39 DEVELOPMENTAL COMPETENCE OF RECONSTRUCTED EMBRYOS PRODUCED BY NUCLEAR TRANSFER BETWEEN CYNOMOLGUS MONKEY FIBROBLAST CELLS AND RABBIT OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 128 ◽  
Author(s):  
Y. Hosoi ◽  
T. Yamochi ◽  
N. Kawata ◽  
M. Takenoshita ◽  
S. Ohta ◽  
...  
Keyword(s):  
Cynomolgus Monkey ◽  
Nuclear Transfer ◽  
Culture Media ◽  
Genetic Material ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Fibroblast Cells ◽  
Cell Stage ◽  
Reconstructed Embryos ◽  
Donor Cells

Interspecies nuclear transfer has been used as an invaluable tool for studying nucleus-cytoplasm interactions and it may also be used for rescuing endangered species whose oocytes are difficult to obtain. In this study, we investigated interaction of the cynomolgus monkey cell as a nuclear donor with the rabbit oocyte as a host cytoplasm. Whole cynomolgus fibroblast cells were injected into the rabbit enucleated oocytes (cynomolgus-rabbit cloned embryos) and cultured in TCM-199 and RPMI 1640 culture media. Rabbit-rabbit cloned embryos we used as control in this study. Karyotype analyses confirmed that genetic material of blastocysts was derived from the cynomolgus donor cells at blastocyst stage. Mitochondrial constitution analysis of the cynomolgus-rabbit cloned embryos indicated that mitochondria from both donor cells and enucleated oocytes coexisted. After culture for 168 h post-nuclear transfer, all cynomolgus-rabbit cloned embryos in TCM-199 were arrested at the 8-cell stage, but some of them developed to the blastocyst stage in RPMI 1640 (11/59, 18.6%). In this experiment, the nutrition requirement in vitro and the cleavage rate at each 24 h were examined. When TCM-199 was supplemented with lactate, some of these embryos developed to the blastocyst stage (15.3%, 2/13). This means that cynomolgus-rabbit cloned embryos might be controlled by the donor nucleus even in these early developmental stages. However, the timing of cleavage of cynomolgus-rabbit cloned embryos is very similar to that of the rabbit-rabbit cloned embryos. Time of cleavage may depend on the protein accumulated in the cytoplasm. In the prolonged culture of reconstructed embryos on feeder cells, adhesion cells were observed. These cells are also very similar to the cells derived from cynomolgus embryos by the same method. Our results suggest that: (1) a cynomolgus nucleus can co-ordinate with rabbit oocyte cytoplasm in early embryo development, (2) the 8- to 16-cell stage block in the cynomolgus-rabbit cloned embryos may due to the same reason as that in the cynomolgus embryos, and (3) ooplasmic factors that control time of cleavage are highly conservative between the species.

scholarly journals 57 INVESTIGATION OF CYNOMOLGUS MONKEY (MACACA FASCICULARIS) FETUS FIBROBLAST CELL NUCLEAR TRANSFER

2005 ◽  
Vol 17 (2) ◽  
pp. 178
Author(s):  
J. Narita ◽  
H. Tsuchiya ◽  
T. Takada ◽  
R. Torii
Keyword(s):  
Embryo Development ◽  
Cynomolgus Monkey ◽  
Nuclear Transfer ◽  
Es Cells ◽  
Fibroblast Cells ◽  
Post Injection ◽  
Embryo Production ◽  
Cell Stage ◽  
Donor Cells ◽  
Whole Mount

Use of nuclear transfer (NT) in the cynomolgus monkey to establish tailor-made ES cells with the final goal of cloned embryo production was investigated. Activation stimulus conditions previously confirmed in parthenote production were used. Injection method NT was conducted using cynomolgus monkey fetus fibroblast cells in order to investigated the time it takes, from injection to activation, to reprogram the donor nuclei. Oocytes were collected under laparoscopic observation from mature cynomolgus monkeys 40 h after hCG (400 IU/kg) administration 9 days after follicle stimulation by i.m. injection of FSH (25 IU/kg). Donor cells, 40-day-old fetus fibroblast cells, were cultivated and synchronized at G0/G1 phase. After mature (MII) oocytes were enucleated using a Piezo-drive unit, donor cells were injected. At 2 h (Experiment 1, E1) and 4 h (Experiment 2, E2) after donor cell injection, activation was carried out by 2-min treatment with ionomycin and cultivation by 6-dimethylaminopurine for 4 h. As a control, parthenote production was carried out under the same activation conditions as NT. After activation, in vitro culture was carried out for about 9 days under conditions of 38°C, 5% CO2, and 5% O2. Whole-mount specimens of NT embryos were made immediately post-injection, 2 and 4 h post-injection, and 2 h after activation. Pronuclear formation (PN) and cleavage rates of NT embryos were 82.1% and 95.7% for E1, and 53.8% and 92.8% for E2, respectively. Control PN and cleavage rates were each 100%. Subsequent embryo development arrested at the 6-cell stage (8.7%) in E1 and 5-cell stage (7.1%) in E2 but proceeded to blastocyst stage (27.3%) in the control. For whole mount specimens, donor nuclei caused premature chromosome condensation in enucleated oocyte cytoplasm, and decondensation due to activation was seen, so injected donor nuclei reconstruction had occurred. No difference was seen between E2 and E1 embryo development and whole mount specimens, but E1 PN rate was clearly higher than that of E2. So 2 h of reprogramming time is more appropriate than 4 h. In this study, most NT embryos arrested at the 4-cell stage. These results suggest that development did not proceed beyond MET (maternal-embryonic transition) which is believed to occur between the 4- and 8-cell stage in cynomolgus monkey. Further study will be necessary to find the condition that completely reprograms injected donor nuclei for cloned embryo production. Table 1. Development of cynomolgus monkey fibroblast nuclear transfer embryos This work was supported by grants from the ministry of Education, Science, Sports, and Culture (13358014, 14380382).

58 HISTONE DEACETYLASES INHIBITOR, SCRIPTAID, IS BENEFICIAL TO THE REPROGRAMMING OF SOMATIC NUCLEI FOLLOWING NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 129
Author(s):  
J. G. Zhao ◽  
J. W. Ross ◽  
Y. H. Hao ◽  
D. M. Wax ◽  
L. D. Spate ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylases ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Untreated Group ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology with potential applications in both agriculture and regenerative medicine. The reprogramming of differentiated somatic nuclei into totipotent embryonic state following NT is not efficient and the mechanism is currently unknown. However, accumulating evidence suggests that faulty epigenetic reprogramming is likely to be the major cause of low success rates observed in all mammals produced through SCNT. It has been demonstrated that increased histone acetylation in reconstructed embryos by applying histone deacetylases inhibitor (HDACi) such as trychostatin A (TSA) significantly enhanced the developmental competence in several species in vitro and in vivo. However TSA has been known to be teratogenic. Compared with TSA, Scriptaid is a low toxic but more efficient HDACi (Su GH et al. 2000 Cancer Res. 60, 3137–3142). The objectives of this study were: 1) to investigate and optimize the application Scriptaid to the NT using Landrace fetal fibroblast cells (FFCs) as donor; 2) investigate the effect of increased histone acetylation on the developmental competence of reconstructed embryos from NIH mini inbred FFCs in vitro and in vivo. The reconstructed embryos were treated with Scriptaid at different concentrations (0 nm, 250 nm, 500 nm and 1000 nm) after activation for 14 to 16 h. IVF embryos without treatment were produced as an additional control. Developmental rates to the 2-cell and blastocyst stage were determined. Developmental potential was determined by transferring Day 1 NT zygotes to the oviducts of surrogates on the day of, or one day after, the onset of estrus. Experiments were repeated at least 3 times and data were analyzed with chi-square tests using SAS 6.12 program (SAS institute, Inc., Cary, NC, USA). The percentage blastocyst of cloned embryos using Landrace FFCs as donors treated with 500 nm Scriptaid was the highest and was significantly higher than untreated group (25% v. 11%, P < 0.05). Percent cleaved was not different among four treatment groups. We used 500 nm Scriptaid for 14 to 16 h after activation for all subsequent experiments. Developmental rate to the blastocyst stage was significantly increased in cloned embryos derived from NIH mini inbred FFCs after treating with Scriptaid (21% v. 9%, P < 0.05), while the blastocyst rate in IVF group was 30%. Embryo transfer (ET) results showed that 5/6 (Transferred embryos No. were 190, 109, 154, 174, 152, and 190, respectively) surrogates (83%) became pregnant resulting in 2 healthy piglets from 2 litters (recipients received 190 and 154 embryos, respectively) in the Scriptaid treatment group, while no pregnancies were obtained in the untreated group from 5 ET (Embryos transferred No. are 140, 163, 161, 151 and 151, respectively). These results suggest that 500 nm Scriptaid treatment following activation increase both the in vitro and in vivo development of porcine SCNT embryos from NIH mini inbred FFCs and the hyperacetylation might actually improve reprogramming of the somatic nuclei after NT. Funding from the National Institutes of Health National Center for Research Resources RR018877.

Generation of parthenogenetic goat blastocysts: effects of different activation methods and culture media

Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 327-335 ◽  
Author(s):  
Hruda Nanda Malik ◽  
Dinesh Kumar Singhal ◽  
Shrabani Saugandhika ◽  
Amit Dubey ◽  
Ayan Mukherjee ◽  
...  
Keyword(s):  
Culture Media ◽  
Combined Treatment ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Quantitative Expression ◽  
Pattern Of Variation ◽  
Better Than

SummaryThe present study was carried out to investigate the effects of different activation methods and culture media on the in vitro development of parthenogenetic goat blastocysts. Calcium (Ca2+) ionophore, ethanol or a combination of the two, used as activating reagents, and embryo development medium (EDM), modified Charles Rosenkrans (mCR2a) medium and research vitro cleave (RVCL) medium were used to evaluate the developmental competence of goat blastocysts. Quantitative expression of apoptosis, stress and developmental competence-related genes were analysed in different stages of embryos. In RVCL medium, the cleavage rate of Ca2+ ionophore-treated oocytes (79.61 ± 0.86) was significantly (P < 0.05) higher than in ethanol (74.90 ± 1.51) or in the combination of both Ca2+ ionophore and ethanol. In mCR2a or EDM, hatched blastocyst production rate of Ca2+ ionophore-treated oocytes (8.33 ± 1.44) was significantly higher than in ethanol (6.46 ± 0.11) or in the combined treatment (6.70 ± 0.24). In ethanol, the cleavage, blastocyst and hatched blastocyst production rates in RVCL medium (74.90 ± 1.51, 18.30 ± 1.52 and 8.24 ± 0.15, respectively) were significantly higher than in EDM (67.81 ± 3.21, 14.59 ± 0.27 and 5.59 ± 0.42) or mCR2a medium (65.09 ± 1.57, 15.36 ± 0.52 and 6.46 ± 0.11). The expression of BAX, Oct-4 and GlUT1 transcripts increased gradually from 2-cell stage to blastocyst-stage embryos, whereas the transcript levels of Bcl-2 and MnSOD were significantly lower in blastocysts. In addition, different activation methods and culture media had little effect on the pattern of variation and relative abundance of the above genes in different stages of parthenogenetic activated goat embryos. In conclusion, Ca2+ ionophore as the activating agent, and RVCL as the culture medium are better than other tested options for development of parthenogenetic activated goat blastocysts.

scholarly journals Birth of rats following nuclear exchange at the 2-cell stage

Zygote ◽  
2003 ◽  
Vol 11 (4) ◽  
pp. 317-321 ◽  
Author(s):  
Sangho Roh ◽  
Jitong Guo ◽  
Nakisa Malakooti ◽  
John R. Morrison ◽  
Alan O. Trounson ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Perivitelline Space ◽  
Sprague Dawley ◽  
Cell Stage ◽  
Rat Embryos ◽  
Sprague Dawley Rats ◽  
Nuclear Exchange

We report full-term development of nuclear transfer embryos following nuclear exchange at the 2-cell stage. Nuclei from 2-cell rat embryos were transferred into enucleated 2-cell embryos and developed to term after transfer to recipients (NT2). Pronuclear exchange in zygotes was used for comparison (NT1). Zygotes and 2-cell embryos were harvested from 4-week-old female Sprague-Dawley rats. Nuclear transfer was performed by transferring the pronuclei or karyoplasts into the perivitelline space of recipient embryos followed by electrofusion to reconstruct embryos. Fused couplets were cultured for 4 or 24 h before being transferred into day 1 pseudopregnant recipients (Hooded Wistar) at the 1- or 2-cell stage. In vitro culture was also carried out to check the developmental competence of the embryos. In vitro development to the blastocyst stage was not significantly different between the two groups (NT1, 34.3%; NT2, 45.0%). Two of three recipients from NT1 and two of five recipients from NT2 became pregnant. Six pups (3 from NT1, 3 from NT2) were delivered from the four foster mothers. Three female pups survived; 2 from NT1 and 1 from NT2. At 2 months of age these pups appeared healthy, and were mated with Sprague-Dawley males. One rat derived from NT1 delivered 15 pups (5 males, 10 females) as did the rat from NT2 (7 males, 8 females). Our results show that by using karyoplasts from 2-cell stage embryos as nuclear donors and reconstructing them with enucleated 2-cell embryos, healthy rats can be produced.

52 EFFECT OF DONOR CELLS WITH VARIOUS DIFFERENTIATED STATUS ON THE DEVELOPMENTAL COMPETENCE OF PORCINE NUCLEAR TRANSFER EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 144
Author(s):  
J. G. Kim ◽  
E. J. Kang ◽  
M. K. Kim ◽  
S. Y. Choe ◽  
G. J. Rho
Keyword(s):  
Stem Cells ◽  
Nuclear Transfer ◽  
Statistical Significance ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Differentiation Potential ◽  
Developmental Potential ◽  
Differentiated Cells ◽  
Donor Cells ◽  
Fetal Fibroblasts

Adult stem cells are more desirable than somatic cells for nuclear transfer (NT) because of their easy reprogrammability to resemble the genome of the zygote (Zhu et al. 2004 Biol. Reprod. 70, 1088–1095). Mesenchymal stem cells (MSCs) are a heterogeneous population of uncommitted and lineage-committed cells and have a more flexible potential as donor cells for NT. The aim of this study was to compare the developmental potential of NT embryos using undifferentiated (MSCs) and differentiated cells in the same lineage (osteocyte, adipocyte, and chondrocyte) by assessing the cleavage and blastocyst rates. Fetal fibroblasts were used as NT control. MSCs obtained from the aspirated bone marrow of a neonatal pig were cultured in advanced-DMEM (ADMEM) supplemented with 5% FCS. The differentiation potential was demonstrated by culture of MSCs at passage 3 under the conditions that were favorable for adipogenic, osteogenic, and chondrogenic development (Pittenger et al. 1999 Science 284, 143–147). For NT, cells from passages 3–5 were transferred into the perivitelline space of enucleated MII oocytes that had been in vitro-matured after collection from slaughterhouse-derived ovaries. After fusion with a needle-type electrode, eggs were cultured in 7.5 µg mL−1 cytochalasin B for 3 h, and subsequently cultured in PZM-3 medium for 6 days. Statistical significance was tested using ANOVA with Bonferroni and Duncan tests. The results are presented in Table 1. The rates of cleavage and development to blastocyst stage of NT embryos varied among donor cell sources. Most eggs (92.2 ± 2.7%) cloned with MSCs cleaved, and 47.8% of eggs developed to the blastocyst stage. In contrast, NT eggs using differentiated MSCs—osteocytes, adipocytes, chondrocytes, and controls (fetal fibroblasts)—revealed significantly (P &lt; 0.05) lower cleavage (74.5, 63.4, 74.3, and 66.4%, respectively) and blastocyst development (33.7, 30.1, 36.5, and 25.5%, respectively) rates than those using undifferentiated MSCs. The results demonstrate that the genome of donor cells with different differentiated status supports embryonic development to various degrees, and multipotent MSCs might have a greater potential in producing viable cloned porcine embryos. Table 1.Development of NT embryos with undifferentiated and differentiated cells This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

185 NONSURGICAL EMBRYO TRANSFER FOR PRODUCTION OF PIGS BY IN VITRO FERTILIZATION AND SOMATIC CELL NUCLEAR TRANSFER

2010 ◽  
Vol 22 (1) ◽  
pp. 251
Author(s):  
J.-G. Yoo ◽  
M.-R. Park ◽  
H.-N. Kim ◽  
Y.-G. Ko ◽  
J.-Y. Lee ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryo Transfer ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Fibroblast Cells ◽  
Vitro Fertilization ◽  
Miniature Pigs ◽  
Donor Cells

Instead of surgical embryo transfer (ET) in the pig, nonsurgical ET is a hopeful method to increase the efficiency of biotechnology applications such as cloning and transgenesis. In this study, we conducted surgical and nonsurgical ET methods after somatic cell nuclear transfer (SCNT) with MHC miniature pig cells to find out the best condition for production of cloned miniature pigs. Ovaries were obtained from prepubertal crossbred gilts at a local slaughterhouse. Oocytes were matured for 40 to 44 h at 38.5°C under 5% CO2 in air. As donor cells, fibroblast cells were cultured from ear skin tissue of 8-month-old MHC inbred miniature pigs. Fibroblast cells were cultured, passaged (3 to 8 passages), and used as donor cells for NT. After the enucleation and injection process, eggs were held in TCM-199. For fusion, 2 DC pulses of 1.2 kV cm-1 were applied for 30 μs. Both IVF and SCNT embryos were cultured in PZM-3 medium. After IVF, 84.9% (411/484) of embryos cleaved and 27.3% (132/484) of embryos reached the blastocyst stage. In the SCNT group, 80.8% (231/286) of eggs fused and 25.9% (60/286) of embryos developed to blastocysts. For surgical ET, approximately 200 SCNT embryos were transferred into oviducts of each synchronized recipient. For nonsurgical ET, embryos were cultured in PZM-3 for 6 days after SCNT and IVF, and then good quality blastocyst stage embryos were selected for ET. The pregnancy status of recipients at Day 30 was determined by ultrasound scanning. Using Day 30 of gestation as an endpoint, the nonsurgical ET method (47.3%, 9/19) had a similar pregnancy rate as the surgical ET method (56.5%, 13/23). Further study is needed to optimize the nonsurgical ET method especially for SCNT eggs. This work received grant support from the Agenda Program (no. 200901FHT010305535), Rural Development Administration, Republic of Korea.

P–806 Neogametogenesis via oocyte replication

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A Petrini ◽  
P Xie ◽  
A Trout ◽  
Z Rosenwaks ◽  
G Palermo
Keyword(s):  
Survival Rate ◽  
Nuclear Transfer ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Preimplantation Embryo Development ◽  
Sibling Oocytes ◽  
Reconstructed Embryos ◽  
Oocyte Yield ◽  
Experimental Group

Abstract Study question Can full preimplantation embryo development be achieved from artificial oocytes created through nuclear transfer of a haploid pseudo-blastomere (HpB) into a recipient ooplast? Summary answer It is feasible to replicate the female genome and generate novel sibling oocytes that can yield full preimplantation embryo development, albeit at a reduced rate. What is known already A limitation of assisted reproduction is the number of available oocytes for embryo creation. It is feasible to utilize a somatic cell nucleus to construct novel oocytes through a process known as haploidization, in which a reverse meiosis occurs after SCNT. Similarly, producing haploid parthenogenetic constructs can generate HpBs, useful for genetic testing at the pre-fertilization level or for reproduction. It is feasible to use a HpB as a nuclear donor since it has already completed homologue segregation. Study design, size, duration This is prospective translational animal model study. Over 6 months, 556 oocytes were manipulated for the experimental group, and 158 control oocytes were employed. B6D2F1 HpBs were used to establish the procedure and acquire expertise. FVB HpBs were subsequently introduced for genetic variance. Experimental and control embryos were cultured in a time-lapse incubator (up to 96h). Cleavage parameters were compared to control. Two-sample T-tests and one-way ANOVA with Bonferroni correction were employed for statistical analysis. Participants/materials, setting, methods A cohort of oocytes was harvested from B6D2F1 or FVB superovulated mice and artificially activated by 8% ethanol. At the 8-cell stage, HpBs were exposed to nocodazole. Another cohort of B6D2F1 oocytes was enucleated for recipient ooplasts. HpBs were individually transferred into the perivitelline space of the ooplasts alongside inactivated Sendai virus. After fusion, reconstructed oocytes with spindle development were fertilized by piezo-actuated ICSI using B6D2F1 spermatozoa. Unmanipulated and fertilized B6D2F1 oocytes served as control. Main results and the role of chance A total of 158 control oocytes underwent ICSI with a 67.7% survival rate; of these, 65.4% developed to the blastocyst stage. For artificial oocyte activation (AOA), up to 10 oocytes were activated for each experiment, yielding 8 HpBs per activated oocyte. For the experimental group, 556 oocytes underwent enucleation with a 96.4% survival rate. Nuclear transfer of HpBs resulted in a 93.2% survival rate, consistent for those derived from BDF and FVB. Reconstructed oocytes showed appropriate development of a novel pseudo-meoitic spindle at a rate of 63.7% for B6D2F1 HpBs and 75.5% for FVB HpBs, and ICSI yielded a 67.1% and 57.7% survival rate, respectively. The fertilization rate for the reconstructed oocytes was 64%. Control oocytes underwent ICSI with a 67.7% survival rate. When evaluating time-lapse parameters, reconstructed embryos created via blastomere nuclear transfer showed asynchrony compared to controls beginning as early as the stage of pronuclear fading. While the majority of reconstructed embryos arrested at the 4-cell stage, of those that progressed, 11.3% of those using BDF HpBs and 14.6% of those using FVB HpBs developed to the fully expanded blastocyst stage. This corresponds to a total of 23 reconstructed embryos that developed to the morula or blastocyst stage. Limitations, reasons for caution While we used single-well embryoscope culture for morphokinetic data collection, group culture is superior to single-embryo culture for mice. Thus, developmental rates may be underestimated by this protocol. Implantation and successful pregnancy are also needed to support the clinical utility of this method in generating gametes. Wider implications of the findings: For women with diminished ovarian reserve, oocyte yield and age-related aneuploidy are limitations to achieving genotyped offspring. Nuclear transfer of HpB can generate sibling oocytes while maintaining genetic information. This model represents a promising path for expanding oocyte yield, allowing genetic assessment of sibling oocytes, and enhancing chances of procreation. Trial registration number none

In vitro development of mouse somatic nuclear transfer embryos: effects of donor cell passages and electrofusion

Zygote ◽  
2008 ◽  
Vol 16 (3) ◽  
pp. 223-227 ◽  
Author(s):  
Gang Zhang ◽  
Qing-Yuan Sun ◽  
Da-Yuan Chen
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Fibroblast Cells ◽  
Negative Effects ◽  
Cell Stage ◽  
Kunming Mouse ◽  
Donor Cells ◽  
Significant Difference ◽  
Developmental Rates

SummaryIn this study, C57BL/6 adult male mouse ear fibroblast cells and Kunming mouse M2 oocytes were used as donors and recipients, respectively, to investigate the effect of passage number on donor cells and electrofusion times on the in vitro development of nuclear transfer (NT) embryos. The results demonstrated firstly that when the ear fibroblast cells from either 2–4, 5–7 or 8–10 passages were used as donors, respectively, to produce NT embryos, the number of passages undergone by the donor cells had no significant effect on the in vitro development of NT embryos. The developmental rates for morula/blastocyst were 15.2, 13.3 and 14.0%, respectively, which were not significantly difference (p > 0.05). Secondly, when the NT embryos were electrofused, there was no significant difference between the fusion ratio for the first electrofusion and the second electrofusion (p > 0.05). The developmental rates of the 2-cell and 4-cell stages that had undergone only one electrofusion, however, were significantly higher than those that had had two electrofusions (65.7% compared with 18.4% and 36.4% compared with 6.1%; p < 0.01), furthermore the NT embryos with two electrofusions could not develop beyond the 4-cell stage. This study suggests that this protocol might be an alternative method for mouse somatic cloning, even though electrofusion can exert negative effects on the development of NT embryos.

69 APPLICATION OF BOVINE AMNIOTIC CELLS FOR PRENATAL GENETIC DIAGNOSIS AND NUCLEAR TRANSFER

2007 ◽  
Vol 19 (1) ◽  
pp. 152
Author(s):  
Y. Nagao ◽  
T. Watanabe ◽  
R. Furutani ◽  
Y. Kato ◽  
R. Takahashi ◽  
...  
Keyword(s):  
Amniotic Fluid ◽  
Nuclear Transfer ◽  
Cultured Cells ◽  
Culture Media ◽  
Genetic Diagnosis ◽  
Reconstructed Embryos ◽  
Prenatal Genetic Diagnosis ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Amniotic Cells

Amniotic fluid includes many cells derived from the fetus called 'amniotic cells'. Although these amniotic cells may have much potential as a reproduction or breeding source of the animals, there has been limited study of the potential applications of these cells. We examined the potential of bovine amniotic cells for biotechnological use. Bovine amniotic cells separated from amniotic fluid obtained from a slaughterhouse were prepared for use in all experiments. First, to examine the culture condition of amniotic cells, the cells were cultured in various culture media. Cytologic normality of the cultured cells was analyzed by chromosomal examination and Papanicolaou examination. Second, to examine the potential of cultured amniotic cells for prenatal genetic diagnosis, the cells were used for sexing by PCR. The coincidence of the results with the gender of the fetus from which the cells were derived was examined. Third, we used the cultured cells as donor cells for nuclear transfer, and examined the developmental ability of reconstructed embryos. The normality of the blastocysts derived from the reconstructed embryos was examined by chromosomal examination and transplantation to the recipient heifer. Bovine amniotic cells were cultured successfully in Amnio-max C-100� (GIBCO, Grand Island, NY, USA), which is marketed as culture medium for human amniotic cells. In all cases, the sex of cultured amniotic cells analyzed by PCR was coincident with that of the fetus from which the amniotic cells were derived. The frequencies of cleavage and development to the blastocyst stage of embryos reconstructed from amniotic cells were the same as those of fetal fibroblasts. There were no differences in the normality of chromosomal number between blastocysts derived from amniotic cells and fetal fibroblasts. A blastocyst derived from amniotic cells developed into a fetus after transplantation. DNA microsatellite analysis of the fetus at Day 64 was coincident with that of the fetus from which the amniotic cells were derived. These results indicate that bovine amniotic cells can be successfully cultured in vitro, and the cultured cells precisely reflect the genetic information of the fetus from which the cells were derived. The cultured cells also have developmental ability as donor cells for nuclear transfer. Amniotic cells may have the potential for effective reproduction and breeding using genetic and biotechnological sources.

36 TREATMENT OF PORCINE FETAL FIBROBLASTS WITH DNA METHYLATION INHIBITORS OR HISTONE DEACETYLATION INHIBITORS IMPROVES DEVELOPMENT OF NUCLEAR TRANSFER EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 118 ◽  
Author(s):  
D. I. Jin ◽  
N. Kenji ◽  
R. X. Han ◽  
S. M. Choi ◽  
M. Y. Kim ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Nuclear Transfer ◽  
Histone Deacetylation ◽  
Control Group ◽  
Fibroblast Cells ◽  
Fetal Fibroblast ◽  
Reconstructed Embryos ◽  
Donor Cells ◽  
Dna Methylation Inhibitors ◽  
Porcine Fetal Fibroblast

Epigenetic status of the genome of a donor nucleus has an important effect on the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). DNA methylation inhibitors [such as 5-aza-2′-deoxyctidine (5-aza-dC), zebularine, and RG108] and histone deacetylase inhibitors [such as trichostatin A (TSA), sodium butyrate (NaBu), and scriptaid (SCR)] have been widely used for the alteration of the levels of the epigenetic modification of somatic cells. This study was designed to investigate the DNA methylation status of porcine fetal fibroblast cells treated with TSA or 5-aza-dC and to determine whether treatments with DNA methylation inhibitors or histone deacetylation inhibitors could improve the in vitro development of porcine reconstructed embryos. When the levels of DNA methylation in the PRE-1 sequence (repeat sequence in a euchromatic region) were examined by bisulfite sequencing following treatment of porcine fetal fibroblast cells with TSA or 5-aza-dC for 1 h, DNA methylation was decreased in 5-nm or 50-nm concentrations even if they were not significantly different. To evaluate the effect of DNA methylation inhibitors and histone deacetylation inhibitors on development of porcine nuclear transfer embryos, porcine fetal fibroblast cells were treated with 5 nm of 5-aza-dC, zebularine, or RG108 for 1 h, or with 50 nm of TSA, NaBu, or SCR for 1 h, or treated with both 50 nm TSA and 5 nm 5-aza-dC for 1 h before NT. The reconstructed embryos were electrically fused and cultured in PZM-3 for 6 days. Developmental rates of the reconstructed embryos from donor cells treated with 5-aza-dC, zebularine, or RG-108 to blastocysts significantly increased compared to the control group (21.4, 23.3, and 22.1 v. 12.3%). Blastocyst rates of the reconstructed embryos from donor cells treated with TSA, SCR, and NaBu also were significantly improved compared to the control group (30.0, 23.9, and 22.4 v. 14.5%), and TSA treatment was the highest in blastocyst rates among the treated groups. However, the development rate to the blastocyst stage was not affected when the combination of TSA and 5-aza-dC was treated. In conclusion, treatment of donor cells with DNA methylation inhibitors or histone deacetylase inhibitors improved the subsequent blastocyst development of porcine reconstructed embryos even though combined treatment with both inhibitors had no beneficial effect.

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