes was inefficient for nuclear remodeling due to a failure of NEBD. However, the reason NEBD is crucial for nuclear remodeling has not been elucidated. Our results showed that successful chromatin remodeling was accompanied by displacement and redistribution of nuclear TBP. In a study on chromatin remodeling of Xenopus somatic nuclei, the displacement of basal transcription factor TBP from somatic nuclei with permeabilized nuclear membrane occurred in S-phase extract of Xenopus egg, where chromosomes remained decondensed [26]. These results suggested that direct interaction of the chromosome with the cytoplasmic environment, but not condensation of the chromosome, is necessary for the chromosome to be remodeled. NEBD may play an important role in reprogramming of differentiated nuclei by allowing direct interactions of chromosomes with cytoplasmic factor(s) in nonactivated oocytes.
Results of these experiments indicate that the successful remodeling of somatic nuclei is brought about by a process similar to that of normally fertilized embryos. Silencing of transcription, accumulation of TBP, and loosening chromatin structure are important events in the process of physiological chromatin remodeling. The failure of the activated oocytes to remodel somatic nuclei is due to the inability of these oocytes to succesfully accomplishing these events. Thus, exploring the mechanism for remodeling transferred somatic nuclei is helpful for understanding the nature of chromatin remodeling that occurs during gametogenesis and early embryogenesis. Furthermore, knowledge of the mechanism underlying chromatin remodeling would facilitate the efficient production of cloned animals. Until now, morphological changes and frequency of development have been used to monitor the remodeling of chromatin. The molecular/biochemical events presented in this study, i.e., silencing of transcription, accumulation of TBP, and increase in DNase I sensitivity, could be used as alternative criteria to efficiently evaluate the extent of chromatin remodeling.
|
FOOTNOTES |
|---|
1 This research was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan, to F.A. and to A.O. and from the Ministry of Health and Welfare and the Human Science Foundation, Japan, to A.O. 
2 Correspondence: Fugaku Aoki, Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Shinryoiki-Seimei Building 302, Chiba 277-8562, Japan. FAX: 81 471 471 3698; aokif{at}k.u-tokyo.ac.jp
3 Current address: The Institute of Physical and Chemical Research (RIKEN), Bioresource Center, 3-1-1 Koyadai, Tsukuba-shi, Ibaraki 305-0074, Japan
Accepted: April 1, 2002.
Received: October 17, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  D J Kwon, C K Park, B K Yang, and H T Cheong Control of nuclear remodelling and subsequent in vitro development and methylation status of porcine nuclear transfer embryos Reproduction, May 1, 2008; 135(5): 649 - 656. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ooga, A. Inoue, S.-i. Kageyama, T. Akiyama, M. Nagata, and F. Aoki Changes in H3K79 Methylation During Preimplantation Development in Mice Biol Reprod, March 1, 2008; 78(3): 413 - 424. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Inoue, S. Noda, N. Ogonuki, H. Miki, S. Inoue, K. Katayama, K. Mekada, H. Miyoshi, and A. Ogura Differential Developmental Ability of Embryos Cloned from Tissue-Specific Stem Cells Stem Cells, May 1, 2007; 25(5): 1279 - 1285. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Hara, K. I. Nakayama, and K. Nakayama Geminin is essential for the development of preimplantation mouse embryos Genes Cells, November 1, 2006; 11(11): 1281 - 1293. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Nakasato, Y. Shirakura, M. Ooga, M. Iwatsuki, M. Ito, S.-i. Kageyama, S. Sakai, M. Nagata, and F. Aoki Involvement of the STAT5 Signaling Pathway in the Regulation of Mouse Preimplantation Development Biol Reprod, October 1, 2006; 75(4): 508 - 517. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ono and T. Kono Irreversible Barrier to the Reprogramming of Donor Cells in Cloning with Mouse Embryos and Embryonic Stem Cells Biol Reprod, August 1, 2006; 75(2): 210 - 216. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Inoue, N. Ogonuki, H. Miki, M. Hirose, S. Noda, J.-M. Kim, F. Aoki, H. Miyoshi, and A. Ogura Inefficient reprogramming of the hematopoietic stem cell genome following nuclear transfer J. Cell Sci., May 15, 2006; 119(10): 1985 - 1991. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kawahara, T. Wakai, K.-I. Yamanaka, J. Kobayashi, S. Sugimura, T. Shimizu, H. Matsumoto, J.-H. Kim, H. Sasada, and E. Sato Caffeine promotes premature chromosome condensation formation and in vitro development in porcine reconstructed embryos via a high level of maturation promoting factor activity during nuclear transfer Reproduction, September 1, 2005; 130(3): 351 - 357. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Fulka, M. Mrazek, O. Tepla, and J. Fulka Jr DNA methylation pattern in human zygotes and developing embryos Reproduction, December 1, 2004; 128(6): 703 - 708. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-I. Kageyama, M. Nagata, and F. Aoki Isolation of Nascent Messenger RNA from Mouse Preimplantation Embryos Biol Reprod, December 1, 2004; 71(6): 1948 - 1955. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Miki, K. Inoue, N. Ogonuki, K. Mochida, H. Nagashima, T. Baba, and A. Ogura Cytoplasmic Asters Are Required for Progression Past the First Cell Cycle in Cloned Mouse Embryos Biol Reprod, December 1, 2004; 71(6): 2022 - 2028. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Liu, J.-M. Kim, and F. Aoki Regulation of histone H3 lysine 9 methylation in oocytes and early pre-implantation embryos Development, May 15, 2004; 131(10): 2269 - 2280. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Gomez, J. A. Jenkins, A. Giraldo, R. F. Harris, A. King, B. L. Dresser, and C. E. Pope Nuclear Transfer of Synchronized African Wild Cat Somatic Cells into Enucleated Domestic Cat Oocytes Biol Reprod, September 1, 2003; 69(3): 1032 - 1041. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Hao, L. Lai, J. Mao, G.-S. Im, A. Bonk, and R. S. Prather Apoptosis and In Vitro Development of Preimplantation Porcine Embryos Derived In Vitro or by Nuclear Transfer Biol Reprod, August 1, 2003; 69(2): 501 - 507. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-M. Kim, H. Liu, M. Tazaki, M. Nagata, and F. Aoki Changes in histone acetylation during mouse oocyte meiosis J. Cell Biol., July 7, 2003; 162(1): 37 - 46. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
