Generation of Dwarf Goat (Capra hircus) Clones Following Nuclear Transfer with Transfected and Nontransfected Fetal Fibroblasts and In Vitro-Matured Oocytes1

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.

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Streptolysin-O Treatment of Fetal Fibroblasts Improves Cell Fusion and In Vitro Development of Porcine Nuclear Transfer Embryos

Journal of Reproduction and Development ◽

10.1262/jrd.20047 ◽

2009 ◽

Vol 55 (3) ◽

pp. 236-239 ◽

Cited By ~ 5

Author(s):

Kenji NARUSE ◽

Yan-Shi QUAN ◽

Baek-Chul KIM ◽

Su-Min CHOI ◽

Chang-Sik PARK ◽

...

Keyword(s):

Cell Fusion ◽

Nuclear Transfer ◽

In Vitro Development ◽

Streptolysin O ◽

Fetal Fibroblasts ◽

Vitro Development

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

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab102 ◽

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 < 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 < 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 < 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 < 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.

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30 NUCLEAR AND MICROTUBULE DYNAMICS IN SOMATIC CELL NUCLEAR TRANSFER SHEEP EMBRYOS ACTIVATED WITH T-DIMETHYLAMINOPURINE AND CYCLOHEXIMIDE

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab30 ◽

2006 ◽

Vol 18 (2) ◽

pp. 123

Author(s):

G. Coppola ◽

B.-G. Jeon ◽

B. Alexander ◽

E. St. John ◽

D. H. Betts ◽

...

Keyword(s):

Cell Cycle ◽

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Laser Scanning ◽

Cell Cycle Progression ◽

Treated Group ◽

Blastocyst Development ◽

Fetal Fibroblasts

The early reprogramming events following somatic cell nuclear transfer (SCNT) determine the fate of the cloned embryo and its development to a healthy viable offspring. In the present study, we undertook a detailed immunocytochemical study of the patterns of both microtubules and chromatin during the first cell cycle of sheep nuclear transfer embryos after fusion and artificial activation using either 6-dimethylaminopurine (6-DMAP) or cycloheximede (CHX). Sheep oocytes were collected from abattoir ovaries and matured in vitro for 18-20 h and enucleated; fetal fibroblasts were transplanted using standard SCNT techniques. Reconstructed cell-cytoplast couplets were fused and activated with ionomycin, followed by culture in two separate groups containing 6-DMAP (2 mM) or CHX (10 �g/mL) for 3 h. Following activation, embryos were cultured in in vitro culture (IVC) medium for blastocyst development. Embryos (n = 15, 3 replicates) were randomly removed from culture at various time points and stained using standard immunocytochemical methods to observe microtubule and nuclear configurations. Images were captured using laser scanning confocal microscopy. Results reveled that at 1 h post-fusion, 63.3% of reconstructed embryos underwent nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) was apparent as chromosomes were situated on a non-polar spindle. The remaining embryos showed abnormal spindle and DNA configurations including chromosome outliers, congression failure, and non-NEBD. At 1 h post-activation (hpa), the embryos treated with 6-DMAP had already formed a clearly visible pronucleus (diameter 6-8 �m), whereas in the CHX-treated group, none of the embryos were at pronuclear stage; instead most of the latter embryos showed two masses of chromatin. At 1 hpa, 6-DMAP- and CHX-treated embryos showed one swelled pronucleus with a mean diameter of 8.4 � 1.3 �m and 25.8 � 0.8 �m, respectively (P < 0.05). At 16 hpa, embryos from both treatment groups still showed one swelled pronucleus. In the 6-DMAP-treated embryos, most of the embryos showed a metaphase spindle with aligned chromosomes of the first mitotic division as early as 18-10 hpa, whereas in the CHX-treated group embryos were still at the pronuclear stage. Typical 2-cell division was seen in most of the 6-DMAP-treated embryos between 24 and 30 hpa, but it was slightly delayed in CHX-treated embryos (32-35 hpa). Blastocyst development rates in the 6-DMAP- and CHX-treated groups were 21.4 � 5.6% and 14.0 � 6.3%, respectively (P < 0.05). In summary, artificial activating agents 6-DMAP and CHX exhibited different effects on chromatin remodeling, cell cycle progression, and the degree of pronuclear swelling which may explain the poor developmental rates and abnormal chromosome complements observed for cloned embryos. This work was funded by NSERC, OMAF, and International Council for Canadian Studies.

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58 EFFICIENCY OF OVUM PICKUP AND EMBRYO PRODUCTION IN VITRO IN CLONED CATTLE

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab58 ◽

2006 ◽

Vol 18 (2) ◽

pp. 137

Author(s):

A. Lucas-Hahn ◽

E. Lemme ◽

K.-G. Hadeler ◽

H.-G. Sander ◽

H. Niemann

Keyword(s):

Nuclear Transfer ◽

Transvaginal Ultrasound ◽

Statistical Significance ◽

Blastocyst Stage ◽

Ultrasound Guided ◽

Embryo Production ◽

Fetal Fibroblasts ◽

Developmental Rates ◽

In Vitro Embryo Production

The reproductive performance of cloned cattle was investigated by assessing the efficiency of transvaginal ultrasound-guided ovum pickup (OPU) and embryo production in vitro. Fetal fibroblasts from the endangered species, German Blackpied Cattle, had been used for nuclear transfer to produce three live cloned offspring (Lucas-Hahn et al. 2002 Theriogenology 57, 433). In the three cloned animals at 12–20 months of age, OPU was performed once per week and the total number of collected oocytes was recorded. In the case of Blondie, the procedure was terminated due to too small ovaries associated with insufficient function. Oocytes suitable for IVF were matured in vitro for 24 h and fertilized in vitro with the semen of a fertile bull. Oocytes derived from abbatoir ovaries were processed in parallel as controls. Embryos were in vitro-cultured in SOFaaBSA medium. Cleavage and developmental rates up to the morula/blastocyst stage were recorded in all groups. Statistical significance was tested using ANOVA and the Student-Newman-Keuls test. The results are presented in Table 1. Embryos from clones had lower cleavage and blastocyst rates compared to those derived from abattoir oocytes. However, results may have been confounded by potential OPU effects. Some of the blastocysts produced from Blacky (n = 5) and Paula (n = 2) were transferred to recipients. Two pregnancies resulted from the Paula transfers. The two male calves were delivered normally. After the completion of this experiment, all three cloned animals were artificially inseminated, became pregnant, delivered healthy calves, and are pregnant again at present. Further studies are needed to explore the fertility of cattle derived from somatic cloning. Table 1. OPU and in vitro embryo production in cloned cattle

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43 COMPARISON OF THE EFFECTS OF PRE-TREATMENT WITH SODIUM CHLORIDE, SUCROSE AND TREHALOSE ON DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab43 ◽

2009 ◽

Vol 21 (1) ◽

pp. 121

Author(s):

L. Lin ◽

P. Kragh ◽

S. Purup ◽

Y. Du ◽

X. Zhang ◽

...

Keyword(s):

Osmotic Stress ◽

Nuclear Transfer ◽

Technical Assistance ◽

Recovery Period ◽

Cell Number ◽

Developmental Competence ◽

Fetal Fibroblasts ◽

Pre Treatment ◽

Maximum Humidity

Modified environmental stress was reported to improve the developmental competence and cryotolerance of porcine oocytes, such as high hydrostatic pressure (HHP; Du et al. 2008 Cloning Stem Cells, Epub ahead of print) and osmotic stress (Lin et al. 2008 Reprod. Biomed. Online, in press). HHP also improved the cryotolerance of bovine and murine blastocysts (Pribenszky et al. 2005a Reprod. Dom. Anim. 40, 338–344; Pribenszky et al. 2005b Anim. Reprod. Sci. 87, 143–150). In the present study we compared the effects of NaCl with that of concentrated solutions of two non-permeable osmotic agents, sucrose and trehalose on in vitro maturated oocytes. A total of 2050 slaughterhouse-derived porcine cumulus–oocyte complexes (COCs) were matured for 41–42 h, and then put into 800 μL T2 (HEPES-buffered TCM-199 [Earle’s salts] with 2% cattle serum) supplemented with additional NaCl, sucrose or trehalose with the same osmotic level (588 mOsmol) in 4-well dishes and incubated for 1 h at 38.5°C in air. COCs incubated in T2 under the same conditions without supplementation were used as controls. Subsequently COCs were incubated in IVM medium for 1 h at 38.5°C in 5% CO2 with maximum humidity. After this recovery period cumulus cells were removed with 1 mg mL–1 hyaluronidase and pipetting, and oocytes were used as recipients for somatic nuclear transfer with handmade cloning (HMC) method. Porcine fetal fibroblasts were used as nuclear donor cells. Embryo culture was performed in PZM-3 medium (Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) in 5% CO2, 5% O2 and 90% N2 and maximum humidity. Cleavage and blastocyst rates were checked on Day 1 and Day 6, respectively. Cell numbers were counted after fixation in glycerol containing 20 μg mL–1 Hoechst 33342 fluorochrome on Day 6. t-test was performed for statistical calculations with SPSS 11.0 program (SPSS, Chicago, IL, USA). Results are shown in Table 1. Osmotic stress with both permeable and non-permeable agents increased developmental competence of porcine IVM oocytes. NaCl seems to be more appropriate for the purpose, as the other two components resulted in decreased cell number in blastocysts after somatic cell nuclear transfer (SCNT). In conclusion, a simple NaCl pre-treatment of oocytes has improved the in vitro efficiency of porcine SCNT. Table 1.Developmental competence of porcine HMC embryos derived from oocytes treated with different agents The authors thank Ruth Kristensen, Anette Pedersen, Janne Adamsen and Klaus Villemoes for their help and excellent technical assistance.

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

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab59 ◽

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 < 0.05) in hDAF-MP (16%) than in LYD (9%) and MP (6%). MP showed higher (P < 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.

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In vitro development of reconstructed ibex (Capra ibex) embryos by nuclear transfer using goat (Capra hircus) oocytes

Small Ruminant Research ◽

10.1016/j.smallrumres.2006.12.009 ◽

2007 ◽

Vol 73 (1-3) ◽

pp. 135-141 ◽

Cited By ~ 8

Author(s):

Liang Wang ◽

Tao Peng ◽

Hai Zhu ◽

Zili Lv ◽

Tingting Liu ◽

...

Keyword(s):

Nuclear Transfer ◽

Capra Hircus ◽

Capra Ibex ◽

In Vitro Development ◽

Vitro Development

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Effect of Fusion/Activation Protocol on In Vitro Development of Porcine Nuclear Transfer Embryos Constructed with Foreign Gene-Transfected Fetal Fibroblasts

Journal of Veterinary Medical Science ◽

10.1292/jvms.65.989 ◽

2003 ◽

Vol 65 (9) ◽

pp. 989-994 ◽

Cited By ~ 5

Author(s):

Mario A. MARTINEZ DIAZ ◽

Tadashi MORI ◽

Masashi NAGANO ◽

Seiji KATAGIRI ◽

Yoshiyuki TAKAHASHI

Keyword(s):

Nuclear Transfer ◽

Foreign Gene ◽

In Vitro Development ◽

Fetal Fibroblasts ◽

Vitro Development

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Metabolic programming of a Warburg effect-like phenotype in donor fibroblasts prior to somatic cell nuclear transfer

10.32469/10355/66732 ◽

2017 ◽

Author(s):

◽

Bethany Rae Mordhorst

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Donor Cell ◽

Nuclear Reprogramming ◽

In Vitro Development ◽

Fetal Fibroblasts ◽

Pharmaceutical Agents ◽

Vitro Development

Gene edited pigs serve as excellent models for biomedicine and agriculture. Currently, the most efficient way to make a reliably-edited transgenic animal is through somatic cell nuclear transfer (SCNT) also known as cloning. This process involves using cells from a donor (which may have been gene edited) that are typically grown in culture and using their nuclear content to reconstruct a new zygote. To do this, the cell may be placed in the perivitelline space of an enucleated oocyte and activated artificially by a calcium-containing media and electrical pulse waves. While it is remarkable that this process works, it is highly inefficient. In pigs the success of transferred embryos becoming live born piglets is only 1-3%. The creation of more cloned pigs enables further study for the benefit of both A) biomedicine in the development of prognosis and treatments and B) agriculture, whether it be for disease resistance, feed efficiency, gas emissions, etc. Two decades of research has not drastically improved the cloning efficiency of most mammals. One of the main impediments to successful cloning is thought to be due to inefficient nuclear reprogramming and remodeling of the donor cell nucleus. In the following chapters we detail our efforts to improve nuclear reprogramming of porcine fetal fibroblasts by altering the metabolism to be more blastomere-like in nature. We used two methods to alter metabolism 1) pharmaceutical agents and 2) hypoxia. After treating donor cells both methods were used in nuclear transfer. Pharmaceutical agents did not improve in vitro development of gestational survival of clones. Hypoxia did improve in vitro development and we are currently awaiting results of gestation.

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