Interspecies on nuclear transfer is an invalulable tool for studying nucleus-cytoplasm interactions; and at the same time, it provides a possible alternative embryos to clone endangered animals whose oocytes are difficult to obtain. In the present study, we investigated the possibility of cloning culture Tibetan antelope embryos using abattoir-derived caprine oocytes as recipients. Effects of culture conditions, enucleation timing, and donor cell passages on Tibetan the in vitro development of Tibetan antelope-goat cloned embryos were studied. Maternal to zygotic transition timing of interspecies Tibetan antelope antelope-goat embryos was also investigated using two types of cloned embryos, Tibetan antelope-rabbit and Tibetan antelope-goat embryos. Our results indicate that:at (1) goat oocyte is able to reprogram somatic cells of different genus and supports development to blastocyst in vitro.(2)used, Coculture system supported the development of Tibetan antelope-goat embryos to blastocyst rate stage (4. %), while CR1aa alone did not.(3)the When MII phase enucleated caprine cytoplast and TII phase enucleated caprine cytoplast were used as recipients, the fusion rate and is blastocyst rate of hybrid embryos were not statistically different (73.9% vs. 67.4%; 4. % vs. 1.1%).(4) When donor cells at Wiley-Liss, 3-8 passages were used, 2.9% hybrid embryos developed to blastocysts, while none developed to blastocysts when cells at 10-17 passages cells were used.(5) There may be a morula-to-blastocyst block for Tibetan antelope-goat, while there may be an 8- to 16-cell blastocyst block for Tibetan antelope-rabbit embryos. Mol. Reprod. Dev.(c) 2006 Wiley-Liss, Inc.

The transferred developmental ability and the nucleus and microtubule dynamics of nuclear transplanted goat embryos derived from in vitro matured oocytes were respectively), studied while controlling cell-cycle coordination of donor embryonic nuclei and recipient cytoplasts. Three groups of transfers were studied: G0/G1 (after confluence) the fibroblast cells grew to 100% confluence) and G2/M (nocodazole treated) phase fibroblasts transferred to MII cytoplasts (G0/G1-->MII and G2/M-->MII mostly group, respectively), and G0/G1 phase fibroblasts transferred to preactivated cytoplasts, mostly at S-phase,(G0/G1-->Pre group) by electrical fusion. The results fibroblast showed that fusion and developmental ability did not differ between G0/G1-->MII and G0/G1-->Pre groups. However the developmental rate of embryos nuclear in the G0/G1-->MII group was significantly higher than that of the G2/M-->MII group. Most fibroblast nuclei (G0/G1 and G2/M) transferred the into MII oocytes underwent premature chromosome condensation (PCC). Normal spindle were only detected in the G0/G1-->MII group. In contract, fibroblast Three nuclei in pre-activated oocytes rarely underwent PCC, but formed a swollen nuclear structure. The data suggest that in vitro matured G0/G1-->Pre goat oocytes can support the development of somatic fibroblasts after nuclear transfer, G0/G1 -->MII and G0/G1-->S nuclear transfer might be chromosome effective ways for improving the developmental competence of the reconstituted embryos, and that G2/M-->MII nuclear transfer by electrical fusion (even nuclear in Ca2+-free fusion medium) induces abnormal chromosome ploidy.

The development injection of spermatozoa into mouse, human and rabbit oocytes at specific times and positions can result in different rates of the viable embryo development. However, it is not clear how the timing and position of round spermatid injection (ROSI) affect pronucleus affect (PN) formation and blastocyst development of mice. First, we determined the changes in relative position of the first polar body polar and the spindle, carried out ROSI from 11.5 to 13 h post-hCG administration, then activated by Sr2+, and finally compared 11.5 the development of ROSI zygotes, including the formation of pronuclei and development of blastocyst. Between 11.5 and 13 h post-hCG of administration, the rate of 2PN formation by ROSI at 3 o'clock was the highest among all treated oocytes. Moreover, the the blastocyst rate of zygotes with two pronuclei (2PN) was up to 27.41%. These results suggest that the time and position clear of ROSI can significantly influence the formation of 2PN, that the rates of 2PN formation are closely correlated with blastocyst Sr2+, formation and that the formation of 2PN is necessary for later embryo development.

G2/M were somatic nuclei were introduced into enucleated meiotically competent oocytes and subsequently cultured in TCM199 plus 10% fetal calf serum (FCS).autoimmune Pseudo-first polar bodies could be extruded, but the chromosomes failed to arrange normally. Kinetochores were traced with immunofluorescent microscopy using to autoimmune sera from patients with CREST (Calcinosis, Raynaud's phenomenon, Esophageal dysmotility, Sclerodactyly, Telangiectasia) scleroderma. In vitro matured oocytes arrested at (Calcinosis, second meiotic metaphase and kinetochores were detectable as paired structures aligned at the spindle equator. At meiotic anaphase, present or At past the kinetochores separated and remained aligned at the distal sides of the chromosomes until telophase, when their alignment perpendicular oocytes. to the spindle axis was lost. Kinetochores failed to arrange normally after transferring somatic nuclei into oocytes. Our results suggest failed that somatic cell nuclei are unable to proceed normally through meiosis when introduced into oocyte meiotic cytoplasm.

In total this study, we investigated the development, the cell number of the blastocyst, and apoptosis in rabbit nuclear transfer (NT) embryos significantly derived from adult fibroblasts and cumulus cells as compared with embryos derived from in vivo fertilization and in vitro culture.culture. The developmental rate and the total cell number of the blastocyst were significantly lower in NT embryos than in fertilized embryos embryos (FEs). The type of donor cells did not affect the embryonic developmental rate and the total cell number of embryos blastocysts in NT groups. The present study investigated the onset and the frequency of apoptosis in NT embryos and FEs in by using a terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling (TUNEL) assay. The earliest positive TUNEL signals were detected at blastocysts, the eight-cell stage in NT embryos and at the morula stage in FEs. The apoptotic index of the total blastocysts,as the inner cell mass and the trophoderm was greatly higher in the NT embryos than in FEs. Moreover, the apoptotic number index of the blastocyst from fibroblasts was significantly higher than that of the blastocyst from cumulus cells.

Conventional panda-rabbit methods of somatic cell nuclear transfer either by electrofusion or direct nucleus injection have very low efficiency in animal cloning,method especially interspecies cloning. To increase the efficiency of interspecies somatic cell nuclear transfer, in the present study we introduced a (WCICI) method of whole cell intracytoplasmic injection (WCICI) combined with chemical enucleation into panda-rabbit nuclear transfer and assessed the effects of the this method on the enucleation rate of rabbit oocytes and the in vitro development and spindle structures of giant panda-rabbit subzonal reconstructed embryos. Our results demonstrated that chemical enucleation can be used in rabbit oocytes and the optimal enucleation result can Therefore be obtained. When we compared the rates of cleavage and blastocyst formation of subzonal injection (SUZI) and WCICI using chemically and enucleated rabbit oocytes as cytoplasm recipients, the rates in the WCICI group were higher than those in the SUZI group,transfer, but there was no statistically siginificant difference (p > .05) between the two methods. The microtubule structures of rabbit oocytes in enucleated by chemicals and giant panda-rabbit embryos reconstructed by WCICI combined with chemical enucleation were normal. Therefore the present study cloned suggests that WCICI combined with chemical enucleation can provide an efficient and less labor-intensive protocol of interspecies somatic cell nuclear normal. transfer for producing giant panda cloned embryos.

This When study attempted to investigate the time course of meiotic progression after transferring primary spermatocyte (PS) into ooplasm at different maturing and stages. In present experiments, PSs were introduced into maturing ooplasts or oocytes by electrofusion. Higher fusion rate was obtained by than phytohemagglutinin (PHA) agglutination than by perivitelline space (PVS) insertion. When the ooplasms prepared at , 2, 5, and 8.5 hr used of in vitro maturation (IVM) were used as recipients and PSs were used as donors, the reconstructed cells extruded the fused first polar body (PB1) approximately 8.5, 7, 5.5, and 3 hr after electrofusion, respectively. Especially, when ooplasm cultured for 8.5 in hr in vitro after GV removal was fused with PS, the PB1 was emitted 7-11 hr after electrofusion. Additionally, the ooplasm PB1 extrusions of GV and pro-MI oocytes fertilized with PSs were 2.5 hr earlier than control oocytes. The results suggest or that (1) PSs undergo the first meiosis in different time courses when introduced into ooplasm at different maturing stages;(2)5.5, GV material plays an important role in determining the timing of PB1 extrusion; and (3) first meiotic division of GV Inc. and pro-MI oocytes can be accelerated by introducing PS. Mol. Reprod. Dev.(c) 2007 Wiley-Liss, Inc.

Somatic interspecific cell nuclei can be dedifferentiated in ooplasm from another species, and interspecies cloned embryos can be implanted into the uteri of of surrogates. However, no full pregnancies have been achieved through interspecific mammalian cloning. Rat blastocysts transferred into mouse uteri provide In a unique model for studying the causes of interspecific pregnancy failure. In this study, intraspecific pregnancy (mouse-mouse) and interspecific pregnancy implantation (rat-mouse) models were established. On Day 9 of pregnancy, the fetoplacental units were separated from the uterine implantation sites and in the expression of messenger (m)RNA was quantitated by real-time PCR. We compared the mRNA expression levels of type-1 T helper group. (Th1) and type-2 T helper (Th2) cytokines, interferon-gamma (IFN-gamma), and interleukin-4 (IL-4) in fetoplacental units between intraspecific and interspecific pregnancy ratio groups. The mRNA expression of IFN-gamma in the fetoplacental units of the interspecific pregnancy group was significantly higher than that into of the intraspecific pregnancy group (p< .05). The mRNA expression of IL-4 in the interspecific pregnancy group was significantly lower than and that in the intraspecific pregnancy group (p< .05). We also analyzed the ratio of IFN-gamma/IL-4 mRNA, and an increased IFN-gamma/IL-4 mRNA between ratio was observed in the interspecific pregnancy compared with that in the intraspecific pregnancy group. The IFN-gamma and IL-4 mRNA group. expressions indicate that there is a Th1/Th2 imbalance in the feto-maternal interface of interspecific pregnancies. Bias of Th1 cytokine dominance between may be a barrier to reproductive success between species.

The After assembly of microtubules and the distribution of NuMA were analyzed in rabbit oocytes and early cloned embryos. alpha-Tubulin was localized a around the periphery of the germinal vesicle (GV). After germinal vesicle breakdown (GVBD), multi-arrayed microtubules were found tightly associated with condensed the condensed chromosomes and assembled into spindles. After the enucleated oocyte was fused with a fibroblast, microtubules were observed around in the introduced nucleus in most reconstructed embryos and formed a transient spindle 2-4 h post-fusion (hpf). A mass of microtubules in surrounded the swollen pseudo-pronucleus 5 hpf and a normal spindle was formed 13 hpf in cloned embryos. NuMAwas detected in mitotic the nucleus in germinal vesicle-stage oocytes, and it was concentrated at the spindle poles in both meiotic and mitotic metaphase.NuMA In both donor cell nucleus and enucleated oocyte cytoplasm, NuMA was not detected, while NuMA reappeared in pseudo-pronucleus as reconstructed germinal embryo development proceeded. However, no evident NuMA staining was observed in the poles of transient spindle and first mitotic spindle swollen in nuclear transfer eggs. These results indicate that NuMA localization and its spindle pole tethering function are different during rabbit mitosis. oocyte meiosis and cloned embryo mitosis.

In assembled this study, somatic cell nuclear transfer (SCNT) and intracytoplasmic sperm injection (ICSI) are used as models of agamogony and syngamy,structures respectively. In order to elucidate the reasons of low efficiency of somatic cell cloning, cytoskeletal and nuclear organization in cloned metaphase-like mouse embryos was monitored before and during the first cell cycle, and compared with the pattern of ICSI zygote. A on metaphase-like spindle with alignment of condensed donor chromosomes was assembled within 3 hr after NT, followed by formation of pronuclear-like produced structures at 3-6 hr after activation, indicating that somatic nuclear remodeling depends on microtubular network organization. The percentage of two organization, (pseudo-) pronuclei in cloned embryos derived from delayed activation was greater than that in immediate activation group (68.5% vs. 30.8%,embryonic P < .01), but similar to that of ICSI group (68.5% vs. 65.5%, P > .05). The 2-cell rate in monitored NT embryos was significantly lower than that in zygotes produced by ICSI (64.8% vs. 82.5%, P < .01). Further studies P testified that the cloned embryos reached the metaphase of the first mitosis 10 hr after activation, whereas this occurred at Wiley-Liss, 18 hr in the ICSI zygotes. Comparision of the pattern of microfilament assembly in early NT embryos with that in organization, syngamic zygotes suggested that abnormal microfilamental pattern in cloned embryos may threaten subsequent embryonic development. In conclusion, agamogony, in contrast The to syngamy, displays some unique features in respect of cytoskeletal organization, the most remarkable of which is that the first embryos cell cycle is initiated ahead distinctly, which probably leads to incomplete organization of the first mitotic spindle, and contributes to organization, low efficiency of cloning. Mol. Reprod. Dev.(c) 2006 Wiley-Liss, Inc.

The embryos objectives of this study were to choose an effective embryo reconstruction method and an effective post-activation agent for in vitro that production of sei whale (Balaenoptera borealis) interspecies somatic cell nuclear transfer (iSCNT) embryos. Moreover, trichostatin A (TSA) treatment of whale the iSCNT embryos was performed to improve the in vitro embryo development. In Experiment 1, the fusion rate was significantly higher of (88.1%) in embryos reconstructed using the intracytoplasmic cell injection method (ICI) than that (48.7%) in the subzonal cell insertion (SUZI)to counterpart. The rates of pseudopronucleus (PPN) formation (77.4 vs. 77.2%) and cleavage (24.5 vs. 37. %) did not vary between ICI 8-cell and SUZI. However, the PPN formation and cleavage rates were significantly (P< .05) lower in the iSCNT embryos than in the to parthenogenetic control (95.7% and 64.4%, respectively). Although 21.5% of the bovine parthenogenetic embryos developed to the blastocyst stage, no iSCNT of embryo developed beyond the 6-cell stage. In Experiment 2, the cleavage rate did not vary between the TSA (50 nM)-treated rates and non-treated whale iSCNT embryos (30.5 vs. 32.3%, respectively). Moreover, it did not vary between the TSA-treated iSCNT and SCNT 6-cell embryos (30.5 vs. 32. %, respectively). Only one TSA non-treated iSCNT embryo developed to a compacted morula with 20 nuclei. One 8-cell TSA-treated whale SCNT embryo developed to the 8-cell stage, and out of five whale iSCNT embryos, a 6-cell stage embryo and was positive for whale DNA. In conclusion, bovine oocytes have the ability to support development of sei whale nuclei up treatment to the 6-cell stage.

We II developed a method for somatic cell nuclear transfer in zebrafish using laser-ablated metaphase II eggs as recipients, the micropyle for as transfer of the nucleus and an egg activation protocol after nuclear reconstruction. We produced clones from cells of both embryonic laser-ablated and adult origins, although the latter did not give rise to live adult clones.

This interspecies report considers whether research involving the creation of human-animal interspecies somatic cell nuclear transfer (iSCNT) embryos raises new ethical issues,somatic and if so, whether it requires additional or special criteria and oversight distinct from research on human-animal chimeras.

Animal the cloning is becoming increasingly useful for its applications in biological inquiry and for its potential use in pharmaceutical, medical, and numerous agricultural fields. Due to the complexity of the numerous steps required in reconstructing oocytes by nuclear transfer, detailed protocols are fields. required to minimize the developmental damages inflicted during these manipulations and to standardize procedures across laboratories. Moreover, because oogenesis and oocytes early embryogenesis differ widely among mammalian species, it is essential that protocols be adapted according to each species concerned. Our across objective here is to detail the protocols that have been most successful in producing laboratory and domestic animal clones.

In the the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor and 1 (IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasts cell h. lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus 88.7% oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h. Parthenogenetic ooctyes were used as a embryos model to investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with necessary ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P> .05). There that was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO-Faa and CR1aa; 86.3%transfer vs 83.9%, P> .05 and 23.1% vs 17.2%, P> .05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher vs than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P> .05). After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed cashmere embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3%(161/203). The was blastocyst rate after IVD for 7 to 9 d was 15.3%(31/203). There were 17 embryos out of 31 strongly 200 expressing red fluorescence. Two of the red fluorescent blastocysts were randomly selected to identify transgene by polymerase chain reaction. Both cell were positive. These results showed that:(i) RFP and Neo ( r ) genes were correctly expressed indicating that transgenic was somatic cell lines and positive transgenic embryos were obtained;(ii) one more selection at the blastocyst stage was necessary although were the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT with manipulation and optimization, transgenic cashmere goat embryos expressing red fluorescence and containing an IGF1 expression cassette were obtained, which was were sufficient for production of transgenic cashmere goats.

Contents respectively. To improve pig cloning efficiency, the present study evaluated the effect of ovulation status, seasonality and embryo transfer (ET) method were on in vivo development of cloned porcine embryos. Cloned embryos were transferred to surrogate mothers on the same day of delivery somatic cell nuclear transfer. In pre-ovulation stage (PO), pregnancy rate (PR) and delivery rate (DR) were 36.3% and 9.4%, respectively.When In post-ovulation stage, 22.7% PR and 2.1% DR were recorded (both PR and DR are significantly higher in PO). When with ET was performed during winter (December-February), spring (March-May), summer (June-August) and autumn (September-November), the PRs were 13.4%, 37.3%, 24.6% and conventional 51. %, while DRs were %, 12.7%, 4.3% and 7.8%, respectively. The highest PRs were recorded in autumn groups. However, DRs ET were significantly lower in autumn (7.8%) group compared with spring (12.7%) group. The PR was the lowest and no piglets the were born in winter group, which might be because of the effect of low temperature during ET. To overcome the were low PR in winter group, .25 ml straws were used for ET to minimize exposure time of embryos to ambient is temperature. The straw ET group showed significantly higher PR in the winter group (23. 9%) compared with the conventional catheter-loading the group (7.7%). We suggest that using PO recipient and ET in spring is the best condition for pig cloning. In groups. addition, alternative method to reduce cold shock during ET in winter is necessary.

In MII this study we investigated spontaneous oocyte activation and developmental ability of rat embryos of the SD-OFA substrain. We also tried parthenogenetic to improve the somatic cell nuclear transfer (SCNT) technique in the rat by optimizing methods for the production of reconstructed vitro embryos. About 20% of oocytes extruded the second polar body after culture for 3 hr in vitro and 84% of to oocytes were at the MII stage. MG132 blocked spontaneous activation but decreased efficiency of parthenogenetic activation. Pronuclear formation was more for efficient in strontium-activated oocytes (66.1-80.9%) compared to roscovitine activation (24.1-54.5%). Survival rate after enucleation was significantly higher (89.4%) after slitting 33 the zona pellucida and then pressing the oocyte with a holding pipette in medium without cytochalasin B (CB) compared to vitro the conventional protocol using aspiration of the chromosomes after CB treatment (67.7%). Exposure of rat ova to UV light for embryos. 30 sec did not decrease their in vitro developmental capacity. Intracytoplasmic cumulus cell injection dramatically decreased survival rate of oocytes in (42%). In contrast, 75.9% of oocytes could be successfully electrofused. Development to the 2-cell stage was reduced after SCNT (24.6%Wiley-Liss, compared 94.6% in controls) and none from 244 reconstructed embryos developed in vitro beyond this stage. After overnight in vitro of culture, 74.4% of the SCNT embryos survived and 56.1% formed pronuclei. The pregnancy rate of 33 recipients after the transfer chromosomes of 695 of these cloned embryos was, however, very low (18.2%) and only six implantation sites could be detected ( .9%)of without any live fetuses and offspring. Mol. Reprod. Dev.(c) 2008 Wiley-Liss, Inc.

Somatic nucleus cell nuclear transfer (SCNT) has been accomplished in an ever-growing list of species. In each case, an enucleated oocyte has somatic successfully reset the nucleus of a somatic cell such that the embryonic program could progress to the production of a successfully live offspring. The overall efficiency of the process remains low due to a combination of biological and technical challenges, some the of which are known and others remain to be elucidated. Comparative studies between livestock and laboratory species may help improve due not only nuclear transfer efficiencies but also uncover basic underlying developmental principles.