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.

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.

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

65 CLONED EMBRYOS FROM HORSE SOMATIC CELLS AND ENUCLEATED BOVINE AND OVINE OOCYTES AND THEIR DEVELOPMENTAL COMPETENCE

2008 ◽  
Vol 20 (1) ◽  
pp. 113
Author(s):  
H. M. Zhou ◽  
B. S. Li ◽  
L. J. Zhang
Keyword(s):  
Somatic Cell ◽  
Somatic Cells ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Electrical Pulses ◽  
Mongolian Horse

The objective of this study was to investigate the reprogramming potential of equine somatic cell donor nuclei in either bovine or ovine recipient oocyte cytoplasmic environments. Heterogeneous embryos were reconstructed by somatic cell nuclear transfer (NT). The percentage of fusion and developmental competence, assessed by rates of cleavage and morula and blastocyst formation, were determined. Skin fibroblast cells, obtained from the ear of an adult female Mongolian horse, were dissociated using 0.25% trypsin and cultured in vitro in a humidified atmosphere of 5% CO2 in air at 37°C. Donor somatic cells were serum-starved before NT and used between passages 4 and 6. Bovine and ovine oocytes derived from slaughterhouse ovaries were matured in vitro for 17–19 and 22–24 h, respectively, in a humidified atmosphere of 5% CO2 in air at 38.5°C, before they were enucleated and used as recipient cytoplasts. The fibroblasts were injected under the zona pellucida of the cytoplasts and electrically fused by 2 DC electrical pulses of 1.58 kV cm–1 for 10 μs, with an interval of 0.13 s. The reconstructed embryos were then activated with 5 μm ionomycin in H-M199 for 5 min and then in 2 mm 6-DMAP for 4 h. The equine-bovine and equine-ovine reconstructed embryos were co-cultured, respectively, with bovine and ovine cumulus cells in synthetic oviduct fluid supplemented with amino acids (SOFaa) and 10% fetal calf serum (FCS) for 168 h. The data were analyzed with ANOVA and differences among the groups were evaluated with t-test. The results of the percentages of fusion, cleavage, and development to morula (8 to 64 cells) and blastocyst stages of equine-bovine and equine-ovine heterogeneous embryos are shown in Table 1. This study demonstrates that heterogeneous embryos can undergo early embryonic divisions and that reprogramming of equine fibroblast nuclei can be initiated in foreign cytoplasts. It appears that embryos reconstructed with equine somatic nuclei and ovine cytoplasts have a higher developmental potential than those using bovine cytoplasts. Table 1. Developmental competence of equine-bovine and equine-ovine reconstructed 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.

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 28 CLONED EMBRYOS CAN BE PRODUCED USING DONOR CELLS OBTAINED FROM A 72-HOUR COOLED CARCASS

2005 ◽  
Vol 17 (2) ◽  
pp. 164 ◽  
Author(s):  
S. Arat ◽  
H. Bagis ◽  
H. Odaman Mercan ◽  
A. Dinnyes
Keyword(s):  
Calf Serum ◽  
Calcium Ionophore ◽  
Cumulus Cells ◽  
Penicillin G ◽  
Developmental Potential ◽  
Viable Cells ◽  
Donor Cells ◽  
A Cell ◽  
Vitro Development

There are few reports on the use of cells from a dead mammal for nuclear transfer (NT). So far, most calves have been cloned from live adult cows or fresh fetal samples. The ability to produce cloned animals using postmortem tissue can provide an additional application to the field of NT. This study was conducted to investigate whether viable cells could be obtained from tissues chilled for 72 h and whether these cells could be used for NT. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM199 supplemented with 10% fetal calf serum (FBS), 50 μg/mL sodium pyruvate, 1% v:v penicillin-streptomycin (10,000 U/mL penicillin G, 10,000 μg/mL streptomycin), 10 ng/mL EGF, 0.5 μg/mL FSH, and 5 μg/mL LH. A cell line (MC) was established from leg muscle of a cow carcass stored at 0°C for 72 h. Tissues from muscle were cut into small pieces. Tissue explants were cultured in DMEM-F12 supplemented with 10% FBS at 37°C in 5% CO2 in air. Bovine granulosa cells (GC) were isolated from ovarian follicles and used for NT as control cells. Prior to NT, all somatic cells were allowed to grow to confluency (G1/G0) in DMEM-F12 medium supplemented with 10% FBS. Cumulus cells were removed by vortexing with hyaluronidase at 18 h after the start of maturation. Matured oocytes labeled with DNA fluorochrome Hoechst 33342 were enucleated under UV to ensure full removal of the chromatin. A single cell was inserted into the perivitelline space of the enucleated oocyte. Oocyte-cell couples were fused by a DC pulse of 133V/500 μm for 25 μs. After fusion, NT units were activated using a combination of calcium ionophore (5 μM), cytochalasin D (2.5 μg/mL) and cycloheximide (10 μg/mL) and cultured for 7 days in BARC or G1.3-G2.3 medium. Differences (developmental potential and cell numbers) among groups were analyzed by one-way ANOVA after arcsin square transformation. The results are summarized in Table 1. The results suggest that viable cells can be obtained from muscle of a cow carcass stored at cold temperature for 72 h and that these cells have ability to generate NT blastocysts at rates similar to those obtained with fresh GCs. In addition, G1.3 and G2.3 culture medium supported embryo development better than BARC medium. Table 1. In vitro development of NT embryos This study was supported by a grant from TUBITAK, Turkey (VHAG-1908 and Turkey-Hungary bilateral project VHAG-2022).

scholarly journals 61 CLONED MOUSE PRODUCED USING A ZONA FREE METHOD OF NUCLEAR TRANSFER

2005 ◽  
Vol 17 (2) ◽  
pp. 180
Author(s):  
R. Ribas ◽  
B. Oback ◽  
J. Taylor ◽  
A. Maurício ◽  
M. Sousa ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Uv Light ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Strontium Chloride ◽  
Embryonic Cells ◽  
Reconstructed Embryos

Mice have been cloned from somatic and embryonic cells; however, only 0–3% of the reconstructed embryos develop into viable offspring. In addition, the piezo microinjection method widely used for mouse nuclear transfer (NT) is difficult to master. Our objective was to compare cumulus and ES cells as nuclear donors using a simplified method of zona-free NT. In cattle, zona-free NT is simpler, faster, easier to learn and more reproducible than zona-intact NT (Oback et al. 2003 Cloning Stem Cells 5, 3–12). Oocytes were recovered at metaphase II stage (13 h after hCG injection) from the oviducts of C57BL/6J × DBA/2 F1 females (8–10 weeks of age). Cumulus cells were removed with hyaluronidase (300 units/mL) and the zona pellucida digested with pronase (0.5%) at 37°C for 3 min. Oocytes were then enucleated under UV light in cytochalasin B (5 μg/mL) after a 5-min staining with Hoechst (5 μL/mL). The metaphase DNA was removed in an enucleation pipette (16–20 μm, perpendicular break) by separating karyoplast and cytoplast with a simple separation pipette (60–80 μm, perpendicular break, closed round tip). Embryonic stem (ES) cells were cultured for 3 days and serum-starved for 16 h before use. Cells from this line had yielded offspring by the piezo procedure. Cumulus cells were used freshly. Donor cells were attached to the cytoplasts with phytohemagglutinin (10 μg/mL) and couplets were electrically fused in 0.2 mM mannitol buffer. Reconstructed embryos were activated 1–2 h after fusion for 5–6 h in CZB medium containing 10 mM strontium chloride and 5 μg/mL of cytochalasin B. Embryos were cultured individually in 5-μL droplets in CZB. Morulae and blastocysts were transferred into the uteri (Day 2.5) of pseudopregnant surrogate mothers (C57BL/6J × CBA/2J). Recipient mothers were sacrificed at 19.5 days postcoitum and pups removed. Airways were cleaned to remove fluid and the pups were held in a warm box before being fostered by a lactating mother. During development of the technique, we assessed the frequency of fusion, cleavage of reconstructed embryos, and development to morula/blastocyst stage. Fusion (58.1 ± 6.7% vs. 24.2 ± 1.7%, P < 0.001) and cleavage (66.4 ± 4.2% vs. 50.5 ± 5.4%, P < 0.05), all respectively, were higher when cumulus cells were used as donors, as compared with ES cells. However, the percentage of embryos developing to morula/blastocyst stage was greater when ES cells were used (22.2 ± 4.2% vs. 5.3 ± 2.7%, P < 0.01). Using ES cells as donors, 19/94 (20.2%) reconstructed embryos reached compacted morula/blastocyst stage. After transfer to five recipients, one pup was born (5.2%). It was larger and heavier than uncloned pups of the same age. The pup is healthy and now 12 weeks old. Genotype was confirmed by microsatellite analysis. The birth of a healthy cloned mouse pup from zona-free NT provides “proof of principle” of a technology that promises to increase throughput, ease of operation, and reproducibility of mouse cloning.

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.

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