62 RECOVERY OF CELL NUCLEI FROM 15 000-YEAR-OLD MAMMOTH TISSUES AND INJECTION INTO MOUSE ENUCLEATED MATURED OOCYTES

Animal cells frozen with suitable cryoprotectants have been successfully cryopreserved for long periods of time, maintaining viability upon thawing. Animal cells frozen without cryoprotectant, however, may suffer serious damage and not be useful as donors in somatic cell nuclear transfer (SCNT). However, in some cases, old animal samples were frozen only as a whole body or a piece of tissue without cryoprotectant. If the cells from such old samples could be useful for SCNT, then there are potentially many candidates where individual animals could be reproduced. In this study, we examined the possibility of using mouse bone marrow cells frozen without cryoprotectant as nuclear donors in SCNT. Thigh bones were collected from B6C3F1 mice and frozen in either a –25�C or a –80�C freezer for more than one month. Thawing of frozen bones was performed by placing them in an incubator at 37�C. Bone marrow cells were collected by washing the bone cavity with saline. Recipient oocytes for SCNT were collected from B6D2F1 female mice. The enucleation of recipient oocytes and the injection of nuclei were performed as previously reported (Wakayama et al. 1998 Nature 394, 369–374) with a piezo-actuated micromanipulator system. In this study, 4 groups of mouse cells (fresh bone marrow cells, bone marrow cells frozen at –25�C, bone marrow cells frozen at –80�C, and fresh cumulus cells) were used as the nuclear donors in SCNT. After nuclear injection, embryos were kept in mCZB medium for 1 h at 37�C. Subsequently, embryos were cultured for 3 h with 5 µg mL–1 cytochalasin B and 10 mm SrCl2 for activation and cultured for an additional 20 h in mKSOM medium. The nuclear dynamics of SCNT embryos in each donor cell group was observed using 42,6-diamidino-2-phenylindole (DAPI) staining and a fluorescent microscope at 0, 1, 7, and 24 h after nuclear injection. Data were analyzed by Student's t-test. The cell viability after thawing by trypan blue vital staining was about 20% regardless of freezing temperature. At 7 h after nuclear injection, the SCNT embryos injected with frozen bone marrow cells, regardless of freezing temperature, had more single pronuclei (67%, 54/81; P < 0.05) than SCNT embryos injected with either fresh bone marrow cells (36%, 26/73) or cumulus cells (28%, 67/236). At 24 h after nuclear injection, fewer SCNT embryos injected with bone marrow cells, either fresh or frozen, developed to the 2-cell stage (fresh: 11%, 6/56; frozen at –25�C: 21%, 5/24; frozen at –80�C: 20%, 10/49) than SCNT embryos injected with cumulus cells (58%, 185/319; P < 0.05). There was no difference in the embryonic development to the 2-cell stage among SCNT embryos injected with either fresh or frozen bone marrow cells. Further studies are required to determine whether cells frozen without cryoprotectant are capable of resulting in viable clones.

P43. Several factors affect the developmental potential of rat somatic cell nuclear-transferred oocytes in vitro

Pregnancies obtained via somatic cell nuclear transfer (SCNT) show an increased incidence of pregnancy complications, including poor placental vascularization, hydrops placenta, and abnormal placental morphology. In a preliminary study of hydrops placentas from SCNT clones v. control cow placentas close to term, we observed a thickening of the endometrial epithelium, which appeared pseudostratified; in addition, the subepithelial endometrial vascular bed consisted of large, rare arterioles with thick basement membranes compared with the normal capillary bed observed in control animals. The fetal membranes of hydrops placenta from bovine SCNT clones also exhibited macroscopic morphological differences, which included edema, increased membrane thickness, and a gelatinous appearance. All of these morphological differences were similar to those we have reported for sheep SCNT clones (Palmieri et al. 2007 Placenta 28, 577–584). In the search for biomarkers that may indicate abnormal placental development early in pregnancy, placental tissues were collected on day 40 from pregnancies established after transfer of embryos created through SCNT (n = 17) or IVF (n = 6). Nuclear donor cells used to obtain SCNT embryos were those we have previously found to be highly susceptible to hydrops (from 5 to 19%), whereas the incidence of hydrops in IVF and controls in our laboratories did not exceed 0.5%. A sample of fetal membranes was fixed in 10% buffered formalin for histological analysis; another sample was snap-frozen in liquid nitrogen for analysis of mRNA levels for vascular endothelial growth factor (VEGF) and its receptors (KDR and FLT) by quantitative, real-time RT-PCR. Histological analysis of cell proliferation rate was performed using immunohistochemical localization of proliferating cell nuclear antigen and image analysis software (Image-Pro Plus). There were no differences (P = 0.39) in the rate of cell proliferation of the trophoblast in SCNT v. IVF (29.4 ± 4.9% v. 22.1 ± 3%). However, VEGF mRNA expression was less (P < 0.04) in the fetal membranes of SCNT than IVF on day 40 of pregnancy (0.30 ± 0.04 v. 0.53 ± 0.12, normalized to α-actin mRNA levels). The mRNA expression for KDR and FLT were similar in SCNT and IVF. These data suggest that the lower expression of VEGF, a major placental angiogenic factor, in fetal membranes of early pregnant SCNT fetuses may be an early biomarker for the development of hydrops placenta later in pregnancy. NDAES Proj. ND01727.

Successful reprogramming of somatic cell nuclei after nuclear transfer requires active remodeling by factors present in the nonactivated cytoplast. High levels of maturation promoting factor (MPF) activity are associated with this remodeling process which includes nuclear envelope breakdown (NEBD), premature chromosome condensation (PCC), and spindle formation. In this study, we examined the extent of nuclear remodeling in monkey somatic cell nuclear transfer (SCNT) embryos by monitoring the dynamics of lamin A/C appearance, as detected immunocytochemically, following fusion of donor cells with recipient cytoplasts. In the control, intracytoplasmic sperm injection (ICSI) fertilized embryos, lamin A/C was readily detected at the pronuclear stage but disappeared in early cleaving embryos only to reappear by the morula stage in association with the activation of the embryonic genome. We initially documented lack or incomplete NEBD and PCC in SCNT embryos in the form of retention of lamin A/C signal emanating from the donor nucleus. This observation was consistent with premature cytoplast activation due to the manipulation procedures. SCNT embryos produced by this approach typically arrested at the morula stage. Significant modifications in nuclear transfer protocols were then employed. Optimization of procedures resulted in robust NEBD and PCC, as indicated by loss of lamin A/C signal from the donor cell. Also, significant improvement of SCNT embryo development in vitro was observed, with a markedly improved blastocyst formation rate (21%). Several different fetal and adult somatic cell types screened as nuclear donors supported blastocyst development. SCNT blastocysts displayed a pattern of Oct-4 expression similar to that of sperm fertilized counterparts, indicative of efficient nuclear reprogramming. However, no pregnancies were established following a preliminary trial of 8 embryo transfers with 48 cloned embryos. Nevertheless, our results represent a breakthrough in efforts to produce cloned monkeys and should provide the resources required for the derivation of embryonic stem cells from SCNT blastocysts.

It was proposed that arresting nuclear donor cells in G0/G1 phase facilitates the development of embryos that are derived from somatic cell nuclear transfer (SCNT). Full confluency or serum starvation is commonly used to arrest in vitro cultured somatic cells in G0/G1 phase. However, it is controversial as to whether these two methods have the same efficiency in arresting somatic cells in G0/G1 phase. Moreover, it is unclear whether the cloned embryos have comparable developmental ability after somatic cells are subjected to one of these methods and then used as nuclear donors in SCNT. In the present study, in vitro cultured sheep skin fibroblasts were divided into four groups: (1) cultured to 70–80% confluency (control group), (2) cultured to full confluency, (3) starved in low serum medium for 4 d, or (4) cultured to full confluency and then further starved for 4 d. Flow cytometry was used to assay the percentage of fibroblasts in G0/G1 phase, and cell counting was used to assay the viability of the fibroblasts. Then, real-time reverse transcription PCR was used to determine the levels of expression of several cell cycle-related genes. Subsequently, the four groups of fibroblasts were separately used as nuclear donors in SCNT, and the developmental ability and the quality of the cloned embryos were compared. The results showed that the percentage of fibroblasts in G0/G1 phase, the viability of fibroblasts, and the expression levels of cell cycle-related genes was different among the four groups of fibroblasts. Moreover, the quality of the cloned embryos was comparable after these four groups of fibroblasts were separately used as nuclear donors in SCNT. However, cloned embryos derived from fibroblasts that were cultured to full confluency combined with serum starvation had the highest developmental ability. The results of the present study indicate that there are synergistic effects of full confluency and serum starvation on arresting fibroblasts in G0/G1 phase, and the short-term treatment of nuclear donor cells with these two methods could improve the efficiency of SCNT.

Serum starvation is a routine protocol for synchronizing nuclear donor cells to G0/G1 phase during somatic cell nuclear transfer (SCNT). However, abrupt serum deprivation can cause serious stress to the cells cultured in vitro, which might result in endoplasmic reticulum (ER) stress, chromosome damage, and finally reduce the success rate of SCNT. In the present study, the effects of tauroursodeoxycholic acid (TUDCA), an effective ER stress-relieving drug, on the nuclear donor cells under serum deprivation condition as well as following SCNT procedures were first assessed in the bovine. The results showed that TUDCA significantly reduced ER stress and cell apoptosis in those nuclear donor cells. Moreover, it significantly decreased the expression of Hdac1 and Dnmt1, and increased the level of H3K9 acetylation in nuclear donor cells compared with control group. SCNT reconstructed embryos cloned from TUDCA-treated donor cells showed significantly higher fusion, cleavage, blastocyst formation rate, total cell number in day 7 blastocysts, and lower apoptotic index than that from control group. In addition, the expression of Hdac1, Dnmt1 and Bax was significantly lower in blastocysts derived from TUDCA-treated donor cells than that from control group. In conclusion, TUDCA significantly reduced the ER stress of nuclear donor cells under serum starvation condition, and significantly improved the developmental competence of following SCNT reconstructed embryos when these TUDCA-treated cells were used as the nuclear donors.

In embryos reconstructed by somatic cell nuclear transfer (SCNT), components of the oocyte cytoplasm are capable of reprogramming the somatic genome to control subsequent development. Although the mechanisms that control nuclear reprogramming are unknown, we have previously hypothesized that the occurrence of nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) in the donor nucleus are beneficial. In previous studies we have demonstrated that treatment of ovine oocytes with caffeine (10 mM), a protein phosphatase inhibitor, increased the activities of both MPF and MAPK in enucleated oocytes (Lee and Campbell 2004 Reprod. Fertil. Dev. 16, 125) and additionally resulted in a significant increase in the occurrence of NEBD and PCC in donor nuclei. Furthermore, SCNT embryos reconstructed following caffeine treatment had significantly increased cell numbers at the blastocyst stage. More recently we have demonstrated that the use of caffeine treated ovine oocytes as cytoplast recipients can regulate the expression of several developmentally important genes in SCNT embryos, including Oct-4 and interferon-tau (Choi et al. 2006 Reprod. Fertil. Dev. 18, in press). This study was designed to establish the developmental potential of NT embryos reconstructed using caffeine treated oocytes as cytoplast recipients. Ear skin fibroblast cells established from a Merino ram were quiesced in DMEM containing 0.1% fetal bovine serum (FBS) for 3 days. Oocyte maturation and embryo reconstruction and culture were performed as previously described (Lee and Campbell 2004 Reprod. Fertil. Dev. 16, 125) with the exception that ovaries from Merino � Romney Marsh cross ewes were stimulated with FSH sponge (Folltropin�-V; Bioniche Animal Health, Beltsville, Ontario, Canada) and were collected at slaughter on Day 13 following sponging. Blastocyst stage embryos were surgically transferred to the uterine horn of synchronized Merino � Romney Marsh cross recipients (three blastocysts per recipient). Recipient ewes were scanned by ultrasonography at Days 30, 60, and 90 following embryo transfer. All data were analyzed by chi-square test. There were no differences in fusion (145/167; 86.8% vs. 174/205; 84.9%), cleavage (123/145; 84.8% vs. 135/174; 77.6%), or the development to blastocyst (33/145; 22.8% vs. 34/174; 19.5%) between control SCNT embryos and caffeine treated SCNT embryos. However, although the frequency of pregnancy between control and caffeine-treated NT groups (5/15; 33.3% vs. 7/14; 50.0%) at 30 days was not significantly different, control SCNT embryos showed significantly lower pregnancies (1/15; 6.7%) than caffeine treated SCNT embryos (4/14; 28.6%) at both 60 and 90 days. In conclusion, embryos reconstructed using caffeine-treated cytoplasts can induce pregnancy at the same frequency as untreated controls; furthermore, the results suggest that SCNT embryos produced in this way are more able to maintain pregnancy.

In vitro fertilisation (IVF) and somatic cell nuclear transfer (SCNT) have been implicated in a variety of developmental abnormalities. Aberrant gene expression is likely to account for much of the diminished viability and developmental abnormalities observed. In the present study, the expression of multiple genes in IVF and SCNT bovine blastocyst-stage embryos were evaluated and compared with in vivo-produced embryos. Eleven genes expressed at and following maternal-zygotic transcription transition were evaluated in individual blastocysts by real-time polymerase chain reaction following RNA amplification. A subset of those genes was also evaluated in individual IVF and SCNT eight-cell embryos. A fibroblast-specific gene, expressed by nuclear donor cells, was also evaluated in IVF and SCNT embryos. The observed gene expression pattern at the eight-cell stage was not different between IVF and SCNT embryos (P > 0.05). In vitro fertilisation and SCNT blastocyst expression was lower (P < 0.01) for all genes compared with their in vivo-produced counterparts, except for lactate dehydrogenase isoenzyme A (P < 0.001). The patterns of gene expression of the IVF and SCNT blastocysts were indistinguishable. Neither SCNT eight-cell nor blastocyst-stage embryos expressed the gene used as a fibroblast marker (collagen VIalpha1). For the genes evaluated, the level of expression was influenced more by the environment than by the method used to produce the embryos. These results support the notion that if developmental differences observed in IVF- and SCNT-produced fetuses and neonates are the result of aberrant gene expression during the preimplantation stage, those differences in expression are subtle.

Production of second-generation sheep clones via somatic cell nuclear transfer using amniotic cells as nuclear donors

Somatic cell nuclear transfer (SCNT) requires cytoplast-mediated reprogramming of the donor nucleus. Cytoplast factors such as maturation promoting factor are implicated based on their involvement in nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC). Given prior difficulties in SCNT in primates using conventional protocols, we hypothesized that the ability of cytoplasts to induce nuclear remodeling was instrumental in efficient reprogramming. NEBD and PCC in monkey (Macaca mulatta) SCNT embryos were monitored by lamin A/C immunolabeling. Initially, a persistent lamin A/C signal from donor cell nuclei after fusion with cytoplasts was observed indicative of incomplete NEBD following SCNT and predictive of developmental arrest. We then identified fluorochrome-assisted enucleation and donor cell electrofusion as likely candidates for inducing premature cytoplast activation and a consequent lack of nuclear remodeling. Modified protocols designed to prevent premature cytoplast activation during SCNT showed robust NEBD and PCC. Coincidently, over 20% of SCNT embryos reconstructed with fetal fibroblasts progressed to blastocysts. Similar results were obtained with other somatic cells. Reconstructed blastocysts displayed patterns of Oct-4 expression similar to fertilized embryos reflecting successful reprogramming. Our results represent a significant breakthrough in elucidating the role of nuclear remodeling events in reprogramming following SCNT.

The nonhuman primate is a highly relevant model for the study of human diseases, and currently there is a significant need for populations of animals with specific genotypes that can not be satisfied by the capture of animals from the wild or by conventional breeding. There is an even greater need for genetically identical animals in vaccine development or tissue transplantation research, where immune system function is under study. Efficient somatic cell nuclear transfer (SCNT) procedures could provide a source for genetically identical nonhuman primates for biomedical research. SCNT offers the possibility of cloning animals using cultured cells and potentially provides an alternative approach for the genetic modification of primates. The opportunity to introduce precise genetic modifications into cultured cells by gene targeting procedures, and then use these cells as nuclear donors in SCNT, has potential application in the production of loss-of-function monkey models of human diseases. We were initially successful in producing monkeys by NT using embryonic blastomeres as the source of donor nuclei and have repeated that success. However, when somatic cells are used as nuclear donor cells, the developmental potential of monkey SCNT embryos is limited, and somatic cell cloning has not yet been accomplished in primates. High rates of in vitro development to blastocysts, comparable with in vitro fertilization controls, and successful production of rhesus monkeys by NT from embryonic blastomeres suggests that basic cloning procedures, including enucleation, fusion, and activation, are consistent with the production of viable embryos. Although modifications or additional steps in SCNT are clearly warranted, the basic procedures will likely be similar to those extant for embryonic cell NT. In this chapter, we describe detailed protocols for rhesus macaque embryonic cell NT, including oocyte and embryo production, micromanipulation, and embryo transfer in nonhuman primates.

Despite the potential utility of primate somatic cell nuclear transfer (SCNT) to biomedical research and to the production of autologous embryonic stem (ES) cells for cell- or tissue-based therapy, a reliable method for SCNT is not yet available. Employing the rhesus monkey as a clinically relevant animal model, we have compared a conventional electrofusion method for SCNT with a one-step micromanipulation (OSM) method. A prospective, randomized trial was conducted using only oocytes that were mature [metaphase II (MII)] at collection and a fibroblast-like cell line as nuclear donor cells (fetal fibroblasts). The embryos produced were characterized for in vitro developmental potential, cell number, karyotype and expression of nuclear mitotic apparatus (NuMA) and OCT-4. An in vitro blastocyst development rate of 24.4% was achieved with the OSM method, significantly higher than the 12.2% obtained following electrofusion. SCNT-produced embryos expressed normal karyotypes, cell numbers and NuMA and OCT-4 proteins in most cases. SCNT with male nuclear donor cells resulted in the production of male, SCNT blastocysts, eliminating the possibility of a parthenogenetic origin. Of the four fibroblast cell lines tested as nuclear donor cells, two supported the routine production of blastocysts following SCNT. The application of a modified SCNT technique (OSM) followed by embryo culture in hamster embryo culture medium-10 (HECM-10) allows, for the first time, the routine production of SCNT blastocysts, most of which appear normal by immunochemical, cytochemical and in vitro developmental criteria. These embryos will provide a resource for isolating ES cells and for studies of nuclear reprogramming by monkey cytoplasts.

Dux-Mediated Corrections of Aberrant H3K9ac during 2-Cell Genome Activation Optimize Efficiency of Somatic Cell Nuclear Transfer.

Development of bioassays using the bovine model to measure the efficiency of SCNT in humans

To date, production of cloned rats by somatic cell nuclear transfer (NT) has not yet been successful. Inducing premature chromosome condensation (PCC) of injected cell nuclei in recipient cytoplasm is considered essential for successful mouse cloning by the Honolulu method. In the present study, some factors affecting PCC of rat cumulus cell nuclei injected into rat oocytes were examined. Wistar female rats (young: 4 to 5-week-old, mature: > or =10-week-old) were superovulated by injections of eCG and hCG, and oocytes recovered 14 or 17 h after hCG injection were received with cumulus cell nuclei using piezo-driven micromanipulator. When the oocytes were recovered 14 h post-hCG injection from young rats and the nuclear injection into oocytes was completed within 45 min, PCC was observed in 44-49% of NT oocytes. In the case of oocytes from mature rats, PCC occurred in 11-19% of the NT oocytes. Oocytes recovered 17 h post-hCG injection did not support PCC of the injected nuclei (0-7%) regardless of the donor age. Treatment of oocytes with a neutral cysteine protease inhibitor, N-acetylleucylleucylnorleucinal, slightly increased the incidence of PCC (48 vs 37%). Comparison of rat strains for oocyte donors indicated that proportions of NT oocytes undergoing PCC in Wistar and LEW oocytes (41-46%) were higher than those in Donryu and F344 oocytes (17-25%). Thus, ability of rat oocytes to promote PCC of the injected nuclei is dependent on the characteristics of oocytes, such as age or strain of donor rats, and timing of oocyte recovery.