Interspecies Nuclear Transfer Research Articles

60 Evaluation of the production of cloned embryos by interspecies nuclear transfer (bovine-sheep)

60 Evaluation of the production of cloned embryos by interspecies nuclear transfer (bovine-sheep)

25th ANNIVERSARY OF CLONING BY SOMATIC-CELL NUCLEAR TRANSFER: Current applications of SCNT in advanced breeding and genome editing in livestock.

SCNT (somatic cell nuclear transfer) has complemented the toolbox of ARTs offering yet another technique to reproduce animals in an unprecedented way. Despite remarkable achievements, SCNT suffers low efficiency, high pregnancy losses and higher than normal stillbirth rates that makes it an expensive technique to reproduce animals. Moreover, due to welfare issues associated with gestation and the newborn offspring, it is banned in some countries. It has become evident that these problems are of epigenetic nature associated with incomplete genome reprogramming, observed more frequently in ruminants and less often and of minor degree in pigs and horses. Genome editing is enormously benefiting from SCNT to turn genome edited cells into animals, even if zygote microinjection of CRISPR/Cas9 will become an alternative route in some occasions. SCNT will also be a route to reprogram somatic cell to pluripotency since bona fide iPSC in livestock are missing while embryonic stem cells have been now established. This opens the way to other technologies like the development of artificial gametes or interspecies nuclear transfer. To strengthen its commercial applications, SCNT will face three major challenges, that is, intellectual property (extremely unclear in genome editing), regulatory approval by the relevant authorities of the resuting potential products and finally, acceptance by the public who will eventually decide with its behavior the life or the death of the technology.

Assessing the effect of interspecies oocyte nucleolar material dosage on embryonic development

Sequence differences are considered to be the basic cause of developmental failure in interspecies embryos when more distant species are combined. However, other phenomena, such as insufficient or excessive quantity of specific cellular factors, might also influence the outcome. These effects are usually not considered. One of the organelles shown to contain different amount of proteins is the oocyte nucleolus-like body. Here we show that upon interspecies transfer, a single porcine nucleolus-like body is unable to support the development of a mouse parthenogenetic embryo derived from an enucleolated oocyte. However, when the amount of the porcine nucleolar material is increased to equalize the amount of mouse nucleolar material by transferring two nucleolus-like bodies, mouse embryos are able to pass the developmental block elicited by enucleolation. These embryos progress to the blastocyst stage at rates comparable to controls. Thus, using the model of an interspecies nucleolus-like body transplantation between mouse and pig oocytes, we show that an inadequate amount of nucleolar factors, rather than the species origin, affects the development. In a wider context of interspecies nuclear transfer schemes, the observed incompatibility between more distant species might not stem simply from sequence differences but also from improper dosage of key cellular factors.

The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos

Abstract One of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription in oocytes and early embryos of several species including bovine and porcine zygotes is species-dependent process and the incomplete DNA methylation correlates with the nuclear transfer failure rate in mammals. In this study the transcription of DNA methyltransferase 1 and 3a (DNMT1, DNMT3a) genes in early embryonic stages of interspecies (bovine, porcine) nuclear transfer embryos (iSCNT) by RT-PCR were analyzed. Coming out from the diverse timing of embryonic genome activation (EGA) in porcine and bovine preimplantation embryos, the intense effect of ooplasm on transferred somatic cell nucleus was expected. In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly influenced by the ooplasmic environment.

Interspecies nuclear transfer using fibroblasts from leopard, tiger, and lion ear piece collected postmortem as donor cells and rabbit oocytes as recipients.

Skin fibroblast cells were obtained from a small piece of an ear of leopard, lion, and tiger collected postmortem and attempts were made to synchronize the skin fibroblasts at G0/G1 of cell cycle using three different approaches. Efficiency of the approaches was tested following interspecies nuclear transfer with rabbit oocytes as recipient cytoplasm. Fluorescence-activated cell sorting revealed that the proportion of G0/G1 cells increased significantly (P < 0.05) when cells subjected to serum starvation, contact inhibition, and 3mM sodium butyrate (NaBu) treatment when compared with cycling cells. However, 3mM NaBu treatment caused alterations in cell morphology and increase in dead cells. Thus, interspecies nuclear transfer was carried out using fibroblast cells subjected to contact inhibition for 72h, serum starvation for 48h, and cells treated with 1.0mM NaBu for 48h. The fusion rates, the proportion of fused couplets that cleaved to two-cell and developed to blastocyst, were highest in all three species when the donor cells were treated with 1.0mM NaBu for 48h. But, the blastocyst percentage of interspecies nuclear embryos (5-6%) was significantly lower when compared with rabbit-rabbit nuclear transfer embryos (22.9%). In conclusion, fibroblast cells of leopard, lion, and tiger were successfully synchronized and used for the development of blastocysts using rabbit oocytes as recipient cytoplasm.

Handmade cloning: recent advances, potential and pitfalls.

Handmade cloning (HMC) is the most awaited, simple and micromanipulator-free version of somatic cell nuclear transfer (SCNT). The requirement of expensive micromanipulators and skilled expertise is eliminated in this technique, proving it as a major revolution in the field of embryology. During the past years, many modifications have been incorporated in this technique to boost its efficiency. This alternative approach to micromanipulator based traditional cloning (TC) works wonder in generating comparable or even higher birth rates in addition to declining costs drastically and enabling cryopreservation. This technique is not only applicable to intraspecies nuclear transfer but also to interspecies nuclear transfer (iSCNT) thus permitting conservation of endangered species. It also offers unique possibilities for automation of SCNT which aims at production of transgenic animals that can cure certain human diseases by producing therapeutics hence, providing a healthier future for the wellbeing of humans. The present review aims at highlighting certain aspects of HMC including recent advancements in procedure and factors involved in elevating its efficiency besides covering the potentials and pitfalls of this technique.

Analysis of nuclear reprogramming following nuclear transfer to Xenopus oocyte.

Germinal vesicle of stage V-VI Xenopus Laevis oocytes (at the prophase I stage of meiosis) can be used to transplant mammalian nuclei. In this type of interspecies nuclear transfer no cell division occurs and no new cell types are generated. However, the transplanted nuclei undergo extensive transcriptional reprogramming. Here, it is first explained how to carry out transplantation of multiple mammalian cell nuclei to Xenopus oocytes. It is then described how to perform RT-qPCR, Western Blot, Chromatin Immunoprecipitation, and live imaging analysis to monitor transcriptional reprogramming of the nuclei transplanted to oocytes.

Fusion Competence of Interspecies Nuclear Transfer Embryos Reconstructed from Pedicle Periosteum Cell of Sika Deer (Cervus nippon) and Oocyte of Bovine

Fusion Competence of Interspecies Nuclear Transfer Embryos Reconstructed from Pedicle Periosteum Cell of Sika Deer (Cervus nippon) and Oocyte of Bovine

Cell cycle analysis and interspecies nuclear transfer of cat cells treated with chemical inhibitors

This study investigated the effect of chemical inhibitors on the cell-cycle synchronisation in cat fibroblast cells and evaluated the development of interspecies embryos reconstructed from cat donor cells and enucleated bovine oocytes. Cat fibroblast cells were treated with 15 μg/mL roscovitine or 0.05 μg/mL deme-colcine prior to cell cycle analysis and nuclear transfer. The percentage of cat fibroblast cells arrested at the G0/G1 phase in the roscovitine group was similar to that in the control group without any treatment. The percentage of cells arrested at the G2/M phase was significantly higher in the demecolcine group than in the control group. The fusion rate of interspecies couplets was significantly greater in the roscovitine group than in the control group. Most embryos stopped the development at the 2- or 4-cell stage, and none developed into blastocysts. Chemical inhibitor-induced donor cell cycle synchronisation did not overcome developmental arrest in interspecies cloned embryos.

Development of interspecies nuclear transfer embryos reconstructed with argali (Ovis ammon) somatic cells and sheep ooplasm

Interspecies nuclear transfer has already achieved success in several species, which shows great potential in recovery and conservation of endangered animals. The study was conducted to establish an efficient system for in vitro argali (Ovis ammon)-sheep embryo reconstruction via interspecies somatic cell nuclear transfer (iSCNT). The competence of domestic sheep cytoplasts to reprogram the adult argali fibroblast nuclei was evaluated, and the effects of enucleation methods and donor cell passage and cell state on the in vitro development of argali-sheep cloned embryos were also examined. Sheep oocytes could support argali and sheep fibroblast cell nuclei transfer and develop to blastocysts in vitro. Oocytes matured for 21–23 h and enucleated by chemically assisted enucleation (CAE) had a higher enucleation rate than blind enucleation (BE), but the development rate of iSCNTembryos was the same (P>0.05). Moreover, passage numbers of fibroblast cells <10, as well as the cell cycle stages did not affect the development rate of iSCNT reconstructed embryos. Thus sheep cytoplasm successfully supports argali nucleus development to blastocyst stage after optimising the nuclear transfer procedure, which indicates that iSCNT can be used to conserve endangered argali in the near future.

In vitro development of goat-sheep and goat-goat zona-free cloned embryos in different culture media

The gradual decline in the genetic diversity of farm animals has threatened their survival and risk of their extinction has increased many fold in the recent past. Endangered species could be rescued using interspecies embryo production. The objective of this study was to investigate the effect of three different culture media on the development of Handmade cloned intraspecies (goat-goat) and interspecies (goat-sheep) embryo reconstructs. Research vitro cleave media (RVCL) yielded higher cleavage and morula-blastocyst development in intraspecies and interspecies nuclear transfer groups compared with G1.G2 and modified synthetic oviductal fluid (mSOFaaci). Cleavage frequency of intraspecies cloned embryos in RVCL, mSOFaaci, and G1.G2 did not differ significantly (87.12%, 82.45%, and 92.52%, respectively). However, the morula/blastocyst frequency in RVCL was greater in mSOFaaci and G1.G2 (51.18% vs. 38.28% vs. 36.50%, respectively). Cleavage and morula/blastocyst frequency in interspecies cloned embryos was greater in RVCL than in mSOFaaci and G1.G2 (76.14% and 42.3% vs. 65.9% and 38.3% vs. 58.56% and 33.1%, respectively). Goat oocytes were parthenogenetically activated and cultured in RVCL, mSOFaaci, and G1.G2 and kept as control. Cleavage and morula/blastocyst frequency in this group was greater in RVCL than in mSOFaaci and G1.G2 (89.66% and 65.26% vs. 85.44% and 48.05% vs. 86.58% and 42.06%, respectively). Conclusively, the results suggest that not only can the interspecies embryos of goat be produced using sheep oocytes as donor cytoplast but also the percentages can be improved by using RVCL media for culturing of the embryos.

Amphibian interorder nuclear transfer embryos reveal conserved embryonic gene transcription, but deficient DNA replication or chromosome segregation

Early interspecies nuclear transfer (iNT) experiments suggested that a foreign nucleus may become permanently damaged after a few rounds of cell division in the cytoplasm of another species. That is, in some distant species combinations, nucleocytoplasmic hybrid (cybrid) blastula nuclei can no longer support development, even if they are back-transferred into their own kind of egg cytoplasm. We monitored foreign DNA amplification and RNA production by quantitative PCR (qPCR) and RT-qPCR in interorder amphibian hybrids and cybrids formed by the transfer of newt (Pleurodeles waltl) embryonic nuclei into intact and enucleated frog (Xenopus laevis) eggs. We found a dramatic reduction in the expansion of foreign DNA and cell numbers in developing cybrid embryos that correlated with reduced gene transcription. Interestingly, expansion in cell numbers was rescued by the recipient species (Xenopus) maternal genome in iNT hybrids, but it did not improve P. waltl DNA expansion or gene transcription. Also, foreign gene transcripts, normalized to DNA copy numbers, were mostly normal in both iNT hybrids and cybrids. Thus, incomplete foreign DNA replication and/or chromosome segregation during cell division may be the major form of nuclear damage occurring as a result of nuclear replication in a foreign cytoplasmic environment. It also shows that the mechanisms of embryonic gene transcription are highly conserved across amphibians and may not be a major cause of cybrid lethality.

Nuclear-Cytoplasmic Incompatibility in Inter-Species Cytoplasm Hybrid Embryos: Implications for Inter-Species Nuclear Transfer Hybrid Embryo.

Interspecies somatic cell nuclear transfer (iSCNT) embryos reconstructed between other than closely related species rarely develop to the blastocyst stage and beyond. One explanation for this lack of development is an inability to reprogram the somatic genome. To explore this possibility we evaluated whether using a karyoplast that would require less extensive reprogramming such as an embryonic blastomere or the meiotic spindle from metaphase II oocytes would provide additional insight into the development of iSCNT embryos. Our results showed that karyoplasts of embryonic or oocyte origin are no different from somatic cells; all interspecies nuclear transfer embryos independent of karyoplast origin failed to develop to the blastocyst stage. We speculated that apart from faulty epigenetic reprogramming, nuclear-cytoplasmic incompatibility could be another reason for failure of embryonic development from interspecies nuclear transfer. To address nuclear-cytoplasmic incompatibility we used pig-mouse interspecies cytoplasmic hybrids as a model. Fertilized murine zygotes were reconstructed by fusing with porcine cytoplasts of varying cytoplasmic volumes (1/5 (small) and 1/3 (large) total volume of mouse zygote) produced from activated oocytes. The presence of pig cytoplasm significantly reduced in vitro development of mouse zygotes to the blastocyst stage as compared to control embryos at 120 h Post hCG (41% vs 6% vs 94%, P<0.05), (1/5, 1/3, control respectively). While copy numbers of the mitochondrial genome (mtDNA) remained relatively unchanged in cytoplasmic hybrids and control mouse embryos, genes responsible for mitochondrial replication (Tfam, PlogG, and PlogG2) mitochondrial fusion (mfn2) glucose transport (Glut 3 and Glut1) anti apopotosis (Bcl2 and hspb1) and pluripotency and lineage segregation (Oct4, Nanog, cdx2, Gata3, Tcfap2c) were significantly down regulated in cytoplasmic hybrid embryos. Methylation at promoter regions of Oct4 in cytoplasmic hybrids was slightly increased, however, methylation at LINE1 was not different from control embryos. We speculate that abnormal gene expression in cytoplasmic hybrids may be a consequence of the presence of foreign cytoplasmic components. RNA binding proteins, microRNAS and maternal transcripts of pig cytoplasmic origin may have adversely interfered with precise regulation of maternal murine transcripts and post-transcriptional and/or translation regulation of nascent mRNA synthesized from the mouse embryonic genome. These results demonstrate that the presence of even a small amount of porcine cytoplasm is detrimental to murine embryo development and suggest that a range of factors are likely to contribute to the failure of interspecies nuclear transfer embryos. (poster)

Nuclear–cytoplasmic incompatibility and inefficient development of pig–mouse cytoplasmic hybrid embryos

Inter-species somatic cell nuclear transfer (iSCNT) embryos usually fail to develop to the blastocyst stage and beyond due to incomplete reprogramming of donor cell. We evaluated whether using a karyoplast that would require less extensive reprogramming such as an embryonic blastomere or the meiotic spindle from metaphase II oocytes would provide additional insight into the development of iSCNT embryos. Our results showed that karyoplasts of embryonic or oocyte origin are no different from somatic cells; all iSCNT embryos, irrespective of karyoplast origin, were arrested during early development. We hypothesized that nuclear-cytoplasmic incompatibility could be another reason for failure of embryonic development from iSCNT. We used pig-mouse cytoplasmic hybrids as a model to address nuclear-cytoplasmic incompatibility in iSCNT embryos. Fertilized murine zygotes were reconstructed by fusing with porcine cytoplasts of varying cytoplasmic volumes (1/10 (small) and 1/5 (large) total volume of mouse zygote). The presence of pig cytoplasm significantly reduced the development of mouse zygotes to the blastocyst stage compared with control embryos at 120 h post-human chorionic gondotropin (41 vs 6 vs 94%, P<0.05; 1/10, 1/5, control respectively). While mitochondrial DNA copy numbers remained relatively unchanged, expression of several important genes namely Tfam, Polg, Polg2, Mfn2, Slc2a3 (Glut3), Slc2a1 (Glut1), Bcl2, Hspb1, Pou5f1 (Oct4), Nanog, Cdx2, Gata3, Tcfap2c, mt-Cox1 and mt-Cox2 was significantly reduced in cytoplasmic hybrids compared with control embryos. These results demonstrate that the presence of even a small amount of porcine cytoplasm is detrimental to murine embryo development and suggest that a range of factors are likely to contribute to the failure of inter-species nuclear transfer embryos.

Comparison of intra- and interspecies nuclear transfer techniques in the production of cloned caprine embryos

Among the ARTs that are applied in goat farming industry, reproductive cloning technology in production of cloned goat embryos are foreseen to facilitate the effort of mass goat production in just a short time frame. There are two possible approaches that can be applied to produce cloned embryos, namely intraspecies SCNT (intraSCNT) and interspecies SCNT (interSCNT). The application of interSCNT is known to play vital role in species preservation, livestock propagation and therapeutic cloning. In fact, the application of interSCNT approach to produce cloned caprine embryos has not been reported at present. The prospect of this application can be seen to overcome the relative difficulty of obtaining caprine oocytes to be used as recipient cytoplasm in the cloning protocol. Several studies have shown that ooplasm of bovine can support early development of embryos produced by nuclear transfer using somatic cell nuclei derived from different mammalian species such as sheep, pigs and rats. Therefore, this present study was conducted with the aim to produce cloned caprine embryos using intraSCNT versus interSCNT technique. As a control to this experiment, bovine intraSCNT was conducted. The source of bovine and caprine oocytes was obtained from the abattoir-derived ovaries and also via laparoscopy ovum pick-up (LOPU) technique on superovulated does. The collected oocytes were subsequently cultured in in vitro maturation medium for 18–22h. The matured oocytes were then subjected to enucleation process. The enucleated oocytes were then injected with either a male ear fibroblast cell from caprine or bovine. The couplets were electrofused and chemically activated before in vitro cultured. The results for fusion rate of caprine interSCNT (64.2%) was significantly lower compared to the caprine intraSCNT (81.9%). The reconstructed caprine oocytes derived from interSCNT approach seemed to have the developmental efficiency that is comparable to the intraSCNT approach as the cleavage rate of both caprine intra- (48.9%) and interSCNT (51.3%) embryos did not differ significantly. The in vitro development of caprine interSCNT could not go beyond morula stage. Therefore the comparison of the in vitro developmental rate of cloned bovine and caprine embryos using the intra- and interSCNT approach was made up to morula stage. The percentage of cloned caprine embryos developed to morula using intra- (20.6%) and interSCNT (6.9%) approach did not differ significantly. However, the percentage of cloned bovine morula derived from intraSCNT approach (46.1%) was significantly higher. Generally, caprine embryos are known to have a lower in vitro developmental potential towards the late perimplantation stage. Even the reports of success in producing cloned kids involved the transfer of embryos at early cloned embryos stages from 2 to 8 cell stages. In the nutshell, cloned caprine embryos can be produced via both intraSCNT and interSCNT approach. The efficacy of interSCNT approach is comparable to the intraSCNT approach in an effort to produce early preimplantation stages of cloned caprine embryos.

Formation of nucleoli in interspecies nuclear transfer embryos derived from bovine, porcine, and rabbit oocytes and nuclear donor cells of various species

The most successful development of interspecies somatic cell nuclear transfer (iSCNT) embryos has been achieved in closely related species. The analyses of embryonic gene activity in iSCNT embryos of different species combinations have revealed the existence of significant aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). However, there are many studies with successful blastocyst (BL) development of iSCNT embryos derived from donor cells and oocytes of animal species with distant taxonomical relations (inter-family/inter-class) that should indicate proper EGA at least in terms of RNA polymerase I activation, nucleoli formation, and activation of genes engaged in morula and BL formation. We investigated the ability of bovine, porcine, and rabbit oocytes to activate embryonic nucleoli formation in the nuclei of somatic cells of different mammalian species. In iSCNT embryos, nucleoli precursor bodies originate from the oocyte, while most proteins engaged in the formation of mature nucleoli should be transcribed from genes de novo in the donor nucleus at the time of EGA. Thus, the success of nucleoli formation depends on species compatibility of many components of this complex process. We demonstrate that the time and cell stage of nucleoli formation are under the control of recipient ooplasm. Oocytes of the studied species possess different abilities to support nucleoli formation. Formation of nucleoli, which is a complex but small part of the whole process of EGA, is essential but not absolutely sufficient for the development of iSCNT embryos to the morula and BL stages.

Two-staged nuclear transfer can enhance the developmental ability of goat–sheep interspecies nuclear transfer embryos in vitro

The technique of interspecies somatic cell nuclear transfer, in which interspecies cloned embryos can be reconstructed by using domestic animal oocytes as nuclear recipients and endangered animal or human somatic cells as nuclear donors, can afford more opportunities in endangered animal rescue and human tissue transplantation, but the application of this technique is limited by extremely low efficiency which may be attributed to donor nucleus not fully reprogrammed by xenogenic cytoplasm. In this study, goat fetal fibroblasts (GFFs) were used as nuclear donors, in vitro-matured sheep oocytes were used as nuclear recipients, and a two-stage nuclear transfer procedure was performed to improve the developmental ability of goat-sheep interspecies clone embryos. In the first stage nuclear transfer (FSNT), GFFs were injected into the ooplasm of enucleated sheep metaphase-II oocytes, then non-activated reconstructed embryos were cultured in vitro, so that the donor nucleus could be exposed to the ooplasm for a period of time. Subsequently, in the second stage nuclear transfer, FSNT-derived non-activated reconstructed embryo was centrifuged, and the donor nucleus was then transferred into another freshly enucleated sheep oocyte. Compared with the one-stage nuclear transfer, two-stage nuclear transfer could significantly enhance the blastocyst rate of goat-sheep interspecies clone embryos, and this result indicated that longtime exposure to xenogenic ooplasm benefits the donor nucleus to be reprogrammed. The two-stage nuclear transfer procedure has two advantages, one is that the donor nucleus can be exposed to the ooplasm for a long time, the other is that the problem of oocyte aging can be solved.

Development of Interspecies Somatic Cell Nuclear Transfer Embryos Reconstructed from Bactrian Camel (Camelus bactrianus) Fibroblasts and Dromedary Camel (Camelus dromedarius) Ooplasm.

Interspecies somatic cell nucleus transfer could not only be a useful bioassay system for assessing the ability of mammalian somatic cells to develop into embryos but can also be used as a possible approach to conserve endangered species. It could provide a useful technique to preserve the wild Bactrian camels, which are threatened with extinction. In the present study embryos were reconstructed by using skin fibroblast cells from a Bactrian camel (Camelus bactrianus) as donor karyoplasts and dromedary camel (Camelus dromedarius) oocytes as recipient cytoplasts for investigating the developmental potential of the these embryos. Mature oocytes were collected from super-stimulated dromedary camels by ultrasound guided transvaginal ovum pick-up, 26 h after GnRH administration. Serum-starved adult Bactrian camel skin fibroblast cells were injected in to the perivitalline space of enucleated dromedary oocytes. The fibroblast cells and recipient cytoplasm were fused by two DC pulses of 100 V/cm for 15 μs each using an Eppendorf electroporator at room temperature. Reconstructs were activated 1 h postfusion with 5μM ionomycine followed by exposure to 6-dimethylaminopurine for 4 h. The activated oocytes were then transferred to 500 μL of embryo culture medium I (modified potassium simplex optimization medium with essential and non-essential amino acids [KSOMaa] supplemented with 1% BSA) and cultured at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 in air. On Day 3 (Day 0 = day of activation) the cleaved embryos were transferred into 500μL of embryo culture medium II (modified KSOMaa supplemented with 10% FCS) and cultured under the same conditions until Day 7. The proportion of oocytes that cleaved was recorded on Day 3, and those that reached morula and blastocyst stages were recorded on Day 7 of culture. The embryos reconstructed from dromedary camel (Camelus dromedarius) fibroblasts and oocytes were treated in similar manner and worked as control. Out of 58 reconstructed embryos, in 3 replicates, 77.4 ± 2.1 couplets fused successfully and were cultured after activation. The embryos that cleaved (59.1 ± 5.7) and those developed to the blastocyst stage (34.4 ± 3.9) were not different from the control group. This study demonstrated, for the first time, that somatic cells from Bactrian camel can be reprogrammed in the dromedary camel ooplasm and such interspecies nuclear transfer embryos can develop to the blastocyst stage. Further studies are needed to see the feasibility of transferring these embryos into dromedary camel recipients and the possibility of producing Bactrian camel calves from dromedary recipient camels. (platform)

Diversity in Public Views Toward Stem Cell Sources and Policies

Studies of public views on stem cell research have traditionally focused on human embryonic stem cells. With more recent scientific research on developing other stem cell sources, a series of focus group studies was undertaken with Canadian adults to examine their views on different stem cell sources (adult, umbilical cord blood, human embryonic stem cells, somatic cell nuclear transfer or SCNT, and interspecies nuclear transfer, or iSCNT). Views on three different policy models--a permissive, middle-of-the-road and restrictive policy approach--were also explored. Participants were recruited from several different social groups including patients, young adults, seniors, members of two ethnic communities, and a mixed group of adults. Participants were generally supportive of the use of adult stem cell sources. While there was also majority support for the use of hESC and SCNT, this was conditional on strict regulatory oversight. There was also majority support for a permissive policy which allows research on hESC and SCNT. General themes that cut across different groups included the potential cost of new technologies to the health care system, issues around who would gain access to these technologies, and trust in the scientific establishment and regulatory systems. A diversity of viewpoints was found as participants justified their positions on stem cell sources and policy approaches, showing more complexity and nuance than has been generally portrayed.

94 NUCLEAR-CYTOPLASMIC INTERACTION AND DEVELOPMENT OF CLONED EMBRYOS

Although cloned canine offspring have been produced, the efficiency of nuclear transfer (NT) is still far from expectations. The most important factor interfering with the success of NT is the shortage of canine matured oocytes. In the present study, the nuclear-cytoplasmic interaction and in vitro development of porcine oocytes cloned with canine fetal fibroblasts (cFF) and canine mesenchymal stem cells (cMSC) were assessed by examining the developmental rate, embryonic morphology, total cell number, canine genome expression, and in vivo development following the transfer of iNT embryos into the oviducts of surrogate mother. Isolated cMSC from bone marrow were characterized by early transcription factors (Oct3/4, Sox2, and Nanog) and by differentiation into adipogenic, osteogenic, and neuronal cells under controlled in vitro conditions. Porcine cumulus–oocyte complexes (COC) were matured in vitro as described by Kim YS et al. (2005 Mol. Reprod. Dev. 70, 308–313). Porcine NT embryos were used for the control group. Canine MSC showed their characteristic property of attaching to plastic culture flasks and forming a monolayer of spindle-shaped cells. Adherent cells showed moderate expression of Oct3/4, Sox2, and Nanog protein by immunostaining. Following osteogenic cell induction, cMSC transformed into mineralized nodules by von Kossa staining. Oil red O staining revealed that MSC produced lipid droplets after incubation in adipogenic media. Neuronal-like cells with multipolar, round cell bodies organized into a network-like structure were confirmed by immunostaining of neuronal markers (NFM). The tissue-specific mRNA levels were confirmed after differentiation by RT-PCR (osteogenic cell, Osteocalcin; adipogenic cell, PPAR γ; neuronal cell, NFM). The control group blastocysts (porcine oocytes-pMSC) exhibited significantly (P &lt; 0.05) greater development and total cell numbers of than blastocysts of interspecies groups (porcine oocytes-cFF and cMSC). The canine genome GAPDH was detected in cFF, cMSC, and interspecies NT embryos using PCR, whereas there was no detection in porcine oocytes and the negative control. Most of the embryos arrested at the 8- to 16-cell stage and only 5% of embryos developed to blastocyst stage. The interspecies-NT embryos (average 35 embryos/recipient dog) were surgically transferred into the oviducts of 3 recipient dogs. Two of them showed exterior signs of implantation; i.e. a slight thickening of the uterus and small pea-sized lumps. No pregnancies were detected in the recipients after ovariohysterectomy surgery. These results demonstrate the potential of cMSC isolated from bone marrow to differentiate into mesoderm (osteogenic and adipogenic cell) and ectoderm (neuronal cell)-like cells under specific induction conditions. Although interspecies NT embryos could not implant in the canine uterine wall, the results provided valuable information on canine MSC and interspecies NT. This work was supported by Grant No. 20070301034040 from Bio-Organ, Republic of Korea.