67 GENERATION OF PIGS TRANSGENIC FOR hCD59/DAF AND HUMAN THROMBOMODULIN BY SOMATIC NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 142 ◽  
Author(s):  
B. Petersen ◽  
W. Kues ◽  
A. Lucas-Hahn ◽  
A.-L. Queisser ◽  
E. Lemme ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Fluorescein Isothiocyanate ◽  
Coagulation Cascade ◽  
Cell Cycle Synchronization ◽  
Novel Approach ◽  
Donor Cells ◽  
Serum Gonadotropin ◽  
Somatic Nuclear Transfer ◽  
Porcine Organs

After a porcine–to–primate xenotransplantation, hyperacute rejection (HAR) destroys the transplanted organ within minutes. The HAR can be overcome either by a knockout of the gene for α–1,3–galactosyltransferase or by overexpression of human complement regulatory proteins such as hCD59 and DAF. When HAR can be controlled, the next hurdle is acute vascular rejection (AVR) which is primarily due to an incompatibility of human protein C and porcine thrombomodulin, both of which are important factors in the coagulation cascade. This incompatibility leads to thrombosis and finally to a disseminated intravascular coagulation (DIC), causing rejection of the xenotransplant. Human thrombomodulin is a good candidate gene for improving survival time of porcine organs after xenotransplantation and for overcoming AVR. Here, we transfected adult fibroblasts obtained from a double transgenic boar (hCD59/DAF) with a construct for human thrombomodulin (hTM). After selection with 800 μg/mL G418 for 14 days, cells were analyzed for integration of the construct by PCR and were visually selected for expression of the hTM–GFP fusion protein under ultraviolet light with a fluorescein isothiocyanate (FITC) filterset. A total of 39 positve clones were obtained, of which two were used in somatic nuclear transfer. Ovaries were collected from a local slaughterhouse, and follicles of 2–5 mm in diameter were aspirated. After 38–42 h of in vitro maturation oocytes were denuded and enucleated. For cell cycle synchronization, the donor cells were serum–starved for 48 h, subsequently trypsinized and placed into the perivitelline space of the enucleated oocytes. The complexes were fused and activated electrically followed by an incubation in DMAP for 3 h. Puberal gilts were synchronized by treatment with 5 mL Regumate® (Intervet UK, Ltd., Milton Keynes, Buckinghamshire, UK) for 13 days. At the end of treatment, the animals received 1000 IU pregnant mare serum gonadotropin (PMSG) intramuscularly followed by an injection of 500 IU hCG 72 h later. Cloned embryos were transferred surgically 20 h after hCG injection. Maintenance of pregnancy was supported by injections of 1000 IU PMSG on Day 11 and 500 IU hCG on Day 14 of the pregnancy. Out of 1409 reconstructed complexes, 1161 were fused (82.4%) successfully. In total, 1040 embryos were transferred to 8 recipients (∼130 embryos/gilt, range 70–162). Five of the eight recipients (62.5%) became pregnant as determined by ultrasound on Days 25, 36, and 51 and will farrow within the next weeks. These results show that cloning with triple transgenic adult donor cells is compatible with high pregnancy rates. Porcine multitransgenic organs will be used in perfusion experiments to test the effectiveness of this novel approach to overcoming the incompatibilities between the porcine and the human coagulation systems. This project is funded by the Deutsche Forschungsgemeinschaft (FOR 535). The hTM–construct was a gift of Dr. Wu which is gratefully acknowledged.

scholarly journals Refrigeration of donor cells in preparation for bovine somatic nuclear transfer

Reproduction ◽  
2001 ◽  
pp. 801-808 ◽  
Author(s):  
JL Liu ◽  
MK Wang ◽  
QY Sun ◽  
XR Zhang ◽  
LK Jiang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Cultured Cells ◽  
Donor Cell ◽  
Serum Starvation ◽  
Fresh Tissue ◽  
Reconstructed Embryos ◽  
Donor Cells ◽  
Somatic Nuclear Transfer

In mammals, preparation of donor cells for somatic nuclear transfer is very important because the character of the donor cell directly affects the efficiency and outcome of transfer. The protocols used most commonly for donor preparation are (i) disaggregating cells from fresh tissue 1-2 h before micromanipulation or (ii) trypsinizing cultured cells temporarily, after special treatments for 3-8 days (for example, serum starvation). In this study, a new simple protocol was designed, whereby the donor cells (cumulus cells) used in bovine somatic nuclear transfer were refrigerated. In brief, cultured cells at 80-100% confluency were detached using trypsin, washed by centrifugation, aliquoted into different vials and refrigerated at 4 degrees C. The density of viable cells was decreased after day 1 of refrigeration; however, the rate of decrease tended to slow down with increasing duration of refrigeration. Cells refrigerated for 15 days were seeded at a density of 5 x 10(4) ml(-1) and reached 70% confluency after day 2 of culture. Most cells had the normal number of chromosomes (2n = 60). Cells chilled at 4 degrees C for different durations were removed from refrigeration and immediately subjected to micromanipulation. The in vitro development of reconstructed embryos (fusion rates, cleavage rates, morula and blastocyst rates) indicated that there were no significant differences among treatment groups regardless of the duration of refrigeration (0-2 weeks) of the donor cells. Reconstructed embryos were transferred into the uteri of recipient cows. No significant differences were observed in established early pregnancies between embryos derived from the non-refrigerated donor cells and those derived from refrigerated donor cells. This study indicates that refrigeration of donor cells for 1-2 weeks is a feasible protocol for preparing donor cells for bovine somatic nuclear transfer, and does not compromise development in vitro and early development in vivo.

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

2007 ◽  
Vol 19 (1) ◽  
pp. 168
Author(s):  
V. Zakhartchenko ◽  
F. Yang ◽  
R. Hao ◽  
E. Wolf
Keyword(s):  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Epigenetic Mark ◽  
Cloning Efficiency ◽  
Developmental Potential ◽  
Acetylation Status ◽  
Donor Cells ◽  
Fetal Fibroblasts

Epigenetic status of the genome of a donor nucleus is likely to be associated with the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). Prevention of epigenetic errors by manipulation of the epigenetic status of donor cells is expected to result in improvement of cloning efficiency. In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Ali/Bas) into metaphase II (MII) oocytes and analyzed the levels of histone H3K9 acetylation in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with one or two blastomeres from in vitro-fertilized or parthenogenetic embryos. Histone acetylation in donor cells and cloned embryos was detected by anti-acH3K9 antibody using Western immunoblot analysis or immunochemistry, respectively. Data were analyzed by chi-square (developmental rates) or Student-Newman-Keuls (histone acetylation) test. The levels of acetylated histone H3K9 were higher in RCCs than in RFFs (P < 0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC-cloned embryos induced a higher initial pregnancy rate as compared to RFF-cloned embryos (40% vs. 20%; P < 0.05). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed; a live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly (P < 0.05) increased the level of histone H3K9/14 acetylation and the proportion of nuclear transfer embryos developing to blastocyst (49% vs. 33% with non-treated RFF; P < 0.05). The distribution of signals for acH3K9 in either group of cloned embryos did not resemble that in in vivo-fertilized embryos, suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo-derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and can be a useful epigenetic mark to predict efficiency of SCNT rabbits. This work was supported by the Bayerische Forschungsstiftung and by Therapeutic Human Polyclonals, Inc.

52 INITIATION OF PREGNANCIES IN SOUTH AFRICAN RIVERINE RABBIT (BUNOLAGUS MONTICULARES) BY INTERSPECIES NUCLEAR TRANSFER USING ADIPOSE-DERIVED SOMATIC CELLS

2008 ◽  
Vol 20 (1) ◽  
pp. 106
Author(s):  
M. J. Sansinena ◽  
D. Owiny ◽  
R. S. Denniston ◽  
D. Salamone ◽  
D. Barry
Keyword(s):  
In Vitro Culture ◽  
Nuclear Transfer ◽  
Uv Light ◽  
Uv Exposure ◽  
Domestic Rabbit ◽  
Donor Cells ◽  
Significant Difference ◽  
Preliminary Study ◽  
Interspecies Nuclear Transfer

The riverine rabbit (Bunolagus monticulares), one of South Africa's most threatened mammals, with an estimated population size under 250, was upgraded from endangered to critically endangered in 2002. The low number of riverine rabbits precludes any attempts of nuclear transfer (NT) using intraspecific oocytes; therefore, the overall aim of this study was to assess the ability of the domestic rabbit (Oryctolagus cuniculus) oocyte to reprogram the somatic cell of the endangered riverine rabbit by interspecies NT. A preliminary study evaluated the effect of timing of enucleation after induction of ovulation (h post-hCG). A second study assessed the effects of two activation protocols. In addition, since the unique characteristics of the rabbit zona pellucida affect the speed of micromanipulation, different exposure periods to UV light at enucleation were evaluated. Adult domestic Californian rabbits were treated with eCG for 72 h, and ovulation was induced by hCG administration. Oocytes were collected by retrograde flushing at 12–14 h or 16–18 h post-hCG administration and stripped of cumulus investments with 0.5% hyaluronidase in Ca-Mg-free PBS. Metaphase-II oocytes were selected by visualizing the first polar body. Oocytes were stained with 2 mg mL–1 Hoechst 33342 for 5 min, and metaphase plates were removed with a 25–30 μm (O.D.) borosilicate beveled, spiked pipette after exposure to <5 or 30–40 s of UV light. Adult adipose-derived riverine rabbit fibroblasts grown to confluency in DMEM with 10% FCS were used as donor cells and fused with 2 consecutive DC pulses (3.2 kV cm–1, 45 μs). After reconstruction, couplets were randomly assigned for activation by either a second set of electrical pulses or incubation with ionomycin, followed by 1 h of incubation in 2 mm 6-DMAP. Embryos were co-cultured with a bovine oviductal cell monolayer in DMEM with 10% FCS and assessed for cleavage after 36 h of in vitro culture. There was a significant difference in the number of cleaved embryos from oocytes collected at 12–14 h post-hCG (n = 50) or 16–18 h post-hCG (n = 51) administration (57% v. 0% cleaved; P < 0.05). No significant difference was detected in embryos developing after electrofusion v. ionomycin activation treatments. However, a significantly greater number (P < 0.05) of embryos cleaved from oocytes exposed to <5 s UV than from oocytes exposed to 30–40 s UV (Table 1). A total of 20 embryos (4-cell to 16-cell stages) were surgically transferred to the oviducts of 4 adult New Zealand white synchronized recipients after 48 h of in vitro culture. Two recipients (<5 s UV exposure treatment group) were diagnosed pregnant by abdominal palpation at 15 days post-transfer; pregnancies were subsequently lost by Day 30, with placental tissues recovered. This preliminary study indicates the domestic rabbit oocyte is capable of reprogramming riverine rabbit donor cells. In addition, the time of oocyte collection after ovulation induction and the UV exposure period during enucleation have an effect on the efficiency of interspecies NT and embryo development in this species. Table 1. Effect of UV exposure during enucleation on the in vitro development of interspecies nuclear transfer riverine rabbit embryos

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.

28 GENERATION OF REACTIVE OXYGEN SPECIES IN BOVINE CULTURED SOMATIC CELLS AND SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DURING MICROMANIPULATION PROCEDURES AND EARLY IN VITRO DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 120 ◽  
Author(s):  
H. K. Bae ◽  
J. Y. Kim ◽  
I. S. Hwang ◽  
C. K. Park ◽  
B. K. Yang ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Serum Starvation ◽  
Ros Generation ◽  
Oh Radical ◽  
Oxygen Species ◽  
Donor Cells

The present study was conducted to examine the reactive oxygen species (ROS) generation levels in the donor cells, recipient oocytes, and somatic cell nuclear transfer (SCNT) embryos during nuclear transfer procedures. Bovine ear skin cells were classified by serum starvation, confluence, and cycling cells. Bovine metaphase II (MII) oocytes matured in vitro for 22 h and denuded by vortexing were enucleated and electrofused with serum-starved donor cells, then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture for 4 h. In vitro fertilization (IVF) was performed for controls. SCNT and IVF embryos were cultured in CR1aa supplemented with 3 mg mL–1 BSA for ∼36 h. Donor cells, recipient oocytes, and SCNT embryos were stained in 10 μM dichlorohydrofluorescein diacetate (DCHFDA) or 10 μM HPF dye each for 30 min at 39°C to measure the H2O2 or ·OH radical levels after various micromanipulation steps. SCNT and IVF embryos were also stained at the 1-, 2-, and 4-cell stages after 8, 24, and 42 h of fusion or insemination, respectively. The fluorescent emissions from the samples were recorded as JPEG file using a digital camera (F5.0, 4 s) attached to a fluorescent microscope with filters at 450 to 480 nm for excitation and at 515 nm for emission. The images were analysed using ImageJ software 1.37 (NIH) by the intensity of fluorescence (pixels) in each cell (total 70 to 75 cells in each group), oocyte and embryo (total 50 to 60 eggs or embryos in each group). 4 to 7 replicates were performed for each experiment, and data were analysed by Duncan′s multiple-range tests. H2O2 and ·OH radical levels of cultured somatic cells were high in confluence group and significantly low in serum starvation group (P < 0.05). During micromanipulation, H2O2 levels in recipient oocytes and SCNT embryos were increased by enucleation (37.2 pixels), electrofusion (49.7 pixels), and activation (40.6 pixels) treatments (P < 0.05) compared to that in MII oocytes (33.1 pixels), and the level of H2O2 was extremely increased immediately after electrofusion. ·OH radical levels were significantly higher during manipulation procedures (51.6 to 55.7 pixels; P < 0.05) compared to MII oocytes. During in vitro culture, the H2O2 and ·OH radical levels of SCNT embryos were significantly higher (P < 0.05) compared to IVF embryos at 1- (32.4 v. 17.3 and 52.0 v. 29.6 pixels, respectively), 2- (27.2 v. 22.0 and 33.4 v. 26.0 pixels, respectively), and 4-cell (25.1 v. 16.5 and 26.9 v. 20.7 pixels, respectively) stages. These results suggest that the culture type of donor cells can affect the ROS generation level and the cellular stress during micromanipulation procedures also can generate the ROS in bovine SCNT embryos, which may lead the cellular damages in bovine SCNT embryos. This work was supported by National Research Foundation of Korea Grant funded by the Korean Government (KRF-2008–313-F00067).

38 EFFECT OF OOCYTE MATURATION DURATION ON BLASTOCYST RATES AFTER EQUINE SOMATIC CELL NUCLEAR TRANSFER

2015 ◽  
Vol 27 (1) ◽  
pp. 112 ◽  
Author(s):  
Y. H. Choi ◽  
I. C. Velez ◽  
B. Macías-García ◽  
K. Hinrichs
Keyword(s):  
Somatic Cell ◽  
Oocyte Maturation ◽  
Nuclear Transfer ◽  
Transvaginal Ultrasound ◽  
Direct Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Blastocyst Development ◽  
Donor Cells

In equine cloning, the scarcity of equine oocytes places emphasis on development of the most efficient nuclear transfer (NT) methods possible. In other species, using oocytes matured for the shortest duration needed to reach metaphase II has increased NT efficiency. In the present study, we examined the effect of duration of oocyte maturation at the time of enucleation on equine cloned blastocyst production. Oocytes were collected from live mares by transvaginal ultrasound-guided aspiration of all visible follicles ≥5 mm in diameter. The oocytes were held overnight (16–22 h) at room temperature, matured in vitro, and reconstructed with donor cells as described in our previous study (Choi et al. 2013 Theriogenology 79, 791–796). In Experiment 1, oocytes were divided into 2 groups and matured for 20 or 24 h. After enucleation, oocytes were reconstructed by direct injection of donor cells. Reconstructed oocytes were held for 5 h and then activated by treatment with 5 μM ionomycin for 4 min, then injection with sperm extract, followed by incubation in 2 mM 6-DMAP for 4 h. The activated reconstructed oocytes were cultured in global human embryo culture medium under 5% CO2, 6% O2, and 89% N2 at 38.2°C for 7 to 11 days (20 mM glucose was added at Day 5) and blastocyst rate was recorded. Because a low maturation rate was found at 20 h in Experiment 1, in Experiment 2 oocytes were denuded at 20 h and those that were mature were enucleated and used for NT; those that had not cast out a polar body at 20 h were cultured for an additional 3 h (20 + 3h) and then evaluated for polar body formation and used for NT, which was conducted as in Experiment 1. Data were analysed by Fisher's exact test. In Experiment 1, 203 oocytes were collected in 46 aspiration sessions. The rate of oocyte maturation to metaphase II was significantly lower for oocytes cultured for 20 h (35/116, 30%), than for those cultured for 24 h (47/80, 59%). However, the rate of blastocyst development was significantly higher for oocytes cultured for 20 h (11/27, 41%) than for 24 h (2/38, 5%). In Experiment 2, 89 oocytes were collected in 18 aspiration sessions. After 20 h of maturation culture, 22 oocytes were mature (25%). After an additional 3 h of culture, 21 additional oocytes had matured. There were no significant differences between the two treatments (20 and 20 + 3h) in reconstruction rates (77%, 17/22, and 90%, 19/21, respectively) or blastocyst rates (24%, 4/17, and 32%, 6/19, respectively). These results indicate that duration of in vitro maturation, or the duration of presence of cumulus cells, influences blastocyst development after somatic cell NT in the horse. This appears to be due to a benefit of using oocytes immediately after they reach metaphase II; if this is ensured as in Experiment 2, the duration of maturation itself had no effect.This work was supported by the American Quarter Horse Foundation, the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts.

394 CHARACTERIZATION OF BOVINE ADULT-DERIVED ADIPOSE STEM CELLS FOR USE IN NUCLEAR TRANSFER

2010 ◽  
Vol 22 (1) ◽  
pp. 353
Author(s):  
A. A. Picou ◽  
J. Wilson ◽  
B. Dresser ◽  
G. T. Gentry ◽  
R. A. Godke ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Cell Lines ◽  
Nuclear Transfer ◽  
Skin Fibroblasts ◽  
Chi Square ◽  
Adult Cattle ◽  
Donor Cells ◽  
Significant Difference ◽  
Chi Square Analysis

Adipose tissue is an abundant source of adult-derived cells that have displayed multipotent properties in vitro. The goal of this research was to study the characteristics of bovine adipose-derived adult stem (ADAS) cells to determine the feasibility for use in NT. Adipose tissue was isolated from the brisket of adult cattle postmortem. Cells were isolated by incubation for 2 h with 0.25% collagenase solution, separation of stromal cells by centrifugation, and selection by adherence to plastic. The lifespan and growth characteristics for culture conditions were determined by a 2 × 2 factorial with DMEM or DMEM:F12 and with or without growth factor (GF) supplementation.A two-way ANOVA, followed by multiple pair-wise comparisons using Tukey’s test when applicable, was used to detect differences in population doublings (PD) until senescence for media treatments and GF supplementation. Dulbecco’s modified Eagle’s medium with GF supported significantly less (PD) (P > 0.05) than DMEM : F12. The average lifespan was approximately 30 PD, with a cell length of 48 h until passage 8 (P8). As cells approached replicative senescence, the cell cycle length was inconsistent. Two ADAS and one adult-derived skin fibroblast cell lines from different animals were subjected to differentiation conditions for adipocytes, chondrocytes, and osteoblasts at P2, P6, and P11. Differentiation was confirmed by histological staining. Passage 2 ADAS cells differentiated more efficiently than did P6, P11, or skin fibroblasts. Global levels of DNA methylation and histone acetylation were analyzed from P1 to P6 in 3 sets of cell lines consisting of ADAS and skin cells from the same animals by immune staining and flow cytometry. There was no significant difference (P > 0.05) between cell types by one-way ANOVA. Nuclear transfer was performed using ADAS cells as donor cells and commercially supplied oocytes. Mature, enucleated oocytes were reconstructed with either adult skin fibroblasts or ADAS cells. The percentage of cleaved and blastocysts from ADAS cells (62% and 8%, n = 163) and skin fibroblasts cells (42% and 8%, n = 170) were not different (P > 0.05) by chi-square analysis. Interspecies NT was attempted with eland (Taurotragus oryx) ADAS cells and enucleated bovine oocytes. Two groups of enucleated oocytes were reconstructed with bovine (n = 234) and eland (n = 290) ADAS cells. There was no significant difference between the number of cleaved embryos (38% and 39%) or blastocysts formed by chi-square analysis. A total of 3 interspecies embryos (1%) and 5 bovine embryos (14%) developed to blastocysts. Bovine ADAS cells are not more efficient than bovine adult-derived skin fibroblasts as donor cells, but they do represent a viable option for use in NT because of their higher in vitro development. Eland ADAS cells resulted in development to the blastocyst stage after interspecies NT.

Sign in / Sign up

Export Citation Format

Share Document