33 IN VITRO IMMUNOGENICITY OF SOMATIC CELL NUCLEAR TRANSFER-DERIVED TRANSGENIC CLONED DOGS

2012 ◽  
Vol 24 (1) ◽  
pp. 128
Author(s):  
G. Kim ◽  
H. J. Oh ◽  
J. E. Park ◽  
M. J. Kim ◽  
E. J. Park ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mononuclear Cells ◽  
Donor Cell ◽  
Foreign Gene ◽  
Beagle Dogs ◽  
Fetal Fibroblasts ◽  
Significant Difference

Histocompatible tissue has been generated by somatic cell nuclear transfer (SCNT) and the resultant tissues were not rejected by the immune system of the nucleus donors. In addition, many transgenic animals combined with SCNT have been produced. However, in vitro immunogenicity of transgenic cloned animals originated from the same donor cell with nontransgenic cloned animals has not been assessed until now. The objective of this study was to evaluate the in vitro immunogenicity of cloned dogs with each other, between cloned dogs and transgenic cloned dogs and between transgenic cloned dogs with each other by mixed lymphocyte reaction. In this study, we used cloned beagles (BG1, 2) derived from SCNT using fetal fibroblasts (BF3). Serially, 4 transgenic cloned beagles (Ruppy 1–3, 5) were also genetically engineered from the same donor cell, BF3, with red fluorescent protein (RFP) gene inserted into their genome. We used 2 age-matched healthy female beagle dogs as control dogs. They have different 3 DLA types with all cloned dogs. Peripheral blood mononuclear cells (PBMC) of 2 cloned beagles and 4 transgenic cloned beagles were isolated from whole bloods using Ficoll gradient solution. PBMC from each dog were mixed to auto PBMC, other transgenic cloned dogs and non-related control dogs under the experimental designs. All the mixtures were incubated at 37°C for 4 days, adding BrdU labeling reagent and re-incubated for 24 h. Results are expressed in absorbance mean value ± standard deviation of 450-nm wavelength read by microplate reader. Each cell combination was assayed in 8 replicates. In Experiment 1, PBMC of cloned beagles were combined with equal concentrations of another cloned beagle's PBMC. In Experiment 2, PBMC suspension of Ruppy 1–3, 5 were mixed with equal concentrations of another transgenic cloned beagle's PBMC suspension. In Experiment 3, PBMC suspensions of cloned beagles were mixed with PBMC suspensions of transgenic cloned beagles and reverse reaction was performed. Statistical analysis was performed by using Mann-Whitney U test. In Experiment 1, whereas the absorbance value of mixture of cloned dogs and control dogs shows apparent proliferation, auto mixture of each dog and allo-mixture of BG1 and BG2 show no proliferation (Table 1), indicating immunological factors exposed to PBMC in 2 cloned dogs were compatible. In Experiment 2 among transgenic cloned dogs, no evidence of proliferations in mixed allo-PBMC was shown (Table 1), suggesting in vitro immunogenicity between transgenic cloned dogs was also not shown. In Experiment 3 among cloned dogs and transgenic cloned dogs, no significant difference was found (Table 1). In conclusion, cloned dogs derived from SCNT shared immunological phenotype. Next, immunogenicity among transgenic cloned beagle dogs was not shown despite random insertion of a foreign gene. Lastly, cloned beagles and transgenic cloned beagles show lymphocyte antigen compatibility irrespective of having a foreign gene or not. Table 1.The absorbance values of mixed lymphocytes of 4 transgenic cloned dogs and 2 cloned dogs This study was supported by RNL BIO (#0468-20110001), IPET, MKE (#10033839-2011-13) and Natural Balance Korea.

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

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.

29 BIRTH OF A FOAL CLONED BY ADULT SOMATIC CELL NUCLEAR TRANSFER WITH ROSCOVITINE-TREATED DONOR CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 123
Author(s):  
Y. H. Choi ◽  
Y. G. Chung ◽  
D. D. Varner ◽  
K. Hinrichs
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Direct Injection ◽  
Donor Cell ◽  
Mixed Gas ◽  
Blastocyst Development ◽  
Donor Cells ◽  
Significant Difference

Only one horse foal produced from adult somatic cell nuclear transfer has been reported in the scientific literature (Galli et al. 2003 Nature 425, 680); a second foal from the same laboratory was reported in the popular press in 2005. In these reports, the blastocyst rates were 3 and 17%, and efficiency to birth of a live foal from total reconstructed oocytes was 0.1 and 0.5%, respectively. In cattle, roscovitine treatment of donor cells has been associated with a decrease in blastocyst development, but an increase in live births (Gibbons et al. 2002 Biol. Reprod. 66, 895-900). The present study was performed to determine the effect of roscovitine treatment of donor cells on blastocyst production after equine nuclear transfer and to evaluate the viability of pregnancies established via this treatment. In Experiment 1, fibroblasts were either grown to confluence or treated with 15 �g/mL roscovitine, for 24 h. Enucleated in vitro-matured oocytes were reconstructed by direct injection of fibroblasts using a piezo drill. Recombined oocytes were activated by injection of stallion sperm extract, followed by culture in the presence of 2 mM 6-DMAP for 4 h. They were then placed in culture in DMEM/F-12 with 10% fetal bovine serum (FBS) under mixed gas for 8 days and evaluated for blastocyst development. In Experiment 2, oocytes recombined with either confluent or roscovitine-treated donor cells were activated as above either alone or with the addition of 10 �g/mL cycloheximide at the time of 6-DMAP treatment. Resulting blastocysts from Experiment 2 were transferred transcervically to the uteri of recipient mares. One embryo was transferred per mare. In Experiment 1, there was no difference in rates of cleavage (73-19%) or blastocyst development between confluence and roscovitine treatments (2/55, 3.6% vs. 2/56, 3.6%, respectively). In Experiment 2, there was no significant difference in rates of cleavage (78-18%) or blastocyst development (0-1%; 4/105, 0/104, 0/106, 2/108) among donor cell or activation treatments. Six blastocysts were transferred to mares: two from confluent donor cells and four from roscovitine-treated donor cells. One mare, which received an embryo from the roscovitine donor/6-DMAP treatment, established pregnancy after transfer. The pregnancy continued normally and the mare delivered a colt with minimal assistance on Day 389. Typing for 13 equine microsatellites confirmed that the colt was of the same DNA type as the donor fibroblasts. The colt has grown and developed normally. Results of these studies show that roscovitine treatment of equine donor cells does not negatively affect the proportion of recombined oocytes that progress to the blastocyst stage. A viable colt resulted from an embryo produced with roscovitine-treated donor cells. More work is needed on methods to increase blastocyst rates after nuclear transfer in this species. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University.

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

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.

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.

Somatic cell nuclear transfer using transported in vitro-matured oocytes in cynomolgus monkey

Zygote ◽  
2007 ◽  
Vol 15 (1) ◽  
pp. 25-33 ◽  
Author(s):  
N. Chen ◽  
S-L. Liow ◽  
R. Bin Abdullah ◽  
WK. Khadijah Wan Embong ◽  
W-Y. Yip ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Oocyte Retrieval ◽  
Development Rate ◽  
Cleavage Rate ◽  
Immature Oocytes ◽  
Polarized Microscopy

SUMMARYSomatic cell nuclear transfer (SCNT) is not successful so far in non-human primates. The objective of this study was to investigate the effects of stimulation cycles (first and repeat) on oocyte retrieval and in vitro maturation (IVM) and to evaluate the effects of stimulation cycles and donor cell type (cumulus and fetal skin fibroblasts) on efficiency of SCNT with transported IVM oocytes. In this study, 369 immature oocytes were collected laparoscopically at 24 h following human chorionic gonadotrophin (hCG) treatment from 12 cynomolgus macaque (Macaca fascicularis) in 24 stimulation cycles, and shipped in pre-equilibrated IVM medium for a 5 h journey, placed in a dry portable incubator (37 °C) without CO2 supplement. A total of 70.6% (247/350) of immature oocytes reached metaphase II (MII) stage at 36 h after hCG administration, MII spindle could be seen clearly in 80.6% (104/129) of matured IVM oocytes under polarized microscopy. A total of 50.0% (37/74) of reconstructive SCNT embryos cleaved after activation; after cleavage, 37.8% (14/37) developed to the 8-cell stage and 8.1% (3/37) developed to morula, but unfortunately none developed to the blastocyst stage. Many more oocytes could be retrieved per cycle from monkeys in the first cycle than in repeated cycles (19.1 vs. 11.7, p < 0.05). There were no significant differences in the maturation rate (70.0 vs. 71.4%, p > 0.05) and MII spindle rate under polarized microscopy (76.4 vs. 86.0%, p > 0.05) between the first and repeat cycles. There were also no significant differences in the cleavage rate, and the 4-cell, 8-cell and morula development rate of SCNT embryos between the first and repeat cycles. When fibroblast cells and cumulus cells were used as the donor cells for SCNT, first cleavage rate was not significantly different, but 4-cell (50.0 vs. 88.9%, p < 0.05) and 8-cell (0 vs. 51.9%, p < 0.01) development rate were significantly lower for the former. In conclusion, the number of stimulation cycles has a significant effect on oocyte retrieval, but has no effect on maturation and SCNT embryo development; however, different donor cell types (cumulus and fibroblast) resulted in different developmental potentials of SCNT embryos.

Porcine somatic cell nuclear transfer donor sources affect transplant success: A meta-analysis

2018 ◽  
Author(s):  
Zhenhua Guo ◽  
Lei Lv ◽  
Di Liu ◽  
Zhongqiu Li
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Meta Analysis ◽  
Donor Cell ◽  
Blastocyst Formation ◽  
Embryo Viability ◽  
Live Births ◽  
Reconstructed Embryo

Herd boars, male domestic pigs used for stud, are economically important, and somatic cell nuclear transfer (SCNT) is a promising technology to expand herd boar yields. However, live births are dictated by donor cell source, and fetal donors may offer more advantages than adult donors. A meta-analysis was conducted to better understand how donor sources affect SCNT outcomes. Of the 1,431 records viewed, 10 were selected for review. Blastocyst formation rates, successful pregnancies, and live births were assessed to measure efficacy. SCNT blastocyst formation differed between adult and fetal donors among the studies. SCNT pigs had more malformed fetuses as well, which negatively affected the post-birth mortality. Organs of porcine fetuses are limited by deficiencies of maternal nutrient and growth hormones, which compromise post-birth adaptations. SCNT pregnancy success is neither determined by donor source nor by live births. Live births are also tied to donor age. Embryos from fetal donors are more frequently healthy likely due to less differentiation and less reprogramming of reconstructed embryos. Adult donors in contrast have more cell differentiation and as such accumulate more mutations and damage. This may reduce reconstructed embryo viability. Finally, SCNT efficiency may be improved with more in vitro passages, but more work is required to validate this concept.

31 FULL-TERM AND LIVE RABBIT CLONES PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 124 ◽  
Author(s):  
F. Du ◽  
J. Xu ◽  
S. Gao ◽  
L. Y. Sung ◽  
D. Stone ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cell Fusion ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Full Term ◽  
Fusion Method ◽  
Nuclear Injection

Transgenic/knockout (KO) rabbits can serve as an excellent animal model for human cardiovascular diseases (CVD) and other diseases. However, the production of transgenic/KO rabbits is hindered by low efficiency of traditional DNA microinjection and the unavailability of embryonic stem cell lines. An alternative approach is to produce transgenic/KO rabbits by somatic cell nuclear transfer (SCNT) using genetically modified somatic cells as nuclear donors. Our initial objective of the study was to prove the feasibility of cloning rabbits by SCNT because rabbit is a difficult species to be cloned. Rabbit oocytes were flushed from the oviducts of superovulated donors treated with the regime of follicle-stimulating hormone (FSH) and human choriani gonadotropin (hCG). Cumulus cells were then denuded from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in M199 + 10% fetal bovine serum (FBS) and confirmed by fluorescence microscopy. Cumulus cells used for nuclear donors were prepared from fresh cumulus-oocytes complexes. The donor nucleus was transferred into a recipient oocyte by either cell fusion or direct nuclear injection method. In the cell fusion method, a small donor cell with the diameter approximately 15–19 µm was transferred into the perivitelline space of an enucleated oocyte; subsequently the somatic cell-cytoplast pair was fused by applying three direct current pulses at 3.2 kV/cm for a duration of 20 µs/pulse. In the direct nuclear injection method, a mechanically lysed donor cell was injected into oocyte cytoplasm with the aid of a piezo-drill system. Fused embryos or injected oocytes were activated by the same electrical stimulation regime described above, and subsequently cultured in M199 + 10% FBS containing 2.0 mM 6-dimethylaminopurine (DMAP) and 5 µg/mL cycloheximide for 2 h. For the in vitro study, cloned embryos were cultured in B2 medium plus 2.5% FBS for 5 days (initiation of activation = day 0) at 38.5°C in 5% CO2 humidified air. For the in vivo study, cloned embryos were cultured for 20–22 h in vitro before transfer into pseudopregnant rabbit recipients. Pregnancy was monitored by palpation and/or ultrasound on Days 14–16 post embryo transfer (ET). The results (Table 1) show that the donor nuclei-introducing rate was higher with nuclear direct injection than with the cell fusion method (P < 0.05). There were no significant differences among subsequent cleavage and development to morula and blastocysts between both methods, although the development rates of cloned embryos via electrically mediated fusion were higher than those derived from the injection group. One recipient in the injection group (1/6, 17%) and six recipients in the fusion group (6/16, 38%) were diagnosed as pregnant. From the fusion group, one full-term but stillborn and one live and healthy clone rabbit were delivered on Days 33 and 31 post-ET, respectively. To our knowledge, this is the second report of full term development of cloned rabbit by somatic nuclear transfer cloning. Our further study is to clone live rabbit offspring with modified transgenic/KO somatic cell lines. Table 1. In vitro development of rabbit cloned embryos with cumulus cells as nuclear donors This work was supported by NIH/NCRR-SBIR grant: 1R43RR020261–11.

20 Aggregation of yak heterospecific somatic cell nuclear transfer embryos improves cloning efficiency

2020 ◽  
Vol 32 (2) ◽  
pp. 135
Author(s):  
M. Yauri Felipe ◽  
M. Duque Rodríguez ◽  
A. De Stéfano ◽  
D. Salamone
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Bos Taurus ◽  
Formation Rate ◽  
Donor Cell ◽  
Blastocyst Formation ◽  
Fluid Medium ◽  
Exact Test ◽  
Significant Difference

Cloning endangered species has the limitation that generally the number of available oocytes is limited. Reprogramming the nuclei heterospecifically using an enucleated oocyte from a different species is an alternative. Aggregation of SCNT (somatic cell nuclear transfer) embryos from the same specie results in improved embryo development. However, after aggregation of heterospecific SCNT embryos from different genera, no effects were observed (Moro et al. 2015 Reproduction 50, 1-10). The objective of this study was to evaluate the influence of aggregation of yak (Bos grunniens) embryos produced by heterospecific SCNT using enucleated oocytes from an animal from the same genus Bos taurus. As control homospecific SCNT of Bos taurus, parthenogenic zone-free embryos and IVF embryos were used. Cumulus-oocyte complexes were recovered from bovine slaughterhouse ovaries by follicular aspiration. The cumulus-oocyte complexes were matured in tissue culture medium 199 containing 10% fetal bovine serum, 10μgmL−1 FSH, 0.3mM sodium pyruvate, 100mM cysteamine, and 2% antibiotic-antimycotic for 22h, at 6.5% CO2 in humidified air and 38.5°C. After denudation, mature oocytes were stripped of the zona pellucida using a protease and then enucleated by micromanipulation. Staining was performed with Hoechst 33342 to observe MII. Enucleated oocytes were placed in phytohemagglutinin to induce adherence with the donor cell followed by electrofusion. All reconstituted embryos were activated using ionomcine. This was followed by a treatment with 6-dimethylaminopurine for 3h. Zona-free reconstituted cloned embryos were cultured in the wells of the well system, placing one (1×) or two (2×) per microwell, in synthetic oviductal fluid medium. The experimental groups were parthenogenic zone free; IVF; reconstituted embryos bull fibroblast-enucleated oocyte from cow (BC1×); reconstituted embryos yak fibroblast-enucleated oocyte from cow (YC1×); and reconstituted embryos aggregated yak fibroblast-enucleated oocyte from cow (YC2×). In all experimental groups, cleavage of at least one embryo in the wells and blastocyst formation at Day 7 were assessed. The effect of cloned embryo aggregation on blastocyst rates was analysed using Fisher exact tests (GraphPad Prisma 8), and results are shown on Table 1. Results demonstrated that aggregation of two SCNT heterospecific embryos increased the blastocyst formation rate of yak (P&lt;0.05). In conclusion aggregation in yak heterospecific SCNT embryos from species of the same genus (Bos) can improve development to blastocyst. Table 1.Aggregation of yak heterospecific somatic cell nuclear transfer embryos Experimental group1 No. of embryos No. of embryos-wells2 Cleavage (%) Blastocyst (%) PZF 68 68 66 (97.06%)a 17 (25.00%)acd IVF 89 - 81 (91.01%)ab 39 (43.82%)b BC1× 45 45 41 (91.11%)b 6 (13.33%)cd YC1× 101 101 77 (76.24%)c 14 (13.86%)c YC2× 134 67 61 (91.04%)ab 21 (31.34%)ab a-dDifferent superscripts in the same column indicate significant difference (Fisher's exact test, P&lt;0.05). 1PZF, parthenogenetic zone free; IFV, IVF fecundation; BC1×, clone of bovine; YC1×, clone of yak-bovine; YC2×, clone of yak-bovine added. 2Wells used with embryos.

Development and spindle formation in rat somatic cell nuclear transfer (SCNT) embryos in vitro using porcine recipient oocytes

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 195-202 ◽  
Author(s):  
Atsushi Sugawara ◽  
Satoshi Sugimura ◽  
Yumi Hoshino ◽  
Eimei Sato
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Spindle Formation ◽  
Fetal Fibroblasts

SummaryCloning that uses somatic cell nuclear transfer (SCNT) technology with gene targeting could be a potential alternative approach to obtain valuable rat models. In the present study, we determined the developmental competence of rat SCNT embryos constructed using murine and porcine oocytes at metaphase II (MII). Further, we assessed the effects of certain factors, such as: (i) the donor cell type (fetal fibroblasts or cumulus cells); and (ii) premature chromosome condensation (PCC) with normal spindle formation, on the developmental competence of rat interspecies SCNT (iSCNT) embryos. iSCNT embryos that had been constructed using porcine oocytes developed to the blastocyst stage, while those embryos made using murine MII oocytes did not. Rat iSCNT embryos constructed with green fluorescent protein (GFP)-expressing fetal fibroblasts injected into porcine oocytes showed considerable PCC with a normal bipolar spindle formation. The total cell number of iSCNT blastocyst derived from GFP-expressing fetal fibroblasts was higher than the number derived from cumulus cells. In addition, these embryos expressed GFP at the blastocyst stage. This paper is the first report to show that rat SCNT embryos constructed using porcine MII oocytes have the potential to develop to the blastocyst stage in vitro. Thus the iSCNT technique, when performed using porcine MII oocytes, could provide a new bioassay system for the evaluatation of the developmental competence of rat somatic cells.

91 PRODUCTION OF A CLONED BOER GOAT (CAPRA HIRCUS) BY SOMATIC CELL NUCLEAR TRANSFER

2007 ◽  
Vol 19 (1) ◽  
pp. 163
Author(s):  
Y. Tao ◽  
W. Han ◽  
M. Zhang ◽  
J. Ding ◽  
X. Zhang
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Capra Hircus ◽  
Blastocyst Development ◽  
Tissue Slices ◽  
Boer Goat ◽  
Reconstructed Embryos ◽  
Significant Difference

We reported the birth of a goat clone produced by somatic cell nuclear transfer. The fusion and activation protocols of reconstructed oocytes and embryo transfer procedure were optimized. The donors of somatic cells were fibroblasts derived from ear skin of a Boer goat while the recipient ooplasm was in vitro-matured oocytes of Huanghuai white goat, an Anhui native goat species. The reconstructed embryos were activated by ionomycin, 6-dimethylaminopurine (6-DMAP), and cytochalasin B (CB) singly or simultaneously (termed as Ionomycin, Ionomycin+6-DMAP, and Ionomycin+6-DMAP+CB). The result showed that the cleavage rate in single ionomycin was significantly lower than that in Ionomycin+6-DMAP and 6-DMAP+CB (34.38% vs. 69.85% and 72.02%; P &lt; 0.05). However, the cleavage rates and blastocyst rates had no significant difference after in vitro culture (P &gt; 0.05). When the cloned embryos were co-cultured with fetal mouse fibroblast monolayer, the blastocyst development rate increased. The reconstructed embryos were equilibrated 1–3 h, 3–6 h, and 6–9 h after fusion, and then activation was undertaken by ionomycin+6-DMAP. We found that the cleavage rates had no significant difference during 1–3 h and 3–6 h (72.58% vs. 72.97%; P &gt; 0.05), but both were significantly higher than during 6–9 h (64.40%) (P &lt; 0.05). A total of 491 reconstructed embryos were surgically transferred into 37 recipient surrogates, Huanghuai white goats with natural estrus. One of those who were treated with hCG after transfer was pregnant and gave birth to a live kid on 153 days. The lamb died accidentally 8 h after birth. The cloned offspring was then dissected and proved well in all organs. Staining of paraffin tissue slices of the viscera suggested that the organs developed well. Microsatellite analysis indicated that the lamb was derived from the somatic cell donor doe genetically.

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