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.

scholarly journals Refrigeration of donor cells in preparation for bovine somatic nuclear transfer

Reproduction ◽  
2001 ◽  
pp. 801-808 ◽  
Author(s):  
JL Liu ◽  
MK Wang ◽  
QY Sun ◽  
XR Zhang ◽  
LK Jiang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Cultured Cells ◽  
Donor Cell ◽  
Serum Starvation ◽  
Fresh Tissue ◽  
Reconstructed Embryos ◽  
Donor Cells ◽  
Somatic Nuclear Transfer

In mammals, preparation of donor cells for somatic nuclear transfer is very important because the character of the donor cell directly affects the efficiency and outcome of transfer. The protocols used most commonly for donor preparation are (i) disaggregating cells from fresh tissue 1-2 h before micromanipulation or (ii) trypsinizing cultured cells temporarily, after special treatments for 3-8 days (for example, serum starvation). In this study, a new simple protocol was designed, whereby the donor cells (cumulus cells) used in bovine somatic nuclear transfer were refrigerated. In brief, cultured cells at 80-100% confluency were detached using trypsin, washed by centrifugation, aliquoted into different vials and refrigerated at 4 degrees C. The density of viable cells was decreased after day 1 of refrigeration; however, the rate of decrease tended to slow down with increasing duration of refrigeration. Cells refrigerated for 15 days were seeded at a density of 5 x 10(4) ml(-1) and reached 70% confluency after day 2 of culture. Most cells had the normal number of chromosomes (2n = 60). Cells chilled at 4 degrees C for different durations were removed from refrigeration and immediately subjected to micromanipulation. The in vitro development of reconstructed embryos (fusion rates, cleavage rates, morula and blastocyst rates) indicated that there were no significant differences among treatment groups regardless of the duration of refrigeration (0-2 weeks) of the donor cells. Reconstructed embryos were transferred into the uteri of recipient cows. No significant differences were observed in established early pregnancies between embryos derived from the non-refrigerated donor cells and those derived from refrigerated donor cells. This study indicates that refrigeration of donor cells for 1-2 weeks is a feasible protocol for preparing donor cells for bovine somatic nuclear transfer, and does not compromise development in vitro and early development in vivo.

scholarly journals 75 RABBIT NUCLEAR TRANSFER WITH CULTURED SOMATIC CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 187 ◽  
Author(s):  
F. Yang ◽  
B. Kessler ◽  
S. Ewerling ◽  
E. Wolf ◽  
V. Zakhartchenko
Keyword(s):  
Nuclear Transfer ◽  
Cultured Cells ◽  
Somatic Cells ◽  
Cumulus Cells ◽  
Cell Stage ◽  
Electric Pulses ◽  
Donor Cells ◽  
Fetal Fibroblasts

Cloned rabbits have been obtained by somatic cell nuclear transfer (SCNT) only with fresh, non-cultured cumulus cells (Chesne et al. 2002 Nat. Biotechnol. 20, 366–369). For the purpose of generating transgenic animals by SCNT, donor cells must be cultured and modified prior to use as nuclear donors. The objective of this study was to optimize the SCNT procedure using cultured cumulus or fibroblast cells. MII oocytes were harvested from superovulated Zika rabbits, and maternal chromosomes were removed by demecolcine-assisted enucleation (Yin et al. 2002 Biol. Reprod. 67, 442–446). Two types of somatic cells originating from Ali/Bass rabbits were used as nuclear donors: cumulus cells collected from in vivo-matured oocytes and cultured for 1–5 passages, and primary fetal fibroblasts obtained from Day 16 fetuses and grown to confluence or starved for 4–5 days. Somatic donor cells and recipient cytoplasts were fused with 2 electric pulses (1.95 kV/cm, 25 μs each, 1 s interval). Twenty to 40 min after fusion, cloned embryos were activated first with the same electropulses as for fusion, and then immediately followed by 1 h incubation in 2 mM 6-dimethylaminopurine and 5 μg/mL cytochalasin B in culture medium (B2 medium supplemented with 10% FCS). Cloned embryos were either transferred at the 2- and 4-cell stage to asynchronized recipients or cultured in vitro for 6 days. Data were compared using chi-square test, and differences were considered significant when P < 0.05. Our results demonstrate that cloned rabbits can be produced by SCNT with cultured cells but the efficiency of this technique is still very low irrespective of the type of donor cells. Table 1. Development of cloned embryos derived from somatic cells This research was supported by the Therapeutic Human Polyclonals, Inc.

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 63 IMPROVED IN VITRO DEVELOPMENT OF PORCINE EMBRYOS PRODUCED BY NUCLEAR TRANSFER, IVF AND PARTHENOGENESIS

2005 ◽  
Vol 17 (2) ◽  
pp. 181 ◽  
Author(s):  
D. Sage ◽  
P. Hassel ◽  
B. Petersen ◽  
W. Mysegades ◽  
P. Westermann ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Culture Media ◽  
Cumulus Cells ◽  
Cell Number ◽  
Foster Mother ◽  
Chi Square ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate ◽  
Parthenogenetic Embryos

Porcine nuclear transfer (NT) is an inefficient process and it is necessary to use as many as 120 NT embryos for each foster mother to obtain small litters of live piglets. In these experiments, we evaluated the effects of culture atmosphere and medium on the development of NT embryos by monitoring blastocyst rate and cell number of Day 6 blastocysts. Age matched IVF and parthenogenetic embryos were also evaluated for comparison. For all experiments a pool of oocytes was aspirated from ovaries collected in a local abattoir. Following aspiration, oocytes were allowed to mature for 40 h in North Carolina State University (NCSU)-37 medium (supplemented with cAMP and hCG/eCG for the first 22 h). After removal of the cumulus cells, denuded oocytes with polar bodies were selected for NT, enucleated, fused with fetal fibroblasts, and sequentially activated electrically and chemically by 3 h of treatment with 6-dimethylaminopurine (6-DMAP). A second group of oocytes from the same denuded pool were maintained in TL-HEPES medium and activated in parallel with the NT group to produce parthenogenetic embryos. A third group was fertilized with frozen-thawed epididymal semen and co-cultured for ∼12 h to give IVF embryos. All three treatment groups were subdivided into a control subgroup and an experimental subgroup. In the first experiment, we compared the effects of atmosphere (20% vs. 5% oxygen) on in vitro embryonic development in NCSU-23 medium. In the second experiment, we used only the 5% oxygen concentration and compared different culture media. One subgroup was maintained in standard NCSU-23 medium and the second subgroup was cultured in a two-step system for the first 58 h in modified NCSU-23 (without glucose but supplemented with 2.0 mM lactate and 0.2 mM pyruvate), followed by addition of glucose to give a final concentration of 5.55 mM. Data were statistically analyzed by analysis of variance and chi square test. Blastocyst rate and mean cell number in all three embryo groups were improved under 5% oxygen. The most dramatic effect was observed in the NT group, in which the blastocyst rate increased significantly (P < 0.001) from 6.7% ± 5.9 (n = 279) to 19.6% ± 8.9 (n = 250) and mean cell number increased from 17.7 ± 12.1 to 25.8 ± 10.3 cells per blastocyst. With 5% oxygen there was also an increase of blastocyst rates and mean cell numbers in both IVF and parthenogenetic groups. In the second experiment, blastocyst rate for NT embryos increased significantly (P < 0.05) from 21.8% ± 7.6 (n = 242) in conventional NCSU-23 to 31.5% ± 11.0 (n = 271) in the modified system whereas there was almost no difference in the mean cell number of both groups (29.2 ± 4.3 vs. 31.5 ± 5.3). In the groups of IVF and parthenogenetic embryos no difference was found. These results indicate that both the reduced oxygen and the modified culture medium are important for pre-implantation development of porcine nuclear transfer embryos.

102 RABBIT CLONING: HISTONE ACETYLATION STATUS OF DONOR CELLS AND CLONED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 168
Author(s):  
V. Zakhartchenko ◽  
F. Yang ◽  
R. Hao ◽  
E. Wolf
Keyword(s):  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Epigenetic Mark ◽  
Cloning Efficiency ◽  
Developmental Potential ◽  
Acetylation Status ◽  
Donor Cells ◽  
Fetal Fibroblasts

Epigenetic status of the genome of a donor nucleus is likely to be associated with the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). Prevention of epigenetic errors by manipulation of the epigenetic status of donor cells is expected to result in improvement of cloning efficiency. In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Ali/Bas) into metaphase II (MII) oocytes and analyzed the levels of histone H3K9 acetylation in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with one or two blastomeres from in vitro-fertilized or parthenogenetic embryos. Histone acetylation in donor cells and cloned embryos was detected by anti-acH3K9 antibody using Western immunoblot analysis or immunochemistry, respectively. Data were analyzed by chi-square (developmental rates) or Student-Newman-Keuls (histone acetylation) test. The levels of acetylated histone H3K9 were higher in RCCs than in RFFs (P &lt; 0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC-cloned embryos induced a higher initial pregnancy rate as compared to RFF-cloned embryos (40% vs. 20%; P &lt; 0.05). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed; a live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly (P &lt; 0.05) increased the level of histone H3K9/14 acetylation and the proportion of nuclear transfer embryos developing to blastocyst (49% vs. 33% with non-treated RFF; P &lt; 0.05). The distribution of signals for acH3K9 in either group of cloned embryos did not resemble that in in vivo-fertilized embryos, suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo-derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and can be a useful epigenetic mark to predict efficiency of SCNT rabbits. This work was supported by the Bayerische Forschungsstiftung and by Therapeutic Human Polyclonals, Inc.

27 SODIUM CHLORIDE TREATMENT OF CELL MEMBRANE-PERMEABILIZED NUCLEAR DONOR CELLS FACILITATES THE DISPLACEMENT OF THE SOMATIC HISTONE H1 AND HMG-17 PROTEINS IN RECONSTRUCTED PORCINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 122
Author(s):  
L. Che ◽  
V. Bordignon
Keyword(s):  
Sodium Chloride ◽  
Cell Membrane ◽  
Chromatin Structure ◽  
Nuclear Transfer ◽  
Chromatin Binding ◽  
Salt Extraction ◽  
Reconstructed Embryos ◽  
High Order Chromatin Structure ◽  
Donor Cells ◽  
High Order Chromatin

Developmental efficiency of somatic cell-reconstructed embryos depends on extensive remodeling of chromatin structural components. Due to their importance for maintaining the high-order chromatin structure and controlling DNA functions, including replication, transcription, repair, and recombination, histones and other chromatin-binding proteins represent leading choice markers to investigate nuclear remodeling in reconstructed embryos. The main objective of this study was to investigate whether or not the exposure of cell membrane permeabilized nuclear donor cells to sodium chloride (salt-extraction) would facilitate the displacement of chromatin-binding proteins in reconstructed porcine embryos. Both linker histone H1 (H1) and high-mobility group (HMG) proteins are known to affect gene expression through the modulation of the high-order chromatin structure. Standard methods of oocyte enucleation and electrofusion were applied for embryo reconstruction using in vitro-matured oocytes and control or salt-extracted fetal fibroblast cells. For salt-extraction, confluent cell cultures were washed with Ca2+/Mg2+-free Hank's balanced salt solution (HBSS); cells were permeabilized by incubation with 1 µg/mL of streptolysin O at 37°C for 30 min in HBSS, and then maintained in Tris-NaCl buffer (10 mM Tris-HCl, 0.5 mM MgCl2, 0.7 M NaCl, 1 M sucrose) for 5 min. After salt-extraction, cells were rinsed and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 2 mM CaCl2 for 1 h at 37.5°C for membrane resealing prior to nuclear transfer. Reconstructed embryos were activated using ionomycin (15 µM/5 min) and strontium chloride (Sr2+; 10 mM/4 h), and then cultured in PZM-3 medium. Immunostaining for H1 and HMG-17 was performed in nuclear donor cells and embryos at different stages after reconstruction. The time required for H1 displacement in transplanted nuclei was reduced by the salt-extraction treatment (Table 1). Salt-extracted cells showed a stronger HMG-17 cytoplasmic signal compared to control cells. The proportion of HMG-17-positive reconstructed embryos at 1, 3, and 6 h was 54 vs. 19, 57 vs. 44, and 75 vs. 62, for control and salt-extracted cells, respectively. These data suggest that salt-extraction prior to nuclear transplantation enhances the remodeling of chromatin structure in embryos reconstructed with somatic cell nuclei. Table 1. Proportion (n) of H1-positive stained embryos after different times from parthenogenetic activation (PA) and nuclear transfer using control (NT-control) or salt-extracted (NT-extracted) cells This work was supported by a NSERC Discovery Grant to VB.

257 EXPRESSION OF DEVELOPMENTAL GENES IN PORCINE NUCLEAR TRANSFER EMBRYOS RECONSTRUCTED WITH BONE MARROW MESENCHYMAL STEM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 236
Author(s):  
B. Mohana Kumar ◽  
H.-F. Jin ◽  
J.-G. Kim ◽  
S. Balasubramanian ◽  
S.-Y. Choe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Nuclear Transfer ◽  
Expression Profiles ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Marrow Mesenchymal Stem Cells

Abnormal gene expression is frequently observed in nuclear transfer (NT) embryos and is one of the suggested causes of the low success rates of this approach. Recent study has suggested that adult stem cells may be better donor cells for NT, as their less differentiated state may ease epigenetic reprogramming by the oocyte (Kato et al. 2004 Biol. Reprod. 70, 415-418). In the present study, we investigated the expression profile of some selected genes involved in the development of the pre-implantation embryos of in vivo- and NT-derived origin using bone marrow mesenchymal stem cells (MSCs) and porcine fetal fibroblasts (pFF) as donors. Isolated population of MSCs from porcine bone marrow were characterized by cell-surface antigen profile (CD13pos, CD105pos, CD45neg, and CD133neg) and by their extensive consistent differentiation to multiple mesenchymal lineages (adipocytic, osteocytic and chondrocytic) under controlled in vitro conditions (Pittenger et al. 1999 Science 284, 143-147). Primary cultures of pFF from a female fetus at <30 days of gestation were established. for NT, donor cells at 3-4 passages were employed. Embryos cloned from MSCs showed enhanced developmental potential compared to pFF cloned embryos, indicated by higher rates of blastocyst formation (15.3% � 4.8 and 9.0% � 3.9, respectively) and total cell number (31.5 � 7.2 and 20.5 � 5.4, respectively) in Day 7 blastocysts. Total RNA was extracted from pools (triplicates) of 10 embryos each of 8-cell, morula, and blastocyst stages of in vivo and NT origin using Dynabeads� mRNA DIRECT" kit (Dynal, Oslo, Norway). Reverse transcription was performed with a Superscript" III cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed on a Light cycler� using FastStart DNA Master SYBR Green I (Roche Diagnostics, Mannheim, Germany). The expression profiles of genes involved in transcription (Oct-4, Stat3), DNA methylation (Dnmt1), de novo methylation (Dnmt3a), histone deacetylation (Hdac2), anti-apoptosis (Bcl-xL), and embryonic growth (Igf2r) were determined. The mRNA of H2a was employed to normalize the levels. Significant differences (P < 0.05) in the relative abundance of Stat3, Dnmt1, Dnmt3a, Bcl2, and Igf2r were observed in pFF NT embryos compared with in vivo-produced embryos, whereas embryos derived from MSCs showed expression patterns similar to those of in vivo-produced embryos. However, Oct-4 and Hdac2 revealed similar expression profiles in NT- and in vivo-produced embryos. These results indicate that MSC-derived NT embryos had enhanced embryonic development and their gene expression pattern more closely resembled that of in vivo-produced embryos. Hence, less differentiated MSCs may have a more flexible potential in improving the efficiency of the porcine NT technique. This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

59 HIGHER BLASTOCYST FORMATION OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DOES NOT GUARANTEE BETTER PREGNANCY IN PIG

2007 ◽  
Vol 19 (1) ◽  
pp. 147
Author(s):  
E. Lee ◽  
K. Song ◽  
Y. Jeong ◽  
S. Hyun
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Skin Fibroblasts ◽  
Miniature Pig ◽  
Donor Cells ◽  
Fetal Fibroblasts

Generally, blastocyst (BL) formation and embryo cell number are used as main parameters to evaluate the viability and quality of in vitro-produced somatic cell nuclear transfer (SCNT) embryos. We investigated whether in vitro development of SCNT pig embryos correlates with in vivo viability after transfer to surrogates. For SCNT, cumulus–oocyte complexes (COCs) were matured in TCM-199 supplemented with follicular fluid, hormones, EGF, cysteine, and insulin for the first 22 h and in a hormone-free medium for 18 h. Three sources of pig skin cells were used as nuclear donor: (1) skin fibroblasts of a cloned piglet that were produced by SCNT of fetal fibroblasts from a Landrace × Yorkshire × Duroc F1 hybrid (LYD), (2) skin fibroblasts of a miniature pig having the human decay accelerating factor gene (hDAF-MP), and (3) skin fibroblasts of a miniature pig with a different strain (MP). MII oocytes were enucleated, subjected to nuclear transfer from a donor cell, electrically fused, and activated 1 h after fusion. SCNT embryos were cultured in a modified NCSU-23 (Park Y et al. 2005 Zygote 13, 269–275) for 6 days or surgically transferred (110–150 fused embryos) into the oviduct of a surrogate that showed standing estrus on the same day as SCNT. Embryos were examined for cleavage and BL formation on Days 2 and 6, respectively (Day 0 = the day of SCNT). BLs were examined for their cell number after staining with Hoechst 33342. Pregnancy was diagnosed by ultrasound 30 and 60 days after embryo transfer. Embryo cleavage was not affected by donor cells (82, 81, and 72% for LYD, hDAF-MP, and MP, respectively), but BL formation was higher (P &lt; 0.05) in hDAF-MP (16%) than in LYD (9%) and MP (6%). MP showed higher (P &lt; 0.05) BL cell number (46 cells/BL) than hDAF-MP (34 cells) but did not show a difference from LYD (37 cells). LYD and MP showed higher pregnancy rates (Table 1) on Days 30 and 60, even though they showed lower BL formation in vitro. Due to a relatively small number of embryo transfers through a limited period, we could not exclude any possible effects by seasonal or operational differences. These results indicated that pregnancy did not correlate with in vitro BL formation of SCNT pig embryos but rather were affected by the source of donor cells. Table 1.In vivo development of somatic cell nuclear transfer pig embryos derived from different sources of donor cells This work was supported by the Research Project on the Production of Bio-organs (No. 200506020601), Ministry of Agriculture and Forestry, Republic of Korea.

29 TREATMENT OF DONOR CELLS WITH XENOPUS OOCYTE EXTRACT ENHANCED SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT

2012 ◽  
Vol 24 (1) ◽  
pp. 126
Author(s):  
X. Yang ◽  
J. Mao ◽  
E. M. Walters ◽  
M. T. Zhao ◽  
K. Lee ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Natural Science ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Control Group ◽  
Blastocyst Formation ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Frog Oocyte

Somatic cell nuclear transfer (SCNT) efficiency in pigs and other species is still very low. This low efficiency and the occurrence of developmental abnormalities in offspring has been attributed to incomplete or incorrect reprogramming. Cytoplasmic extracts from both mammalian and amphibian oocytes can alter the epigenetic state of mammalian somatic nuclei as well as gene expression to more resemble that of pluripotent cells. Rathbone et al. (2010) has showed that pretreating somatic donor cells with frog oocyte extract (FOE) increased live birth in ovine. Liu et al. (2011) also reported that treating donor cells with FOE enhanced handmade clone embryo development in pigs. The aim of this study was to evaluate the early development of cloned embryos produced with porcine GFP fibroblasts pre-treated with a permeabilizing agent, digitonin and matured frog oocyte extract. Frog egg cytoplasmic extract was prepared from one frog's oocytes after being matured in vitro to MII stage. The experiment included 2 groups. In the FOE-treated group, GFP-tagged fetal fibroblasts were permeabilized by digitonin (15 ng mL–1) and incubated in FOE containing an ATP-regenerating system (2.5 mM ATP, 125 μM GTP, 62.5 μg mL–1 of creatine kinase, 25 mM phosphocreatine and 1 mM NTP) at room temperature (24°C) for 2 h; cell membranes were re-sealed by culturing in 10% FBS in DMEM media for 2.5 h at 38.5°C before used as donor cells. In the control group, the same donor cells were treated with digitonin, but without frog oocyte extract incubation. The SCNT embryos were produced by using the 2 groups of donor cells as described above. In total, 305 control and 492 FOE oocytes were enucleated from 8 biological replicates. Two hundred fifty control and 370 FOE couplets were fused and cultured in porcine zygote medium 3. Percent cleavage was recorded on Day 2 and the percent blastocyst formation was determined on Day 7 (SCNT day = 0). In addition, the number of nuclei in the blastocysts was recorded on Day 7. Percent fusion, cleavage, blastocyst formation and number of nuclei in blastocysts were analysed by using SAS software (v9.2), with day and treatment class as main effects. There was no difference in percent fusion (FOE, 76.2 ± 2.5% vs control, 80.8 ± 2.8%) or in cleavage (FOE: 74.8 ± 2.5% vs control: 74.6 ± 2.9%). Only green blastocysts with 16 or more nuclei were considered to be a true SCNT blastocyst. The percent blastocyst was higher in the FOE group than that in the control (13.9 ± 0.8% vs 9.5 ± 0.9%, P < 0.05), whereas the number of nuclei in the blastocysts was not different between the 2 groups (39.7 ± 2.4, 35.9 ± 3.8 for FOE and control, respectively). In conclusion, our study demonstrated that pre-treatment of donor cells with digitonin and Xenopus MII oocyte extract increased porcine SCNT embryo development to blastocyst and cloning efficiency. Funded by the National Natural Science Foundation of China (NO. 31071311), Natural Science Foundation of Fujian Province of China (No. 2009J06017) and NIH U42 RR18877.

36 CLONED BEAGLE DOGS DERIVED FROM FETAL FIBROBLASTS BY NUCLEAR TRANSFER

2008 ◽  
Vol 20 (1) ◽  
pp. 98 ◽  
Author(s):  
S. G. Hong ◽  
G. Jang ◽  
M. K. Kim ◽  
H. J. Oh ◽  
J. E. Park ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Nuclear Transfer ◽  
Coat Color ◽  
Cell Types ◽  
Beagle Dogs ◽  
Black And White ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Identical Nucleus

Somatic cell nuclear transfer (SCNT) has been successfully performed in various mammals including sheep, cow, pig, and mouse using a variety of somatic cell types as nuclear donors. Several reports of livestock SCNT indicate that fetal fibroblasts are superior to adult fibroblasts as donor cells. In canine SCNT, however, only adult ear fibroblasts have been used as donor cells (Lee et al. 2005 Nature 436, 641; Jang et al. 2007 Theriogenology 67, 941–947). Accordingly, in the present study, we evaluated the ability of canine fetal fibroblasts to support fetal development to term after nuclear transfer. For SCNT, in vivo-matured oocytes flushed (approximately 72 h after ovulation) from the oviducts of six estrus females were used. Donor cells (fetal fibroblasts) were isolated from the fetus of a beagle bitch obtained at 28 days after artificial insemination. Before using fetal fibroblasts as donor cells, sex was determined by SRY gene detection using PCR. Oocytes were enucleated, microinjected with a female fetal fibroblast, fused by electrical stimulation, and activated chemically (Jang et al. 2007). A total of 50 cloned presumptive embryos were transferred (Day 0) into the oviducts of two naturally synchronous recipient bitches. One pregnancy, detected by ultrasonography on Day 23, was maintained to term and two healthy female puppies weighing 250 and 260 g were born by natural delivery on Day 60. They were genotypically identical to the donor cells, and had phenotypically similar black and white coat color patterns. Analysis of their mtDNA distribution showed that mtDNA in the two cloned beagles originated from one of the six oocyte donor dogs. In conclusion, our results demonstrate the potential of using fetal fibroblasts to facilitate nuclear transfer in the dog. The cloned beagle dogs, which had identical nucleus and mitochondrial DNA, will be provided for biomedical research as bioresources. This study was financially supported by KOSEF (grant # M10625030005-07N250300510) and the Korean MOE, through the BK21 program for Veterinary Science.

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