Endangered wolves cloned from adult somatic cells

Over the world, canine species, including the gray wolf, have been gradually endangered or extinct. Many efforts have been made to recover and conserve these canids. The aim of this study was to produce the endangered gray wolf with somatic cell nuclear transfer (SCNT) for conservation. Adult ear fibroblasts from a female gray wolf (Canis lupus) were isolated and cultured in vitro as donor cells. Because of limitations in obtaining gray wolf matured oocytes, in vivo matured canine oocytes obtained by flushing the oviducts from the isthmus to the infundibulum were used. After removing the cumulus cells, the oocyte was enucleated, microinjected, fused with a donor cell, and activated. The reconstructed cloned wolf embryos were transferred into the oviducts of the naturally synchronized surrogate mothers. Two pregnancies were detected by ultrasonography at 23 days of gestation in recipient dogs. In each surrogate dog, two fetal sacs were confirmed by early pregnancy diagnosis at 23 days, but only two cloned wolves were delivered. The first cloned wolf was delivered by cesarean section on October 18, 2005, 60 days after embryo transfer. The second cloned wolf was delivered on October 26, 2005, at 61 days postembryo transfer. Microsatellite analysis was performed with genomic DNA from the donor wolf, the two cloned wolves, and the two surrogate female recipients to confirm the genetic identity of the cloned wolves. Analysis of 19 microsatellite loci confirmed that the cloned wolves were genetically identical to the donor wolf. In conclusion, we demonstrated live birth of two cloned gray wolves by nuclear transfer of wolf somatic cells into enucleated canine oocyte, indicating that SCNT is a practical approach for conserving endangered canids.     

A cloned toy poodle produced from somatic cells derived from an aged female dog

To date, dogs have been cloned with somatic cell nuclear transfer (SCNT), using donor cells derived from large-breed dogs 2 months to 3 years of age. The objective of the present study was to use SCNT to produce a small-breed dog from ear fibroblasts of an aged poodle, using large-breed oocyte donors and surrogate females, and to determine the origin of its mitochondrial DNA (mtDNA) and the length of its telomeres. Oocytes were derived from large-breed donors, matured in vivo, collected by flushing oviducts, and reconstructed with somatic cells derived from an aged (14-year-old) female toy poodle. Oocytes and donor cells were fused by electric stimuli, activated chemically, and transferred into the oviducts of large-breed recipient females. Overall, 358 activated couplets were surgically transferred into the oviducts of 20 recipient dogs. Two recipients became pregnant; only one maintained pregnancy to term, and a live puppy (weighing 190 g) was delivered by Caesarean section. The cloned poodle was phenotypically and genetically identical to the nuclear donor dog; however, its mtDNA was from the oocyte donor, and its mean telomere length was not significantly different from that of the nuclear donor. In summary, we demonstrated that a small-breed dog could be cloned by transferring activated couplets produced by fusion of somatic cells from a small-breed, aged donor female with enucleated in-vivo-matured oocytes of large-breed females, and transferred into the oviduct of large-breed recipient female dogs.     

Dogs cloned from fetal fibroblasts by nuclear transfer

Fetal fibroblasts have been considered as the prime candidate donor cells for the canine reproductive cloning by somatic cell nuclear transfer (SCNT) in regard to the future production of transgenic dogs, mainly due to their higher developmental competence and handling advantage in gene targeting. In this study, the cloning efficiency with canine fetal fibroblasts as donor cells was determined. A total of 50 presumptive cloned embryos were reconstructed, activated and transferred into the oviducts of naturally synchronous recipient bitches. While the fusion rate (76.9%) was similar to those of our earlier studies with adult fibroblasts as donor cells (73.9–77.1%), a high cloning efficiency (4.0%; 2 births/50 embryos transferred) was found compared to the previous success rate with adult fibroblasts (0.2–1.8%). The cloned beagles were healthy and genotypically identical to the donor fibroblast cells. This study shows that a fetal fibroblast cell would be an excellent donor for future production of transgenic dogs via gene targeting in this cell followed cloning using SCNT technology.     

Production of cloned dogs by decreasing the interval between fusion and activation during somatic cell nuclear transfer

To improve the efficiency of somatic cell nuclear transfer (SCNT) in dogs, we evaluated whether or not the interval between fusion and activation affects the success rate of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells from a male or female Golden Retriever. In total, 151 and 225 reconstructed oocytes were transferred to 9 and 14 naturally synchronized surrogates for male and female donor cells, respectively. Chromosomal morphology was evaluated in 12 oocytes held for an interval of 2 hr between fusion and activation and 14 oocytes held for an interval of 4 hr. Three hundred seventy-six and 288 embryos were transferred to 23 and 16 surrogates for the 2 and 4 hr interval groups, respectively. Both the male (two pregnant surrogates gave birth to three puppies) and female (one pregnant surrogate gave birth to one puppy) donor cells gave birth to live puppies (P > 0.05). In the 2 hr group, significantly more reconstructed oocytes showed condensed, metaphase-like chromosomes compared to the 4 hr group (P < 0.05). A significantly higher pregnancy rate and a greater number of live born puppies were observed in the 2 hr group (13.0% and 1.1%, respectively) compared to the 4 hr group (0%) (P < 0.05). In total, three surrogate dogs carried pregnancies to term and four puppies were born. These results demonstrate that decreasing the interval between fusion and activation increases the success rate of clone production and pregnancy. These results may increase the overall efficiency of SCNT in the canine family.     

In vitro oocyte culture and somatic cell nuclear transfer used to produce a live-born cloned goat

The use of an in vitro culture system was examined for production of somatic cells suitable for nuclear transfer in the goat. Goat cumulus-oocyte complexes were incubated in tissue culture medium TCM-199 supplemented with 10% fetal bovine serum (FBS) for 20 h. In vitro matured (IVM) oocytes were enucleated and used as karyoplast recipients. Donor cells obtained from the anterior pituitary of an adult male were introduced into the perivitelline space of enucleated IVM oocytes and fused by an electrical pulse. Reconstituted oocytes were cultured in chemically defined medium for 9 days. Two hundred and twenty-eight oocytes (70%) were fused with donor cells. After in vitro culture, seven somatic cell nuclear transfer (SCNT) oocytes (3%) developed to the blastocyst stage. SCNT embryos were transferred to the oviducts of recipient females (four 8-cell embryos per female) or uterine horn (two blastocysts per female). One male clone (NT1) was produced at day 153 from an SCNT blastocyst and died 16 days after birth. This study demonstrates that nuclear transferred goat oocytes produced using an in vitro culture system could develop to term and that donor anterior pituitary cells have the developmental potential to produce term offspring. In this study, it suggested that the artificial control of endocrine system in domestic animal might become possible by the genetic modification to anterior pituitary cells.     

Improvement of a porcine somatic cell nuclear transfer technique by optimizing donor cell and recipient oocyte preparations

This study was conducted to improve a porcine somatic cell nuclear transfer (SCNT) technique by optimizing donor cell and recipient oocyte preparations. Adult and fetal fibroblasts, and cumulus and oviduct cells were used as donor cells, and in vivo- and in vitro-matured oocytes were employed as recipient oocytes. The percentages of fusion and development to the blastocyst stage, the ratio of blastocysts to 2-cell embryos, and cell number of blastocysts were monitored as experimental parameters. In Experiment 1, donor cells of four different types were transferred to enucleated oocytes matured in vitro, and more (P < 0.05) blastocysts were derived from SCNT of fetal fibroblasts than from that of other cells (15.9% versus 3.1-7.9%). For SCNT using fetal fibroblasts, increasing the number of subcultures up to 15 times did not improve developmental competence to the blastocyst stage (12.2-16.7%). In Experiment 2, fetal fibroblasts were transferred to enucleated oocytes that matured in vivo or in vitro. When parthenogenetic activation of both types of oocytes was conducted as a preliminary control treatment, a significant increase in blastocyst formation was found for in vivo-matured compared with in vitro-matured oocytes (36.4% versus 29.5%). However, no improvement was achieved in SCNT using in vivo-matured oocytes. In conclusion, the type of donor somatic cell is important for improving development after porcine SCNT, and fetal fibroblasts were the most effective among examined cells. A system with good reproducibility has been established using fetal fibroblasts as the donor karyoplast after subculturing 1-10 times, and using both in vivo and in vitro-matured oocytes as the recipient cytoplast.     

Production of nuclear transfer-derived piglets using porcine fetal fibroblasts transfected with the enhanced green fluorescent protein

A system for somatic cell nuclear transfer (SCNT) was developed and led to the successful production of GFP-transfected piglets. In experiment 1, two groups of SCNT couplets reconstructed with porcine fetal fibroblasts (PFF) and enucleated sow (S) or gilt oocytes (G): 1). received a simultaneous electrical fusion/activation (S-EFA or G-EFA groups), or 2). were electrically fused followed by activation with ionomycin (S-EFIA or G-EFIA groups), or 3). were subjected to electrical fusion and subsequent activation by ionomycin, followed by 6-dimethylaminopurine treatment (S-EFIAD or G-EFIAD groups). The frequency of blastocyst formation was significantly higher in S-EFA (26%) compared with that observed in the other experimental groups (P < 0.05), but not with S-EFIA (23%). Sow oocytes yielded significantly higher cleavage frequencies (68%-69%) and total cell numbers of blastocysts when compared with gilt oocytes, regardless of fusion/activation methods (P < 0.05). However, the ratio of inner cell mass (ICM)/total cells in G-EFA and S-EFA was significantly lower than in the other groups (P < 0.05). In experiment 2, SCNT couplets reconstructed with PFF cultured in the presence or absence of serum and enucleated sow oocytes were subjected to EFA. There were no effects of serum starvation on cell-cycle synchronization, developmental competence, total cell numbers, and ratio of ICM/total cells. In experiment 3, SCNT couplets reconstructed with PFF transfected with an enhanced green fluorescence protein (EGFP) gene using FuGENE-6 and enucleated sow oocytes were subjected to EFA and cultured for 7 days. Expression frequencies of GFP gene during development were 100%, 78%, 72%, 71%, and 70% in fused, two-cell, four to eight cells, morulae, and blastocysts, respectively. In experiment 4, SCNT embryos derived from different recipient cytoplasts (sows or gilts) and donor karyoplasts (PFF or GFP-transfected) were subjected to EFA and transferred to the oviducts of surrogates. The pregnancy rates in SCNT embryos derived from sow oocytes (66%-69%) were higher than those with gilt oocytes (23%-27%) regardless of donor cell types. One live offspring from GFP-SCNT embryos and two from PFF-SCNT embryos were delivered. Microsatellite analysis confirmed that the clones were genetically identical to the donor cells and polymerase chain reaction (PCR) from genomic DNA of cloned piglets and subsequent southern blot analysis confirmed the integration of EGFP gene into chromosomes.     

Cloning endangered gray wolves (Canis lupus) from somatic cells collected postmortem

The objective of the present study was to investigate whether nuclear transfer of postmortem wolf somatic cells into enucleated dog oocytes, is a feasible method to produce a cloned wolf. In vivo-matured oocytes (from domestic dogs) were enucleated and fused with somatic cells derived from culture of tissue obtained from a male gray wolf 6h after death. The reconstructed embryos were activated and transferred into the oviducts of naturally synchronous domestic bitches. Overall, 372 reconstructed embryos were transferred to 17 recipient dogs; four recipients (23.5%) were confirmed pregnant (ultrasonographically) 23-25 d after embryo transfer. One recipient spontaneously delivered two dead pups and three recipients delivered, by cesarean section, four cloned wolf pups, weighing 450, 190, 300, and 490g, respectively. The pup that weighed 190g died within 12h after birth. The six cloned wolf pups were genetically identical to the donor wolf, and their mitochondrial DNA originated from the oocyte donors. The three live wolf pups had a normal wolf karyotype (78, XY), and the amount of telomeric DNA, assessed by quantitative fluorescence in situ hybridization, was similar to, or lower than, that of the nuclear donor. In conclusion, the present study demonstrated the successful cloning of an endangered male gray wolf via interspecies transfer of somatic cells, isolated postmortem from a wolf, and transferred into enucleated dog oocytes. Therefore, somatic cell nuclear transfer has potential for preservation of canine species in extreme situations, including sudden death.     

Preimplantational embryo development and incidence of blastomere apoptosis in bovine somatic cell nuclear transfer embryos reconstructed with long-term cultured donor cells

This study was performed to investigate whether types and/or age of donor cells affect preimplantational embryo development and the incidence of apoptosis in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine fetal or adult ear fibroblasts were isolated, cultured in vitro and categorized into fresh or long-term cultured cells in terms of population doublings (PD): in fetal fibroblasts, <16 being considered fresh and >50 being long-term cultured; in adult ear fibroblasts, <16 being considered fresh and >30 being long-term cultured. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199, enucleated and reconstructed by SCNT. The reconstructed oocytes were fused, chemically activated, and cultured in modified synthetic oviduct fluid (mSOF) at 39 degrees C in a humidified atmosphere of 5% CO(2) air for 7 days. The early development of SCNT embryos was monitored under a microscope and the quality of blastocysts was assessed by differential counting of inner cell mass (ICM) and trophectoderm (TE) cells and by apoptosis detection in blastomeres using a terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling (TUNEL) assay. As results, types and/or age of donor cells did not affect the rate of blastocyst formation and the number of ICM and TE cells. However, a significant increase in apoptotic blastomeres was observed in SCNT embryos reconstructed with long-term cultured fetal or adult ear fibroblasts compared to those in SCNT embryos derived from fresh fetal or adult ear fibroblasts. In conclusion, these results indicated that the long-term culture of donor cells caused increased the incidence of apoptosis in bovine SCNT embryos but did not affect the developmental competence and the cell number of blastocysts.     

Improvement of canine somatic cell nuclear transfer procedure

The purpose of the present study on canine somatic cell nuclear transfer (SCNT) was to evaluate the effects of fusion strength, type of activation, culture media and site of transfer on developmental potential of SCNT embryos. We also examined the potential of enucleated bovine oocytes to serve as cytoplast recipients of canine somatic cells. Firstly, we evaluated the morphological characteristics of in vivo-matured canine oocytes collected by retrograde flushing of the oviducts 72 h after ovulation. Secondly, the effectiveness of three electrical strengths (1.8, 2.3 and 3.3 kV/cm), used twice for 20 micros, on fusion of canine cytoplasts with somatic cells were compared. Then, we compared: (1) chemical versus electrical activation (a) after parthenogenetic activation or (b) after reconstruction of canine oocytes with somatic cells; (2) culture of resulting intergeneric (IG) embryos in either (a) mSOF or (b) TCM-199. The exposure time to 6-DMAP was standardized by using bovine oocytes reconstructed with canine somatic cells. Bovine oocytes were used for SCNT after a 22 h in vitro maturation interval. The fusion rate was significantly higher in the 3.3 kV/cm group than in the 1.8 and 2.3 kV/cm treatment groups. After parthenogenesis or SCNT with chemical activation, 3.4 and 5.8%, respectively, of the embryos developed to the morula stage, as compared to none of the embryos produced using electrical activation. Later developmental stages (8-16 cells) were transferred to the uterine horn of eight recipients, but no pregnancy was detected. However, IG cloned embryos (bovine cytoplast/canine somatic cell) were capable of in vitro blastocyst development. In vitro developmental competence of IG cloned embryos was improved after exposure to 6-DMAP for 4 h as compared to 0, 2 or 6h exposure, although the increase was not significantly different among culture media. In summary, for production of canine SCNT embryos, we recommend fusion at 3.3 kV/cm, chemical activation, culture in mSOF medium and transfer of presumptive zygotes to the oviduct of recipient animals. The feasibility of IG production of cloned canine embryos using bovine cytoplasts as recipient of canine somatic cells was demonstrated.     

Production of transgenic canine embryos using interspecies somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry 'foreign' DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6?. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8-16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8-16-cell stages without mosaicism. In summary, our results demonstrated that, following successful isolation of canine transgenic cells, iSCNT embryos developed to early pre-implantation stages in vitro, showing stable GFP expression. These canine-bovine iSCNT embryos can be used for further in vitro analysis of canine transgenic cells and will contribute to the production of various transgenic dogs for use as specific human disease models.     

Production of viable cloned miniature pig embryos using oocytes derived from domestic pig ovaries

For production of viable somatic cell nuclear transferred (SCNT) miniature pig embryos, in vitro condition for controlling the quality of recipient oocytes derived from domestic pig ovaries should be evaluated. In the present study, to get information on optimal in vitro maturation (IVM) condition of oocytes, we investigated the effect of IVM duration of recipient oocytes on subsequent development of SCNT miniature pig embryos, the maturation-promoting factor (MPF) activity in recipient oocytes before and after SCNT, and the occurrence of premature chromosome condensation (PCC) and spindle morphologies of donor nuclei following SCNT. The optimal window of the IVM period in terms of in vitro developmental ability of SCNT embryos was determined to be 36-40 h after the start of IVM. The use of recipient oocytes matured for 36 and 40 h resulted in a high level of MPF activity before and after SCNT, and increased the occurrence of PCC in transferred nuclei compared to the use of oocytes matured for 44 and 52 h. The proportion of abnormal spindle-like structures increased as the IVM period was prolonged. In addition, SCNT embryos constructed from recipient cytoplasts obtained after 40 h of maturation by using fetal fibroblasts of miniature pigs were transferred to surrogate miniature pigs, and developed to full term. These results suggest that recipient oocytes matured for 36 h and 40 h effectively induce PCC with a normal cytoskeletal structure because of a high level of MPF activity; furthermore, the 40-h IVM period improves in vitro development of SCNT embryos to the blastocyst stage, resulting in the production of viable cloned miniature pigs.     

Cotransfer of parthenogenetic embryos improves the pregnancy and implantation of nuclear transfer embryos in mouse

The majority of somatic cell nuclear transfer (SCNT) clones dies in the peri- or postimplantation period. Improvement of the full-term healthy pregnancy rates is a key issue for the economical viability and animal welfare profile of SCNT technology. In this study the effects of cotransfer of parthenogenetic or fertilized embryos on the pregnancy and implantation of SCNT mouse embryos have been investigated. SCNT embryos were produced by transferring cumulus cell nuclei into enucleated B6D2F1 mouse oocytes, whereas parthenogenetically activated (PA) and fertilized embryos were derived from ICR mice by artificial activation with strontium and in vivo fertilization, respectively. SCNT embryos were inferior in their developmental capacity to blastocyst compared to either PA or fertilized embryos. SCNT embryos were transferred alone (SCNT), or cotransferred with two to three PA (SCNT + PA) or fertilized (SCNT + Fert) embryos into the oviducts of an ICR recipient. Both pregnancy and implantation rates originating from clones in the SCNT + PA group were significantly higher than those of SCNT group (p < 0.05). The weight of placentas of clones derived from SCNT, SCNT + PA, or SCNT + Fert was in all cases significantly higher than that of fertilized controls (p < 0.001). Most of the clones derived from SCNT embryos cotransferred with PA or fertilized embryos survived to adulthood and were fertile and healthy according to histopathological observations. Our results demonstrate in mouse that cotransfer of PA embryos improves the pregnancy and implantation of SCNT embryos without compromising the overall health of the resulting clones.     

Duration of in vitro maturation of recipient oocytes affects blastocyst development of cloned porcine embryos

This study investigated the effects of different incubation periods for oocyte maturation and contact inhibition of donor cells as well as different osmolarities for storage of recipient oocytes on fusion rates, cleavage rates, and blastocyst yields of porcine somatic nuclear transfer (SCNT) derived embryos. In addition, the in vivo developmental potential of cloned embryos derived from the most promising SCNT protocol was tested by transfer to recipient gilts. Storage of in vitro-matured oocytes for 7.5 h in calcium-free TL-HEPES medium at 295 or 320 mOsmol prior to activation yielded significantly (p < 0.05) higher parthenogenetic blastocyst rates compared to storage in TL-HEPES with an osmolarity of 270 mOsmol (24.4 +/- 3.0% and 26.2 +/- 4.3% vs. 18.3 +/- 6.4%, respectively, mean +/- SD) and improved the visibility of the polar body. Electrical fusion of fibroblasts to enucleated oocytes matured for 38, 40, or 42 h resulted in similar fusion and cleavage rates (74.8-84.4%). However, nuclear transfer with oocytes matured for 40 h in vitro yielded significantly higher (p < 0.05) development to the blastocyst stage after 7 days of culture (14.7 +/- 1.7%) than with oocytes matured for 38 h (9.5 +/- 2.1%) or 42 h (5.1 +/- 2.1%). Contact inhibition for 24, 48, or 72 h significantly (p < 0.05) increased the proportion of cells at G0/G1 compared with cycling fibroblasts. However, duration of contact inhibition of the donor cells for either 24, 48, or 72 h had no effect on blastocyst rates of SCNT embryos. Four gilts received an average of 150 SCNT embryos (range 138-161) reconstructed with oocytes matured for 40 h; two of these became pregnant; one of them went to term and farrowed four piglets on day 115 of pregnancy. Microsatellite analysis confirmed that the clones were genetically identical with the donor cells. These results show that changes of the in vitro maturation protocol may affect in vitro development of reconstructed porcine embryos, while duration of the contact inhibition period plays a minor role for the success of porcine SCNT. The effects on in vivo development are yet to be determined.     

Expression of leptin ligand and receptor and effect of exogenous leptin supplement on in vitro development of porcine embryos

The present study investigated the expression of ligand and receptor for leptin, and the effect of leptin supplementation on preimplantation development of porcine in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. The IVF embryos were produced using frozen boar semen and SCNT embryos were obtained by nuclear transfer of fetal fibroblasts into enucleated oocytes. The protein expression of leptin ligand and receptor was investigated in in vitro matured oocytes, 2-, 4- and 8-cell embryos, morulae and blastocysts derived from IVF and SCNT using immunofluorescence. Both the ligand and receptor were detected in in vitro matured oocytes and all stage of IVF and SCNT embryos. The IVF and SCNT embryos were cultured in modified North Carolina State University (mNCSU)-23 medium supplemented with various concentrations (0, 1, 10, 100 or 1000 ng/mL) of leptin. The rates of cleavage at day 2 and blastocyst formation at day 7, and cell number of blastocysts were monitored as experimental parameters. In SCNT embryos, supplementing with 1000 ng/mL leptin significantly (P<0.05) increased the rate of blastocysts formation (20.2% versus 12.9%) and total cell number (54.6+/-17.4 versus 45.1+/-15.2) compared to the control group. In IVF embryos, leptin supplementation did not affect preimplantation embryo development and cell number in blastocysts. In conclusion, the present study demonstrated the expression of leptin ligand and receptor and the embryotropic effect of leptin in SCNT embryos.     

The effect of donor cell serum starvation and oocyte activation compounds on the development of somatic cell cloned embryos

Two experiments, one comparing nuclear transfer (NT) embryo activation compounds, the other donor cell treatments, were conducted with a goal of identifying factors that improve the in vitro development of cloned bovine embryos. In experiment 1, 539 NT embryos were produced by combining serum starved bovine fetal fibroblasts with enucleated in vitro matured oocytes, activated with ionomycin, then randomly allocated to be incubated for 4 hours in either Butyrolactone-I (BL-I) or 6-dimethylaminopurine (DMAP). There was no significant difference in development to blastocyst or compact morula of fused embryos at Day 6.5 between BL-I and DMAP activated embryos (22.4% vs. 20.2%; p = 0.18). Karyotyping of 20 blastocysts and compact morula from each group determined that 65% of BL1 and 63% of DMAP embryos were diploid with the remainder mixoploid (2n + 4n). In Experiment 2, the development of 389 NT embryos reconstructed from either serum starved or serum fed fetal fibroblasts was assessed. More Day 7 blastocysts and compact morula developed in the serum starved group (34.5% vs. 18.8%; p = 0.008). To verify the viability of BL-I activated embryos, 10 blastocytes from experiment 2 were transferred into 4 recipient cows. Two morphologically normal fetuses, genetically identical to the original fetal cell line, were surgically recovered at day 45 of gestation. In summary, serum starvation of bovine fetal fibroblasts prior to NT significantly improved development to blastocyst. Additionally, we have shown that BL-I is a novel alternative compound for use in combination with ionomycin to activate NT embryos.     

Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes

The developmental potential of caprine fetal fibroblast nuclei after in vitro transfection and nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated. Fetal fibroblasts were isolated from Day 27 to Day 30 fetuses from a dwarf breed of goat (BELE: breed early lactate early). Cells were transfected with constructs containing the enhanced green fluorescent protein (eGFP) and neomycin resistance genes and were selected with G418. Three eGFP lines and one nontransfected line were used as donor cells in NT. Donor cells were cultured in Dulbecco minimum Eagle medium plus 0.5% fetal calf serum for 4-8 days prior to use in NT. Immature oocytes were recovered by laparoscopic ovum pick-up and matured for 24 h prior to enucleation and NT. Reconstructed embryos were transferred as cleaved embryos into synchronized recipients. A total of 27 embryos derived from transgenic cells and 70 embryos derived from nontransgenic cells were transferred into 13 recipients. Five recipients (38%) were confirmed pregnant at Day 35 by ultrasound. Of these, four recipients delivered five male kids (7.1% of embryos transferred) derived from the nontransfected line. One recipient delivered a female kid derived from an eGFP line (7.7% of embryos transferred for that cell line). Presence of the eGFP transgene was confirmed by polymerase chain reaction, Southern blotting, and fluorescent in situ hybridization analyses. Nuclear transfer derivation from the donor cells was confirmed by single-strand confirmation polymorphism analysis. These results demonstrate that both in vitro-transfected and nontransfected caprine fetal fibroblasts can direct full-term development following NT.     

Birth of Beagle dogs by somatic cell nuclear transfer

The present study was undertaken to evaluate two enucleation methods for somatic cell nuclear transfer (SCNT), and to standardize the optimum number of embryos for transfer to each recipient for canines. Oocytes retrieved from outbreed dogs were reconstructed with adult somatic cells from a male Beagle dog. A total of 134 or 267 oocytes were enucleated either by aspiration or squeezing method, fused with two DC pulses of 1.75 kV/cm for 15 μs electrical stimulation, chemically activated after 1 h of fusion using 10 μM calcium ionophore for 4 min and cultured 4 h in 1.9 mM 6-dimethylaminopurine. Finally, 103 or 214 embryos for aspiration or squeezing method were transferred to 6 or 11 naturally synchronized recipients, respectively. A total of 53, 317 and 342 embryos were transferred to 7, 17 and 12 recipients for the group of 4–10, 11–25 and 26–40 embryos, respectively. There was no difference between fusion rate (76.87% vs. 80.15%), full term pregnancy rate (16.66% vs. 27.27%) and percent of live puppies born (0.97% vs. 1.87%) for aspiration and squeezing method (P > 0.05). Production efficiency of cloned dogs was significantly affected by the number of embryos transferred to each recipient. No pregnancy was established for the group of 4–10 embryos (n = 7) and 26–40 embryos (n = 12) while pregnancy was detected in 23.53% recipients received a group of 11–25 embryos (n = 17). Among them, five (1.76%) live puppies were born (P < 0.05). These data show an increase in the overall efficiency of SCNT in canine species.     

Production of second-generation cloned cats by somatic cell nuclear transfer

We successfully produced second-generation cloned cats by somatic cell nuclear transfer (SCNT) using skin cells from a cloned cat. Skin cells from an odd-eyed, all-white male cat (G0 donor cat) were used to generate a cloned cat (G1 cloned cat). At 6 months of age, skin cells from the G1 cloned cat were used for SCNT to produce second-generation cloned cats. We compared the in vitro and in vivo development of SCNT embryos that were derived from the G0 donor and G1 cloned donor cat's skin fibroblasts. The nuclei from the G0 donor and G1 cloned donor cat's skin fibroblasts fused with enucleated oocytes with equal rates of fusion (60.7% vs. 58.8%, respectively) and cleavage (66.3% vs. 63.4%). The 2-4-cell SCNT embryos were then transferred into recipients. One of the five recipients of G0 donor derived NT embryos (20%) delivered one live male cloned kitten, whereas 4 of 15 recipients of the G1 cloned donor cat derived NT embryos (26%) delivered a total of seven male second-generation cloned kittens (four live kittens from one surrogate, plus two stillborn kittens, and one live kitten that died 2d after birth from three other surrogate mothers). The four second-generation cloned kittens from the same surrogate all had a white coat color; three of the four second-generation cloned kittens had two blue eyes, and one of the second-generation cloned kittens had an odd-eye color. Despite low cloning efficiency, cloned cats can be used as donor cats to produce second-generation cloned cats.