Enhanced survivability of cloned calves derived from roscovitine-treated adult somatic cells

Nuclear transfer to produce cattle is inefficient because 1) donor cells are not easily synchronized in the proper phase of the cell cycle, 2) the nucleus of these cells is not effectively reprogrammed, 3) the rate of attrition of late-term pregnancies is high, and 4) the health of early postnatal calves is compromised. The cyclin dependent kinase 2 inhibitor, roscovitine, was used to maximize cell cycle synchrony and to produce cells that responded more reliably to nuclear reprogramming. Roscovitine-treated adult bovine granulosa cells (82.4%) were more efficiently synchronized (P < 0.05) in the quiescent G0/G1 phase of the cell cycle than were serum-starved cells (76.7%). Although blastocyst development following nuclear transfer was elevated (P < 0.05) in the serum-starved group (21.1%) relative to the roscovitine-treated cells (11.8%), the number of cells in the blastocysts derived from roscovitine-treated cells was higher (P < 0.05) than those derived from the serum-starved group (roscovitine-treated group: 142.8 +/- 6.0 cells; serum-starved group: 86.8 +/- 14.5 cells). The resulting fetal and calf survival after embryo transfer was enhanced in the roscovitine-treated group (seven surviving calves from six pregnancies) compared with serum-starved controls (two calves born, one surviving beyond 60 days, from five pregnancies). Roscovitine culture can predictably synchronize the donor cell cycle and increase the nuclear reprogramming capacity of the cells, resulting in enhanced fetal and calf survival and increased cloning efficiency.     

Reproductive characteristics of cloned heifers derived from adult somatic cells.

This study examined the onset of puberty, follicular dynamics, reproductive hormone profiles, and ability to maintain pregnancy in cloned heifers produced by somatic cell nuclear transfer. Four adult somatic cell-cloned heifers, derived from a 13-yr-old Holstein cow, were compared to 4 individual age- and weight-matched heifers produced by artificial insemination (AI). From 7 to 9 mo of age, jugular venous blood samples were collected twice weekly, and from 10 to 11 or 12 mo of age, blood sampling was carried out every other day. After the heifers reached puberty (defined as the first of 3 consecutive blood samples with peripheral plasma progesterone concentrations of >1 ng/ml), ultrasound examination of ovaries and jugular plasma sample collection were carried out daily for 1 estrous cycle. Cloned heifers reached puberty later than controls (mean +/- SEM, 314.7 +/- 9.6 vs. 272 +/- 4.4 days and 336.7 +/- 13 vs. 302.8 +/- 4.5 kg for clones and controls, respectively; P < 0.05). However, cloned and control heifers were not different in estrous cycle length, ovulatory follicle diameter, number of follicular waves, or profiles of hormonal changes (LH, FSH, estradiol, and progesterone). Three of the 4 clones and all 4 control heifers became pregnant after AI. These results demonstrate that clones from an aged adult have normal reproductive development.     

Behavioral observations of adolescent Holstein heifers cloned from adult somatic cells

Cloning using somatic cells offers many potential applications in biomedicine and basic research. The objective of this study was to test whether clones from the same genotype can be used as models to study the genetic influences of behavior. Specifically, several aspects of the behavior of four prepubertal heifers cloned from somatic cells of a 13-year-old Holstein cow along with age-matched control heifers were compared to determine whether juvenile clones from an aged adult behave similarly to their age-matched controls, and whether clones with identical genetic makeup exhibit any behavioral trends. Behavioral observations or behavior challenge tests were conducted to compare the following traits: vocalization, play behavior, movement frequencies, grooming, curiosity, and companion preference, as well as dominance and aggressiveness. From play behavior, movements and vocalization, we observed that these four juvenile clones of an aged genetic donor did not show behavioral indications of aging and were similar to their counterparts of comparable chronological age except that they tended to play less than controls. Behavioral trends were also observed in the clones that indicated that they exhibited higher levels of curiosity, more grooming activities and were more aggressive and dominant than controls. Furthermore, these four clones preferred each other or the donor as companions, which may indicate genetic kin recognition.     

Production of cloned pigs from adult somatic cells by chemically assisted removal of maternal chromosomes

The present study demonstrated that brief treatment of in vitro-matured porcine oocytes with demecolcine results in a membrane protrusion that contains a condensed chromosome mass, which can be easily removed by aspiration. This simple, chemically assisted method for removing maternal chromosomes enabled the production of a large number of nuclear-transferred porcine eggs. The development of eggs whose chromosomes were removed by this procedure following transfer of somatic cell nuclei to the blastocyst stage was not significantly different among groups activated using different procedures (6% to 11%) and was also not different among donor cells of different origins (3% to 9%), except for cumulus cells (0.4%). After transfer of 180 to 341 nuclear-transferred eggs that received somatic cells to 6 recipients, 2 of the recipients produced 8 healthy cloned piglets from the heart cells of a female pig. The chemically assisted method for removing maternal chromosomes was also effective for bovine and rabbit eggs.     

Production of cloned goats after nuclear transfer using adult somatic cells.

The developmental potential of adult somatic nuclei after nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated in a dwarf breed of goat (BELE: Breed Early Lactate Early). Somatic donor cells were obtained from two different sources: 1) adult granulosa cells (GCs) and 2) fetal fibroblasts. Primary GCs were obtained from follicular aspirants after laparoscopic oocyte pick-up (LOPU) and were cryopreserved immediately. Frozen aliquots of cells were thawed and cultured until confluent and were then cultured in low serum for 4 days before use in NT. Immature oocytes were obtained by LOPU and matured before enucleation and NT. Ninety-one adult GC-derived NT embryos were transferred into eight recipients, four of which were confirmed pregnant (50%) at Day 30 by ultrasound. Fifty-four male fetal fibroblast-derived NT embryos were transferred into six recipients, one of which was confirmed pregnant (17%). All pregnancies were maintained through term. Four recipients delivered seven female kids (three sets of twins) derived from the GC cultures (7.7% of embryos transferred). The other recipient delivered two male kids (3.7% of embryos transferred). Birth weights were within the normal range for dwarf goats. One female twin and one male twin died at birth; the remaining kids appeared healthy and normal. DNA analysis confirmed that the kids were genetically identical to their respective donors. These results demonstrated that adult caprine somatic cells could direct normal development after NT.     

Generation of cloned calves from different types of somatic cells

Remarkable progress has been made in animal cloning research since the first mammal was success-fully cloned[1], and the technique of SCNT is now widely used in biological studies. In theory, successful development of live offspring from SCNT embryos demonstrates that a fully differentiated somatic cell can be reprogrammed and restore its totipotency; in practice, animal cloning can be applied for duplication of elite animals, production of transgenic animals, rescue of endangered species …     

Phenotypic characterization of Hanwoo (native Korean cattle) cloned from somatic cells of a single adult

We investigated phenotypic differences in Hanwoo cattle cloned from somatic cells of a single adult. Ten genetically identical Hanwoo were generated by somatic cell nuclear transfer from a single adult. Weights at birth, growing pattern, horn and noseprint patterns were characterized to investigate phenotypic differences. The weights of clones at 6 and 12 months were slightly heavier than that of the donor. A horn pattern analysis revealed that seven clones had exactly the same horn pattern as the donor cow, whereas three were different. Although similarities such as general appearance can often be used to identify individual cloned animals, no study has characterized noseprint patterns for this end. A noseprint pattern analysis of all surviving clones showed that all eight animals had distinct noseprints. Four were similar to the donor, and the remaining four had more secondary-like characteristics.     

Generation of cloned calves from different types of somatic cells

Six types of bovine somatic cell lines, including a granulosa cell line of Chinese red-breed yellow cattle (YGR), a granulosa cell line of Holstein cow (HGR), two skin fibroblast cell lines of two adult Holstein cows respectively (AFB1 and AFB2), a skin fibroblast cell line (FFB) and an oviduct epithelial cell line (FOV) of a Holstein fetus, were established. Somatic cell nuclear transfer (SCNT) was carried out using these cells as nuclei donor, and a total of 12 healthy calves were cloned. The effects of different types of donor cells on developmental potential of bovine SCNT embryos were investigated, (i) There was no significant difference in development rates to the blastocyst stage for SCNT embryos from YGR and HGR (33.2% and 35.1%, respectively). Pregnancy rates of them were 33.3% and 30.2%, respectively; and birth rates were 16.7% and 11.6%, respectively, (ii) Development rates to the blastocyst stage for SCNT embryos from diffetent individuals (AFB1 and AFB2) differed significantly (27.9% and 39.4%, respectively, P < 0.05). Pregnancy rates of them were 36.2% and 36.4%, respectively; and birth rates were 14.9 % and 27.3%, respectively, (iii) There was significant difference in development rates to the blastocyst stage for SCNT embryos from FFB and FOV of the same fetus (37.9% and 41.5%, respectively,P < 0.05). Pregnancy rates of them were 45.7% and 24.1%, respectively; and birth rates were 22.9 % and 10.3%, respectively. Finally, developmental potential of bovine SCNT embryos from all four types of somatic cells from Holstein cows (HGR, AFB, FFB and FOV) were compared. Forin vitro development stage, development rates to the blastocyst stage for SCNT embryos from HGR, AFB, FFB and FOV were 35.1%^A, 29.4%^B, 37.9%^A and 41.5%^C, respectively (P ^ABC < 0.05); forin vivo development stage, pregnancy rates of them were 30.2%, 36.2%, 45.7% and 24.1%, respectively; and birth rates of them were 11.6%, 17.2%, 22.9% and 10.3% respetively.     

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.     

Reproductive fertility of cloned male cats derived from adult somatic cell nuclear transfer

This study was designed to investigate the reproductive fertility by the natural breeding of cloned male cats with domestic female cats, and to measure endocrine hormone concentration related to male reproduction such as testosterone, leutinizing hormone (LH), and follicle stimulating hormone (FSH). Cloned A, B, C, and D cats produced three, two, four, and five kittens after natural mating with four domestic female cats, respectively, despite later puberty of the cloned D cat than those of the other cloned male cats. Three of the 14 kittens expressed an odd eye color, which was produced by 1 and 2 from cloned A and B cats. The eye color of the other F1 kittens varied from nine brown to two blue. Body weight at birth ranged from 72.9 to 134.0 g. Although clone D had a poorer libido and entered puberty later than those of the other cloned male cats, he produced gonadal hormones within the average range. Four of the cloned male cats had normal fertility. The concentration of gonadal hormones in cloned male cats was similar to two control and donor cats. The concentration of testosterone was not significantly different among clones A, B, C, D, and control cats (5.99 +/- 5.68; 3.46 +/- 2.81; 6.41 +/- 2.17; 3.75 +/- 0.34; 4.0 +/- 3.63 ng/mL, p < 0.05). The concentrations of LH and FSH were not significantly different among the cloned cats (p < 0.05). Seven male and seven female (in total 14) kittens were produced by the natural breeding with four domestic female cats. These results indicated that cloned male cats have normal reproductive fertility and lie within the normal range of gonadal hormone production. All F1 kittens were produced by natural breeding and delivery, and are still alive and have normal growth health (27 months age).     

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.     

Mitochondrial DNA heteroplasmy in calves cloned by using adult somatic cell

Adult somatic cell cloned calves were produced by somatic cell nuclear transfer prepared by fusion of cultured ear fibroblast from a Holstein cow into enucleated oocytes of Luxi Yellow cow. In order to determinate the source of mitochondrial DNA of cloned calves, we designed the breed-specific PCR primers by aligning the known D-loop sequences of Bos taurus and analyzed the displacement loop sequences of five live cloned calves by breed-specific primers PCR. The results demonstrated that mtDNA originated from Holstein breed and that from Luxi breed co-exist in all five live calves.     

Cloned rabbits produced by nuclear transfer from adult somatic cells

We have developed a method to produce live somatic clones in the rabbit, one of the mammalian species considered up to now as difficult to clone. To do so, we have modified current cloning protocols proven successful in other species by taking into account both the rapid kinetics of the cell cycle of rabbit embryos and the narrow window of time for their implantation after transfer into foster recipients. Although our method still has a low level of efficiency, it has produced several clones now proven to be fertile. Our work indicates that cloning can probably be carried out successfully in any mammalian species by taking into account physiological features of their oocytes and embryos. Our results will contribute to extending the use of rabbit models for biomedical research.     

Chromosomal aneuploidy in African Wildcat somatic cells and cloned embryos

In the present study, we compared the incidence of aneuploidy in in vitro fertilized domestic cat embryos (DSH-IVF) with that of African Wildcat (AWC) cloned embryos reconstructed with AWC fibroblast donor cells from different passages (AWC-NT). Fibroblast cells were cultured to passages 1 (P1), 3 (P3), 4 (P4), and 9 (P9), after which cells at each passage were karyotyped and serum-starved before being frozen for nuclear transfer. AWC-NT embryos were produced by fusion of a single AWC somatic cell at P1, P3, P4, or P9 to enucleated domestic cat cytoplast derived from in vitro matured (IVU) oocytes. DSH-IVF embryos were produced after IVU oocytes were fertilized in vitro with domestic cat spermatozoa. To determine chromosome numbers, embryos (2-4-cell) or fibroblast cells were cultured in medium containing 0.28 microg/mL of Colcemid for 22-24 h or 15-24 h, respectively. Subsequently, embryos and cells were placed in hypotonic solution, fixed, and stained for analysis of chromosome spreads by bright field microscopy. Chromosomal abnormalities in AWC fibroblast cells increased progressively during culture in vitro: P1 (43%), P3 (46%), P4 (62%), and P9 (59%). In fibroblast cells, hypoploidy (94/202, 46%) was the major chromosomal abnormality, and it occurred more frequently than hyperploidy (14/202, 7%; p < 0.05). While the percentage of hyperploid cells remained stable during all passages, the proportion of hypoploidy in fibroblast cells increased significantly after P4. The overall incidence of chromosomal abnormalities in AWC-NT embryos at P1 (45%), P3 (60%), and P4 (50%) was similar to that of the fibroblast cells from which they were derived; however, the incidence was higher for embryos reconstructed with donor fibroblasts at P9 (89%). Hypoploidy was the most common chromosomal abnormality observed in either AWC-NT or DSH-IVF embryos. AWCNT embryos reconstructed with donor cells at early passages (P1, P3, and P4) had similar frequencies of chromosomal diploidy, as did DSH-IVF embryos. Accordingly, based on the present results, for NT we are currently using cat donor cells at early passages, when the percentage of cells with chromosomal abnormalities is low. It is recommended that the chromosomal stability of each cell line be analyzed before use as NT donor cells to reduce the incidence of chromosomal anomalies in reconstructed embryos and to possibly produce a subsequent increase in cloning efficiency.     

Postnatal growth and behavioral development of mice cloned from adult cumulus cells

Since the first successful cloning of mammals from adult somatic cells, there has been no examination of the learning or behavior of cloned offspring. The possibility of adverse effects on animals produced through adult somatic cell cloning is high because many natural biological processes are bypassed and DNA from adult cells, which presumably contain mutations, are used. In this study, we compared cloned mice produced by microinjection transfer of cumulus cell nuclei into enucleated oocytes, to control mice that were specifically generated to eliminate confounding factors that are unique to our cloning procedure. Postnatal weight gain of clones was significantly greater than that of controls. Preweaning development observations revealed that first appearance or performance of 3 out of 10 measures was delayed in cloned mice; however, results of subsequent tests of learning and memory, activity level, and motor skills were comparable for both groups. Together, these data suggest that nuclear transfer of adult somatic cell nuclei to produce cloned mice may delay the appearance of a few developmental milestones but it does not adversely affect the overall postnatal behavior of mice. In addition, this procedure may cause late onset of significantly increased body weight in cloned offspring, the cause or causes of which are being further examined.     

DNA methylation status in somatic and placenta cells of cloned cats

We recently produced 11 cloned kittens by somatic cell nuclear transfer (SCNT) using fibroblasts from a feline fetus (donor A, three kittens), an adult domestic cat (donor B, one kitten), and a deaf adult Turkish Angora cat (donor C, seven kittens). Two kittens were stillborn and three died a month after birth. The donor C-derived kittens did not share their donor's eye color or deafness. To test whether this and the low cloning success rate are due to epigenetic modifications, we compared the methylation of somatic and placental cells from the cloned cats and domestic normal cats by bisulfite mutagenesis sequencing analysis. The DNA methylation of somatic cells from the cloned kittens ranged from 78.0% to 88.9%, and did not differ significantly depending on whether they were stillborn, died early after birth, or were healthy. Donors B and C showed similar levels of methylation (77.0% and 79.1%, respectively), as did somatic cells from normal domestic and Turkish Angora cats (range, 75.7-88.0%). However, donor A showed less methylation (70.6%) than the somatic cells from the kittens derived from it (range, 82.2-88.9%). Moreover, placental cells from three donor C-derived kittens showed significantly higher DNA methylation (range, 76.7-80.5%) than placental cells from normal domestic cats (range, 64.2-74.9%). Thus, methylation of satellite regions in somatic cells may not be responsible for the stillbirth, early death, or different eye and hearing attributes of cloned cats. However, hypermethylation in the placenta of cloned cats may be responsible for low success rates in cloning cats.     

Growth and fertility of bulls cloned from the somatic cells of an aged and infertile bull

In the present study, somatic cell cloning technology was used to produce eight newborn calves from an aged, infertile bull. Average birth weight of these calves was significantly higher than that of calves produced using AI. Four of the cloned calves died during the peripartum period; the remaining four (Clones A-D) survived and were used in this study. Two of the surviving calves (Clones C and D) were castrated; growth rates of the intact and castrated clones were similar to those of intact and castrated bulls, respectively, that had been derived by AI. Both uncastrated bulls (Clones A and B) began to produce normal semen at approximately 12 months of age. Semen produced by these clones, and their nuclear donor, was subsequently used for IVF; the proportion of IVM-IVF oocytes developing to the blastocyst stage was 23.4% (50/214), 28.4% (52/183) and 30.9% (63/204), respectively. Conception rates for AI were 54.5% (12/22) and 62.7% (64/102) for semen derived from Clone A and from the nuclear donor, respectively. The length of pregnancy and birth weight of the calves derived from semen collected from clones were similar to those of calves obtained by conventional AI using semen from their nuclear donor. Therefore, sires cloned from the somatic cells of an aged and infertile bull had normal fertility.