scholarly journals 340PRODUCTION OF TRANSGENIC PORCINE BLASTOCYSTS BY HANDMADE CLONING

2004 ◽  
Vol 16 (2) ◽  
pp. 290 ◽  
Author(s):  
P.M. Kragh ◽  
G. Vajta ◽  
T.J. Corydon ◽  
L. Bolund ◽  
H. Callesen
Keyword(s):  
Nuclear Transfer ◽  
Culture Medium ◽  
Fluorescent Protein ◽  
Uv Light ◽  
Calcium Ionophore ◽  
Ionophore A23187 ◽  
Transgenic Pigs ◽  
Reconstructed Embryos ◽  
Cattle Serum ◽  
Handmade Cloning

The present study demonstrates the application of the recently developed handmade cloning (HMC) technique in production of transgenic porcine blastocysts. The HMC technique was originally established for bovine nuclear transfer (Vajta et al., 2003, Biol. Reprod. 68, 571–578), and has the advantages of being less demanding and more productive than traditional nuclear transfer techniques. Cumulus-oocyte complexes were aspirated from slaughterhouse ovaries and matured for 41h. Subsequently, the cumulus cells were removed by pipetting in 1mgmL−1 hyaluronidase in HEPES-buffered TCM-199; zonae pellucidae were removed by incubation in 2mgmL−1 pronase in HEPES-buffered TCM-199 supplemented with 2% cattle serum (T2) for 1min. Bisection was performed by hand under a stereomicroscope using a microblade in 5μgmL−1 cytochalasin B in TCM-199 supplemented with 20% cattle serum (T20). Demi-oocytes were incubated in 5μgmL−1 Hoechst 33342 in T20 for 10min, followed by examination under UV light to select the halves containing no chromatin, i.e., the cytoplasts. Porcine fibroblasts harvested from an ear skin biopsy were transfected with pN1-EGFP (Clontech) using Lipofectamine (Gibco, Life Technologies). G418 selection (0.8mgmL−1) was applied 48h after transfection, and well separated G418-resistant cell colonies originating from a single transfected cell were isolated, expanded, and cryopreserved. Days before, nuclear transfer cells were grown to a confluent monolayer in DMEM supplemented with 10% FCS. Fusions were performed 43h after start of maturation. One cytoplast was attached to one fibroblast in 500μgmL−1 phytohemagglutinin dissolved in T2. In the fusion chamber, covered with fusion medium (0.3M mannitol, 0.1mM MgSO4, 0.05mM CaCl2, and 0.01% PVA), one cytoplast-fibroblast pair was fused with one cytoplast in a single step. The fusions were performed with a double DC pulse of 65V, each pulse for 20μs and 0.1s apart from each other. Successfully fused embryos were activated 1h after the end of fusion by incubation in 2μM calcium ionophore A23187 in T20 for 5min followed by 3-h incubation in microdrops of culture medium (NCSU-23 with 4mgmL BSA) containing 2mM 6-dimethylaminopurine. Activated embryos were cultured individually in microdrops of culture medium for 7 days. In four independent experiments, 93% of attempted reconstructed embryos fused and survived activation (31/31, 15/23, 28/28, and 37/37, respectively). On Day 7 after activation, the blastocyst rates (per successfully reconstructed embryos) were 6% (2/31), and 7% (1/15), 7% (2/28), and 3% (1/37), respectively. Green Fluorescent Protein was expressed in all cells of the developing blastocysts. The results show that transgenic porcine blastocysts can be produced using HMC, and the technique may also be applied for the production of transgenic pigs.

Production of transgenic porcine blastocysts by hand-made cloning

2004 ◽  
Vol 16 (3) ◽  
pp. 315 ◽  
Author(s):  
P. M. Kragh ◽  
G. Vajta ◽  
T. J. Corydon ◽  
S. Purup ◽  
L. Bolund ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Nuclear Transfer ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Reconstructed Embryos ◽  
Green Fluorescent

Recently, a zona-free technique for bovine somatic cell nuclear transfer (NT) with no requirement for micromanipulation (i.e. hand-made cloning (HMC)) has been described. The present study demonstrates the application of the HMC technique in the production of transgenic porcine blastocysts. In vitro-matured zona-free porcine oocytes were bisected manually using a microblade and halves containing no chromatin (i.e. the cytoplasts) were selected. Two cytoplasts were electrofused with one transgenic fibroblast expressing enhanced green fluorescent protein and reconstructed embryos were activated in calcium ionophore (A23187) followed by 6-dimethylaminopurine. Subsequently, embryos were cultured in NCSU-23 medium supplemented with 4 mg mL–1 bovine serum albumin for 7 days. In five replicates, 93.0 ± 7.0% (mean ± s.e.m.) of attempted reconstructed embryos fused and survived activation (31/31, 15/23, 28/28, 37/37 and 28/28). On Day 7 after activation, the respective blastocyst rates (per successfully reconstructed embryos) were 6% (2/31), 7% (1/15), 7% (2/28), 3% (1/37) and 7% (2/28), resulting in an average of 6.0 ± 0.8%. Enhanced green fluorescent protein was expressed in all cells of all eight developing blastocysts. Efforts are now directed towards the production of offspring from such transgenic NT blastocysts.

scholarly journals 270 COMBINED ELECTRICAL AND CHEMICAL ACTIVATION OF ZONA-FREE PORCINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 284
Author(s):  
P.M. Kragh ◽  
N.R. Mtango ◽  
T.J. Corydon ◽  
L. Bolund ◽  
H. Callesen ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Culture Medium ◽  
Cytochalasin B ◽  
Chemical Activation ◽  
Cumulus Cells ◽  
Control Model ◽  
Parthenogenetic Development ◽  
Cloning Technique ◽  
Cattle Serum ◽  
Handmade Cloning

Activation is a crucial step in mammalian somatic cell nuclear transfer (SCNT). Recently we described the application of the handmade cloning technique for porcine SCNT that uses oocytes without zonaa pellucidae (zona-free) in a micromanipulation-independent procedure (Kragh et al. 2004 Reprod. Fertil. Dev. 16, 315–18). The purpose of the present study was to investigate the effect of a combined electrical and chemical activation of zona-free porcine oocytes. Cumulus-oocyte complexes were aspirated from ovaries of sows and matured for 41 h. Subsequently, the cumulus cells were removed by the addition of 1 mg/mL hyaluronidase in HEPES-buffered TCM-199. For zonae pellucidae removal, oocytes were incubated in 8 mg/mL pronase in HEPES-buffered TCM-199 supplemented with 20% cattle serum for 10 s. Three independent experiments with four treatments were conducted, and oocytes were activated by a combined electrical and chemical activation. Oocytes were washed once in activation medium (0.3 M mannitol, 0.1 mM MgSO4, 0.1 mM CaCl2, and 0.01% polyvinyl alcohol) and transferred to a chamber with two wires (0.5-mm separation) covered with activation medium. After the electrical pulse, oocytes were incubated in culture medium (NCSU-37 containing 4 mg/mL BSA) supplemented with 5 μg/mL cytochalasin B and 10 μg/mL cycloheximide for 6 h. Activated oocytes were cultured in culture medium using the wells of wells system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–64) in the submarine incubation system (Vajta et al. 1997 Theriogenology 48, 1379–85). The rate of development into blastocysts was checked on Day 7 of culture. In treatment 1, zona pellucida-intact oocytes were first activated by a single DC pulse of 1.25 kV/cm for 80 μs, and subsequently cultured in cytochalasin B and cycloheximide for 6 h. In treatments 2 and 3, oocytes without zonae pellucidae were activated by a single DC pulse of 1.25 and 0.85 kV/cm for 80 μs, respectively, and subsequently cultured in cytochalasin B and cycloheximide for 6 h. In treatment 4, oocytes without zonae pellucidae were bisected by hand under a stereomicroscope using a microblade in 5 μg/mL cytochalasin B in TCM-199 supplemented with 15 mg/mL BSA, re-fused/activated by a single DC pulse of 1.25 kV/cm for 80 μs in activation medium, and cultured in cytochalasin B and cycloheximide for 6 h. The rates of blastocyst formation from activated oocytes (mean ± SEM) in treatments 1, 2, 3, and 4 were 55 ± 4%, 40 ± 2%, 49 ± 1%, and 41 ± 8%, respectively. In conclusion, the combined electrical and chemical activation efficiently induced parthenogenetic development of zona-free oocytes. Also, a more authentic control model for activation during SCNT was established by activating and producing blasctocysts from re-fused bisected oocytes.

scholarly journals 27BIRTH OF AFRICA'S FIRST NUCLEAR-TRANSFERRED ANIMAL PRODUCED WITH HANDMADE CLONING

2004 ◽  
Vol 16 (2) ◽  
pp. 136 ◽  
Author(s):  
P. Bartels ◽  
J. Joubert ◽  
M. de la Rey ◽  
R. de la Rey ◽  
R. Treadwell ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Calf Serum ◽  
Calcium Ionophore ◽  
Physiological Saline ◽  
Cumulus Cells ◽  
Animal Biotechnology ◽  
Additional Tool ◽  
Reconstructed Embryos ◽  
Cattle Serum ◽  
Hoechst Staining

Cloning technology has the potential to stimulate the development of the animal biotechnology industry in southern Africa, as well as provide conservationists with an additional tool to possibly assist with conserving critically endangered wildlife species sometime in the future. The aim of this study was to determine whether cloning could produce blastocysts and possibly live progeny in a field-type laboratory without micromanipulators and CO2 incubator. Approx. 1×1-cm ear skin notches were surgically removed from a physically immobilized 9-year-old Holstein cow, a former South African milk production record holder. The tissues were placed into physiological saline and transported to the laboratory at 4°C within 2h, cleaned with chlorohexidine gluconate and sliced finely in Minimal Essential Medium supplemented with 10% fetal calf serum. The resultant tissue explants were treated as previously described (Bartels et al., 2003 Theriogenology 59, 387) and actively growing fibroblast cultures were made available for the nuclear transfer process. Bovine oocytes from slaughterhouse-derived ovaries were collected and matured for 21h in modified TCM-199 medium supplemented with 15% cattle serum, 10IUmL−1 eCG and 15IUmL−1 hCG. Nuclear transfer was performed using the HMC technique (Vajta et al., 2003 Biol. Reprod. 68, 571–578). At 21h after the start of maturation, cumulus cells and zonae pellucidae were removed and oocytes were randomly bisected by hand. Cytoplasts were selected using Hoechst staining and a fluorescent microscope. After a two-step fusion, reconstructed embryos were activated with calcium ionophore and dimethylaminopurine. Culture was performed in SOFaaci medium supplemented with 5% cattle serum using WOWs (Vajta et al., Mol. Reprod. Dev. 50, 185–191). All incubations including culture of donor cells were performed in the submarine incubator system (SIS; Vajta et al., 1997 Theriogenology 48, 1379–1385). In two consecutive experiments, 6 blastocysts were produced from 52 reconstructed embryos. On Day 7, 5 blastocysts were selected for transfer into 3 previously synchronized recipients. All three recipients became pregnant, but two of the recipients aborted at six and seven months, respectively. Post-mortem examination on the first aborted fetus did not reveal any identifiable etiology, but coincided with 6 abortions from natural pregnancies during a heat wave, while the organism Brucella abortis was isolated from the second aborted fetus. The third pregnancy went to term, and a healthy calf, weighing 27kg, was delivered by Caesarean section. The three-month-old calf is being raised by a surrogate Jersey cow under standard dairy conditions and is expected to join the dairy in eighteen months’ time. The birth of ‘Futhi’, meaning ‘replicate’ in Zulu, is Africa’s first cloned animal and signifies an important milestone in the development of animal biotechnology in Africa.

60 EFFECTS OF CAFFEINE ON THE IN VITRO DEVELOPMENT OF BOVINE NUCLEAR TRANSFER EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 138
Author(s):  
W. E. Maalouf ◽  
J. H. Lee ◽  
K. H. S. Campbell
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Calcium Ionophore ◽  
Cell Number ◽  
Developmental Competence ◽  
Mitogen Activated Protein Kinase ◽  
Ionophore A23187 ◽  
Cell Cycle Regulators ◽  
Reconstructed Embryos

Previous studies have demonstrated that treating ovine oocytes with caffeine increases the activities of both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). When such oocytes are used as cytoplast recipients for nuclear transfer (NT), there is an increase in cell numbers at the blastocyst stage (Lee and Campbell 2004 Rep. Fert. Dev. 16, 125). The objective of this study was to determine the effects of caffeine on MPF and MAPK activities and the development of bovine NT embryos. Oocytes were matured in maturation medium (MM) composed of TCM199, 10% fetal bovine serum (FBS), 5 �g mL-1 follicle-stimulating hormone FSH, 5 �g mL-1 lutcinizing hormone (LH) and 1 �g mL-1 estradiol for 24 h. Subsequently, oocytes were cultured in MM supplemented with 0, 5, 10, and 15 mM caffeine for 6 h. Groups of 10 oocytes were sampled and analyzed for MPF and MAPK activities as previously described (Ye et al. 2003 Reproduction 125, 645-656). Treatment with 15 mM caffeine significantly increased the levels of MPF and MAPK activities in MII oocytes. To study development potential, oocytes at 16 h post-onset of maturation (hpm) were stripped of cumulus cells and enucleated in HSOF containing 5 �g mL-1 Hoechst 33342 and 7.5 �g mL-1 cytochalasin B; enucleation was achieved using a blunt (25-�m i.d.) pipette after cutting a hole in the zona pellucida with a XYClone laser (Hamilton Thorne Research, Beverly, MA, USA). Enucleated oocytes were then cultured in MM �15 mM caffeine for a further 6 h. For NT, quiesced primary bovine foetal fibroblasts were used. Cell fusion was induced with two DC pulses of 35 V for 65 �s at 24 hpm. At 2 h post-fusion, all reconstructed embryos were briefly exposed to ultraviolet light under a fluorescence microscope (Leica Microsystems AG, Wetzler, Germany) in order to assess nuclear morphology, and then activated in HSOF containing 5 �g mL-1 calcium ionophore (A23187), cultured in SOF with 10 �g mL-1 cycloheximide and 7.5 �g mL-1 cytochalasin B for 5 h, and transferred to mSOFaaBSA medium. On Day 2, cleavage was assessed and 10% FBS added to the medium. Development to blastocyst was assessed on Day 7. All data were analyzed using the chi-square test. There was a significant increase in the number of reconstructed embryos that underwent nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) when caffeine-treated cytoplast recipients were used (28.6 � 9.9% and 60.0 � 11.0% for control and caffeine groups respectively, P < 0.05). Cleavage rates (47.6 � 10.9% and 50.0 � 11.1%), development to blastocyst (20.0 � 4.0% and 30.0 � 4.6%), and mean cell number (85.0 � 7.1 and 122.5 � 3.5) were not statistically different between control and caffeine treated groups, respectively. In summary, treatment of bovine oocytes with 15 mM caffeine increased the activities of two key cell-cycle regulators MPF and MAPK, and statistically increased the occurrence of NEBD and PCC in the donor nuclei. We previously hypothesized that the occurrence and extent of NEBD and PCC may increase nuclear reprogramming in NT embryos (Lee and Campbell 2004 Rep. Fert. Dev. 16, 125; Campbell et al. 2005 Rep. Dom. Anim. 40, 256-268); however, further studies are required to determine the developmental competence of these embryos.

39 Embryo Development of Kazakh Argali (Ovis ammon collium) by Handmade Cloning

2018 ◽  
Vol 30 (1) ◽  
pp. 159
Author(s):  
Y. Toishibekov ◽  
E. Asanova ◽  
M. Yermekova ◽  
A. Seisenbayeva ◽  
D. Toishybek ◽  
...  
Keyword(s):  
Embryo Development ◽  
Nuclear Transfer ◽  
Culture Medium ◽  
Calcium Ionophore ◽  
Blastocyst Stage ◽  
Critically Endangered ◽  
Domestic Sheep ◽  
Wildlife Species ◽  
Cattle Serum ◽  
Ovis Ammon

Wildlife conservation requires innovative preservation methods in order to preserve gene and species biodiversity. Nuclear transfer has the potential to preserve genes from critically endangered wildlife species where few or no oocytes are available from the endangered species, and where cryopreserved cell lines have been conserved in cryobanks. The purpose of this study was to investigate the developmental ability of embryos reconstructed with transfer of cryopreserved somatic cells from the Kazakh argali (Ovis ammon collium) to enucleated domestic sheep (Ovies aries) oocytes. Frozen-thawed fibroblasts were diluted with DMEM (1:5) and centrifuged at 300g for 7 to 10 min. Supernatants were removed, and cells were diluted with DMEM at a concentration of 2 × 106 cells mL−1. Fibroblasts were placed into culture Petri dishes containing DMEM supplemented with 20% (v/v) fetal bovine serum (FBS), and incubated at 5% CO2, 95% relative humidity, and 37°C. After 21 to 22 days of incubation, a fibroblast monolayer was observed, culture medium was removed, and cells were incubated for 7 to 10 min in presence of Dulbecco’s PBS + 0.25% trypsin. Dissociated fibroblasts were washed with DMEM by centrifugation at 300 × g for 10 min. Cumulus-oocyte complexes were aspirated from slaughterhouse ovaries. Subsequently, the cumulus cells were removed by pipetting in 1 mg mL−1 hyaluronidase in HEPES-buffered TCM-199; zonae pellucidae were removed by incubation in 2 mg mL−1 pronase in HEPES-buffered TCM-199 supplemented with 2% cattle serum (T2) for 1 min. Bisection was performed by hand under a stereomicroscope using a microblade in 5 μg mL−1 cytochalasin B in TCM-199 supplemented with 20% cattle serum (T20). Fusions were performed 24 to 28 h after the start of maturation. One cytoplast was attached to one fibroblast in 500 μg mL−1 phytohemagglutinin dissolved in T2. In the fusion chamber, covered with fusion medium (0.3 M mannitol, 0.1 mM MgSO4, 0.05 mM CaCl2, and 0.01% polyvinyl alcohol), one cytoplast-fibroblast pair was fused with one cytoplast in a single step. The fusions were performed with a single DC pulse of 100V, each pulse for 9 μs. Successfully fused embryos were activated 1 h after the end of fusion by incubation in 2 μM calcium ionophore (Sigma, St. Louis, MO, USA) in T20 for 5 min followed by 3-h incubation in microdrops of culture medium containing 2 mM 6-DMAP. After successful reconstruction, 79 nuclear transferred and activated embryos were cultured in well-of-the-wells in trigas (5% O2, 5% CO2, 90% N2) in Submarine incubation system for 7 days. All except 15 embryos cleaved; 35 (44.3%) developed to compacted morula, and 15 (18.9%) to the blastocyst stage. In conclusion, argali embryos developed from reconstruction using their frozen–thawed fibroblasts combined with domestic sheep cytoplasts; however, in vitro developmental ability to the blastocyst stage was limited. Additional research that establishes the early embryo development with optimising nuclear transfer techniques may have a potential role in the conservation of critically endangered wildlife species.

29 EVALUATION OF DIFFERENT FUSION PARAMETERS IN THE RECONSTRUCTION OF SWINE HANDMADE CLONING EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 95
Author(s):  
C. Feltrin ◽  
A. S. Lima ◽  
M. Monaco ◽  
S. M. Wilson ◽  
D. Kim ◽  
...  
Keyword(s):  
Uv Light ◽  
Polar Body ◽  
Donor Cell ◽  
Fusion Rate ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Reconstructed Embryos ◽  
Cloning Technique ◽  
Handmade Cloning

The goal of this experiment was to compare different fusion parameters in the handmade cloning technique to produce cloned swine embryos. After in vitro maturation of 618 oocytes, 431 (69.8%) presented a visible polar body and were used in the experiment. The next step was the removal of the cumulus oophorus cells and the digestion of the zona pellucida using pronase (5 mg mL–1) in HEPES TCM199. Oocytes were then exposed to a medium containing cytochalasin B (5 µg mL–1) for 15 min before being bisected with a hand-held blade. The bisected oocytes (cytoplasts) were then placed in medium supplemented with Hoechst 33342 and exposed to UV light to select cytoplasts without metaphase II plates. Next, two cytoplasts and a mesenchymal stem cell (nucleus donor) were pushed together in a phytohemagglutinin (550 µg mL–1) solution. Once adhered, these structures were divided into 3 groups (G) to be fused using different parameters: (G1) 2 pulses (DC) of 0.6 kV cm–1 for 30 µs, (G2) 2 pulses (DC) of 0.9 kV cm–1 for 30 µs, and (G3) 2 pulses (DC) of 1.2 kV cm–1 for 30 µs. For all three groups, 0.3 m of mannitol solution (without calcium) was used in the fusion chamber, and an initial pre-pulse (AC) of 10V for 15 s was performed to permit the alignment of 100% of the cytoplast-donor cell structures. After fusion, reconstructed embryos were activated in 0.3 m mannitol and 0.1 mm calcium in the fusion chamber using 2 pulses of 0.9 kV cm–1 for 30 µs followed by incubation in 10 µg mL–1 of cycloheximide solution for 4 h. Afterwards, the reconstructed embryos were transferred to NCSU23 medium supplemented with amino acids (nonessential and essential) and 0.4% bovine serum albumin. The embryos were cultured at 39�C in a 100% humidified atmosphere containing 5% CO2, 5% O2, and 90% N2. Cleavage rates were evaluated after 48 h of culture. For G1, the fusion rate was 43% (25/58) with 72% cleavage (18/25), the G2 fusion rate was 87% (56/64) with 80% cleavage (45/56), and the G3 fusion rate was 79% (53/67) with 69% cleavage (37/53). Statistical analysis was performed using the chi-square test. There were no significant differences in fusion rates between groups G2 and G3, but the fusion rate of these groups was significantly different from that of G1 (P < 0.05). No significant differences in cleavage rate were observed among the three groups. In conclusion, fusion using 2 pulses at either 0.9 or 1.2 kV cm–1 for 30 µs was more efficient for embryo reconstruction in the handmade cloning technique compared to that using 2 pulses at 0.6 kV cm–1 for 30 µs. Further studies need to be performed to improve cleavage rates and assess development to the blastocyst stage.

61 EFFICIENCY OF TWO ENUCLEATION METHODS FOR THE PRODUCTION OF TRANSGENIC PIG EMBRYOS BY HANDMADE CLONING

2007 ◽  
Vol 19 (1) ◽  
pp. 148 ◽  
Author(s):  
J. Li ◽  
Y.-H. Zhang ◽  
Y.-T. Du ◽  
P. M. Kragh ◽  
S. Purup ◽  
...  
Keyword(s):  
High Efficiency ◽  
Fluorescent Protein ◽  
Cumulus Cells ◽  
Transgenic Pig ◽  
Transgenic Pigs ◽  
Uv Illumination ◽  
Polar Bodies ◽  
Transgenic Embryos ◽  
Handmade Cloning

Since the successful production of transgenic pigs by somatic nuclear transfer (Lai et al. 2002 Science 295, 1089–1092), more efficient reproduction technologies for transgenic pigs have been in demand. The purpose of our work was to develop an efficient method for production of transgenic embryos by handmade cloning (HMC; Vajta et al. 2001 Cloning 3, 89–95) connected to oriented enucleation to eliminate potential harm of staining and UV illumination at cytoplast selection. After 41–42 h of in vitro maturation, oocytes were further cultured with or without 0.4 µg mL−1 demecolcine for 45 min (i.e. chemically assisted handmade enucleation (CAHE) vs. oriented handmade enucleation (OHE)). Subsequently, the cumulus cells were removed and zonae pellucidae were partially digested. Oocytes with visible extrusion cones or polar bodies attached to the surface were subjected to oriented bisection. The putative cytoplasts without extrusion cones or polar bodies, containing the major part of cytoplasm, were selected as the recipients. Two cytoplasts were electro-fused with one transgenic fibroblast expressing either amyloid precursor protein (APP) or green fluorescent protein (GFP), while non-transgenic fibroblasts were used as control nuclear donors. After activation (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Cloning Stem Cells 7, 199–205), reconstructed embryos were cultured in porcine zygote medium-3 for 7 days. The rates of cleavage and blastocyst cell numbers were recorded on Day 2 and Day 7, respectively. In 5 replicates, the correct bisection efficiency achieved with CAHE was higher compared to that with the OHE method (93 ± 1% vs. 82 ± 2%, respectively; P &lt; 0.05). Table 1 shows that blastocyst rates with APP and GFP transgenic fibroblasts as nuclear donors after CAHE were lower (P &lt; 0.05) compared to those with the OHE method; in contrast, cleavage rates of embryos from different fibroblast donors were similar and so were blastocyst rates of non-transgenic donors after either CAHE or OHE. Our results show that embryos reconstructed from APP and GFP transgenic donors have compromised in vitro developmental rates after CAHE rather than after the OHE method; however, a high efficiency with both enucleation methods was observed when using non-transgenic somatic cells. Table 1.Comparison of two enucleation methods for the production of transgenic pig embryos

19 Success of handmade cloning in a commercial setting

2019 ◽  
Vol 31 (1) ◽  
pp. 135
Author(s):  
T. Waybright ◽  
S. Sonsteby ◽  
G. Vajta
Keyword(s):  
Direct Current ◽  
Calcium Ionophore ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Reconstructed Embryos ◽  
Commercial Farms ◽  
Blastocyst Rate ◽  
On Farm ◽  
Practicable Method ◽  
Handmade Cloning

The purpose of this field trial was to determine whether handmade cloning could be used in a commercial setting to produce, transport, and implant embryos into recipients and to determine blastocyst and pregnancy rates. Donor animals and recipients were housed on 2 commercial farms, Farm A and Farm B. Ear notches were collected, grown in DMEM 10% FCS and 1% penicillin-streptomycin, and incubated at 38°C. Ovaries from a local abattoir were processed to collect cumulus-oocyte complexes for cloning. The cloning process included the following: (1) addition of demecolcine to maturation media at 22h, (2) bisection at 24 to 26h, (3) fusion at 25 to 27h, (4) activation at 30h, and (5) culture at 36h. After maturation, the cumulus cells were removed from the oocytes by incubating in 0.1% (wt/vol) hyaluronidase in HEPES-buffered TCM-199 with 2% (vol/vol) steer serum (T2) for 5min, followed by vortexing for 3min. The resulting cumulus-free oocytes were incubated in maturation media containing 0.5µg mL−1 of demecolcine for 2h. Next, the zona pellucida was removed with 0.2% (wt/vol) pronase in T2. An ultrasharp cutting blade was used to bisect the oocytes under a stereomicroscope, producing karyoplasts containing extrusion cones and cytoplasts. Fusion of 2 cytoplasts with a fibroblast was performed on a BTX fusion slide (San Diego, CA, USA) using a single direct current pulse of 100V for 9 µs. After fusion, the reconstructed embryos (REC) were incubated in SOFaaci for 3h until activation. The REC were activated with 10µM calcium ionophore for 5min in T2, followed by incubation in SOFaaci containing 2mM DMAP for 6h. Activated REC were individually cultured in well-of-the-wells (Vajta et al. 2000Mol. Reprod. Dev. 55, 256-264) containing SOFaaci without serum in 6% CO2, 5% O2, and 89% N2 for 7 days. For transport, 2-mL transfer tubes were filled with 400 uL of SOFaaci; overlayed with oil; gassed with 6% CO2, 5% O2, and 89% N2; loaded with 1 embryo per tube; and placed into a 39°C portable incubator. On Farm A, 34 REC were produced, with 13 developing to blastocyst stage (38% blastocyst rate). After a 1.5-h transport, 7 grade 1 expanded blastocysts were implanted into 7 synchronized recipients. At the 90-day pregnancy check, 3/7 (42%) were pregnant. On Farm B, 35 REC were produced, with 14 grade 1 morulas or early blastocysts developing (40% blastocyst rate). After a 6-h transport, 9 morulas or early blastocysts were implanted into 9 synchronized recipients. At the 90-day pregnancy check, 2/9 (22%) were pregnant. Overall, 5/16 (31%) of recipients remained pregnant by month 8 of gestation. In conclusion, handmade cloning is a practicable method to produce, transport, and implant embryos into recipients in a commercial setting.

54 CHEMICAL ENUCLEATION OF IN VITRO-MATURED BUFFALO (BUBALUS BUBALIS) OOCYTES INDUCED BY DEMECOLCINE FOR HANDMADE SOMATIC CELL CLONING

2008 ◽  
Vol 20 (1) ◽  
pp. 108
Author(s):  
R. A. Shah ◽  
M. S. Chauhan ◽  
R. S. Manik ◽  
S. K. Singla
Keyword(s):  
Somatic Cell ◽  
Nuclear Transfer ◽  
Uv Light ◽  
Genetic Material ◽  
Bubalus Bubalis ◽  
Significant Loss ◽  
Control Group ◽  
Cone Formation ◽  
Handmade Cloning

Cloning by somatic cell nuclear transfer requires enucleation of the recipient oocyte to remove its genetic material. In handmade cloning, a simplified cloning procedure (Vajta et al. 2001 Cloning 3, 89–95), zona-free oocytes are manually bisected, stained, and exposed to UV light for selection of demi-oocytes devoid of chromosomes. Development of procedures for enucleation which avoid exposure to UV light and conserve most of the cytoplasmic volume are necessary for improving the efficiency of handmade cloning. Chemically assisted enucleation protocols involve treating cumulus–oocyte complexes (COCs) during IVM with cytoskeleton-modifying agents, like demecolcine, which induce protrusion cone formation on the surface of the oocytes (Tani et al. 2006 Cloning Stem Cells 8, 61–66). Such a cone-like structure can then be easily excised with a microblade and enucleation of the oocyte achieved without a significant loss of cytoplasm. The aim of the present study was to establish an efficient protocol for demecolcine treatment of buffalo COCs during IVM to obtain the maximal proportion of oocytes where chromosomes are either expelled into the surface protrusion cone or completely enucleated. In Experiment I, COCs (n = 244), obtained from slaughterhouse buffalo ovaries and matured in vitro in TCM-199 (containing 10% FBS, 5 µg mL–1 pFSH, and 0.81 mm sodium pyruvate) at 38.5�C (in 5% CO2, 90–95% relative humidity), were treated in two groups with demecolcine (0.5 µg mL–1) beginning at either 15 h or 19 h from the start of IVM up to the end of IVM at 22 h, and compared to a control group (without demecolcine). Data were analyzed using ANOVA. The proportion of oocytes where a protrusion cone was observed was greatest (P < 0.05) in the 15-h treatment group (84%, n = 72/86) compared to those in either the 19-h or control groups (58%, n = 46/78, and 60%, n = 48/80, respectively). The presence of demecolcine for the last 7 h of IVM appears to have a significant effect on protrusion cone formation in buffalo oocytes. In Experiment II, COCs (n = 276)were divided into four groups, matured in vitro this time for 24 h and treated with demecolcine (0.5 µg mL–1) from the onset of IVM and up to 18, 21, or 24 h, and compared to a control group without demecolcine. Protrusion cone formation was observed in 72% (52/72), 66% (40/60), 62% (40/64), and 70% (56/80) of oocytes, respectively, in these groups. These percentages did not differ significantly (P < 0.05). However, the 21-h treatment resulted in complete enucleation in 32% (20/64) of oocytes, which was significantly greater (P < 0.05) than that in the other three groups where no such enucleation was observed. It can be hypothesized that demecolcine-free treatment from 21 to 24 h may have assisted in inducing complete enucleation of a significant number of treated oocytes. In conclusion, these results show that demecolcine-assisted and induced enucleation procedures can be used for increasing the efficiency of oocyte enucleation in handmade cloning and other nuclear transfer procedures in buffalo.

56 TRANSGENESIS BY HANDMADE CLONING USING EGFP-TRANSFECTED YUCATAN FIBROBLASTS

2007 ◽  
Vol 19 (1) ◽  
pp. 146
Author(s):  
P. M. Kragh ◽  
Y. Du ◽  
J. Li ◽  
Y. Zhang ◽  
L. Bolund ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Polar Body ◽  
Cumulus Cells ◽  
Miniature Pig ◽  
Egfp Gene ◽  
Fusion Procedure ◽  
Reconstructed Embryos ◽  
Fetal Fibroblasts ◽  
Handmade Cloning

Somatic cell nuclear transfer (SCNT) offers the possibility of pig transgenesis. Importantly, genetic manipulations can be performed in cells isolated from special breeds followed by SCNT into enucleated oocytes isolated from slaughterhouse ovaries. In the present study, we established production of Yucatan blastocysts by the handmade cloning (HMC) technique using non-transgenic fibroblasts from the Yucatan miniature pig, and produced transgenic blastocysts using enhanced green fluorescent protein (EGFP)-positive Yucatan fetal fibroblasts. For transgenesis, Yucatan fibroblasts from a 40-day old fetus were transfected with a vector containing an EGFP gene and a neomycin-resistance selection gene by lipofection. Well separated neomycin-resistant colonies were isolated, expanded, and used for HMC. For HMC, cumulus–oocyte complexes were aspirated from ovaries of slaughterhouse sows and matured for 41 h. Subsequently, the cumulus cells were removed in hyaluronidase, and zonae pellucidae were partially digested by incubation in pronase. Oocytes with a visible polar body (PB) were subjected to oriented bisection. Less than half of the cytoplasm adjacent to the PB was removed with a microblade. The remaining parts, i.e. cytoplasts, were used as recipients for embryo reconstruction. Reconstructed embryos were produced by a two-step fusion procedure. At the first step, one cytoplast was fused with one fibroblast in the absence of Ca2+. After one h, the cytoplast-fibroblast pair and another cytoplast were fused and activated simultaneously in the presence of Ca2+, and subsequently cultured in cytochalasin B and cycloheximide for 4 h. The development of reconstructed embryos to the blastocyst stage was determined after 7 days of in vitro culture. When using non-transgenic and EGFP-positive Yucatan fetal fibroblasts, the rate of blastocyst formation (mean � SEM) were 36 � 7% (36/102) and 42 � 7% (32/77), respectively. In conclusion, the HMC technique was very efficient for production of blastocysts of Yucatan miniature pig origin using both non-transgenic and EGFP-positive fetal fibroblasts.

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