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.

scholarly journals 32 EFFICIENCY OF FEMALE-DERIVED DONOR CELLS ON HIGH POSTNATAL SURVIVAL IN PIG CLONING

2005 ◽  
Vol 17 (2) ◽  
pp. 166
Author(s):  
S.K. Cho ◽  
M.R. Park ◽  
D.N. Kwon ◽  
E.K. Lee ◽  
S.J. Kang ◽  
...  
Keyword(s):  
Adenosine Monophosphate ◽  
Cell Number ◽  
Developmental Competence ◽  
Cell Stage ◽  
Male And Female ◽  
Electric Pulses ◽  
Donor Cells ◽  
Fetal Fibroblasts

The present study was conducted to investigate the developmental competence of male and female somatic cell derived nuclear transfer (NT) porcine embryos and also the production and survival efficiency of cloned male and female piglets. Maturation of porcine COCs was accomplished by incubation in NCSU-23 medium supplemented with 0.6 mM cysteine, 10% porcine follicular fluid, 1 mM dibutyryl cyclic adenosine monophosphate, and 0.1 IU/mL human menopausal gonadotrophin for 20 h and then culture without dbcAMP and hMG for another 18 to 24 h. Fetal cells were isolated from a male fetus and two female fetuses, and cultured in ES-DMEM medium containing 10% FCS. Enucleated oocytes were fused with fetal fibroblasts (passage 4 to 15). Reconstructed embryos were cultured in NCSU-23 with 4 mg/mL BSA under mineral oil at 39°C in 5% CO2 in air for up to 6 days. NT eggs that had been activated with electric pulses and cultured for 1 or 2 days were transported to the experimental station in modified NCSU-23 with antibiotics. NT embryos were surgically transferred into the oviducts of recipients between Day 27 and Day 30; pregnancy was determined by ultrasound. The potential of NT embryos to develop into blastocysts was not different among donor cells of different origins. However, the mean cell number of in vivo female and male blastocysts (83.8 ± 46.2 to 99.2 ± 55.7) was higher than in in vitro culture of NT groups (31.4 ± 8.29 to 33.2 ± 10.15). A total of 11,535 NT embryos (1- to 8-cell stage) were surgically transferred into 66 surrogate gilts. Among fourteen pregnant gilts, four recipients aborted during the period of conception. Five pregnant gilts delivered fifteen female piglets, 1.28 ± 0.33 kg (0.48∼1.83 kg) in female piglets and 0.84±0.25 kg (0.45∼1.25 kg) in male piglets. Nine live cloned female (60.0%) and four male piglets (18.2%) were produced. According to these results, survival rates and birth weights of female cloned piglets were higher than those of cloned male piglets (P < 0.05). This study suggests that use of female, compared with male, fetal fibroblast cells as nuclear donors may increase cloning outcomes. This work was supported in part by a grant program from RDA(Biogreen21) and Cho-A, Republic of Korea.

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.

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.

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.

In vitro development of reconstructed bovine embryos and fate of donor mitochondria following nuclear injection of cumulus cells

Zygote ◽  
2001 ◽  
Vol 9 (3) ◽  
pp. 211-218 ◽  
Author(s):  
Jeong Tae Do ◽  
Kwon Ho Hong ◽  
Bo Yon Lee ◽  
Seung Bo Kim ◽  
Nam-Hyung Kim ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Control Group ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Developmental Potential ◽  
Donor Cells

In this study we examined the developmental potential of reconstructed embryos and the fate of donor mitochondria during preimplantation development after nuclear transfer in cattle. Isolated cumulus cells were used as donor cells in nuclear transfer. Cumulus cells labelled with MitoTracker Green FM fluorochrome were injected into enucleated bovine MII oocytes and cultured in vitro. MitoTracker labelling on donor cells did not have a detrimental effect on blastocyst formation following nuclear transfer. Cleavage rate was about 69% (56/81) and blastocyst formation rate was 6.2% (5/81) at 7 days after nuclear transfer. The labelled mitochondria dispersed to the cytoplasm and became distributed between blastomeres and could be identified up to the 8- to 15-cell stage. Small patches of mitochondria were detected in some 8- to 15-cell stage embryos (5/20). However, donor mitochondria were not detected in embryos at the 16-cell stage and subsequent developmental stages. In the control group, mitochondria could be identified in arrested 1-cell embryos up to 7 days after nuclear transfer. These results suggest that disappearance of the labelled donor mitochondria in nuclear transfer bovine embryos is not due to fading of the fluorochrome marker, but is rather an as yet undefined cytoplasmic event.

scholarly journals Expression of Recombinant Human Alpha-Lactalbumin in the Milk of Transgenic Goats Using a Hybrid Pomoter/Enhancer

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Yu-Guo Yuan ◽  
Liyou An ◽  
Baoli Yu ◽  
Shaozheng Song ◽  
Feng Zhou ◽  
...  
Keyword(s):  
Cell Lines ◽  
Nuclear Transfer ◽  
Goat Milk ◽  
Cell Stage ◽  
Specific Expression ◽  
Transgenic Cell ◽  
Donor Cells ◽  
Transgenic Goats

To improve nutrient content of goat milk, we describe the construction of a vector (pBLAC) containing a hybrid goatβ-lactoglobulin (BLG) promoter/cytomegalovirus (CMV) enhancer. We also describe the generation of transgenic goats expressing rhLA by somatic cell nuclear transfer (SCNT). Of 334 one-cell stage embryos derived from three transgenic cell lines and 99 embryos derived from non-transgenic (NT) cells surgically transferred to the oviducts of 37 recipients, two recipients delivered two kids (2%) from the non-transfected line and five recipients delivered six kids (1.8%) from transgenic cell lines, three of which died within 2 days. Compared to the NT donor cells, transfection of donor cells does not negatively affect the development of nuclear transfer embryos into viable transgenic offspring. However, the clone efficiency in cell line number 1 was lower than that in numbers 2 and 3, and in the NT lines (0.9% versus 1.9% 2.4% and 2%;P<0.05). Two transgenic cloned goats expressed rhLA in the milk at 0.1–0.9 mg/mL. The mammary gland-specific expression vector pBLAC with hybrid BLG/CMV can drive the hLA gene to expressin vitroandin vivo. These data establish the basis for use of a hybrid promoter/enhancer strategy to produce rhLA transgenic goats.

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.

44 IN VITRO AND IN VIVO SURVIVABILITY ON VITRIFIED EMBRYOS OBTAINED BY BOVINE SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 131
Author(s):  
K. Kaneyama ◽  
S. Kobayashi ◽  
S. Matoba ◽  
Y. Hashiyada ◽  
K. Imai ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Survival Rates ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Nuclear Transplantation ◽  
Embryo Cryopreservation

Although many studies have been conducted on somatic cell nuclear transfer, there are only a few reports on cryopreservation of reconstructed embryos after nuclear transplantation. The objective of this study was to examine in vitro or in vivo development of vitrified blastocysts obtained by nuclear transfer. Nuclear transfer was carried out according to the procedure of Goto et al. (1999 Anim. Sci. J. 70, 243–245), and conducted using abattoir-derived oocytes and cumulus cells derived by ovum pickup from Holstein and Japanese Black cows. Embryos were vitrified as described by Saito et al. (1998 Cryobiol. Cryotech. 43, 34–39). The vitrification solution (GESX solution) was based on Dulbecco's PBS containing 20% glycerol (GL), 20% ethylene glycol (EG), 0.3 M sucrose (Suc), 0.3 M xylose (Xyl), and 3% polyethylene glycol (PEG). The blastocysts were equilibrated in three steps, with 10% GL, 0.1 M Suc, 0.1 M Xyl, and 1% PEG for 5 min (1); with 10% GL, 10% EG, 0.2 M Suc, 0.2 M Xyl, and 2% PEG for 5 min (2) and GESX solution (3). After transfer to GESX, equilibrated embryos were loaded to 0.25-mL straws and plunged into liquid nitrogen for 1 min. The vitrified blastocysts were warmed in water (20°C) and diluted in 0.5 M and 0.25 M sucrose for 5 min each. Equilibration and dilution procedures were conducted at room temperature (25–26°C). After dilution, the vitrified blastocysts were cultured in TCM-199 supplemented with 20% fetal calf serum and 0.1 mM β-mercaptoethanol at 38.5°C under gas phase of 5% CO2 in air. In Experiment 1, survival rates after vitrification were compared between the nuclear transfer and the IVF blastocysts. Survival rates of vitrified nuclear transfer blastocysts (n = 60, Day 8) at 24 and 48 h were 70.0% and 56.7%, respectively, and those of vitrified IVF blastocysts (n = 41) were 82.9% and 82.9%, respectively. There were no significant differences in survival rates at 24 and 48 h between the two groups. In Experiment 2, one (VIT-single) or two (VIT-double) vitrified and one (nonVIT-single) or two (nonVIT-double) nonvitrified reconstructed blastocysts per animal were transferred into Holstein dry cows. The result of Experiment 2 is shown in Table 1. This experiment demonstrated that the vitrification method in this study can be used for cloned embryo cryopreservation but the production rate should be improved. Table 1. Comparison of survival rates of vitrified or nonvitrified cloned embryos after transfer

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.

180 IDENTIFICATION AND QUANTIFICATION OF DIFFERENTIALLY REPRESENTED TRANSCRIPTS IN PREIMPLANTATION BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 169 ◽  
Author(s):  
C. E. McHughes ◽  
G. K. Springer ◽  
L. D. Spate ◽  
R. Li ◽  
R. J. Woods ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Nuclear Transfer ◽  
Developmental Stages ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Production Techniques

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. Table 1. Differentially represented transcripts between developmental stages

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