Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.

Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs1, intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref. 2) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51-52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca2+ handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.

Ultra-Structural Alterations in In Vitro Produced Four-Cell Bovine Embryos Following Controlled Slow Freezing or Vitrification.

Cryopreservation is the process of freezing and preserving cells and tissues at low temperatures. Controlled slow freezing and vitrification have successfully been used for cryopreservation of mammalian embryos. We investigated the effect of these two cryopreservation methods on in vitro produced four-cell stage bovine embryos which were classified according to their quality and separated into three groups. The first group was maintained as untreated controls (n = 350). Embryos of the second (n = 385) and the third (n = 385) groups were cryopreserved either by controlled slow freezing or by vitrification. Embryos in groups 2 and 3 were thawed after 1 day. Hundred embryos were randomly selected from the control group, and 100 morphologically intact embryos from the second and third group were thawed after 1 day and cultured to observe the development up to the blastocyst stage. The blastocyst development rate was 22% in the control group, 1% in the slow-freezing group and 3% in the vitrification group. Remaining embryos of all three groups were examined by light microscopy, transmission electron microscopy and immunofluorescence confocal microscopy with subsequent histological staining procedures. Cryopreservation caused degenerative changes at the ultra-structural level. Compared with vitrification, slow freezing caused an increased mitochondrial degeneration, cytoplasmic vacuolization, disruption of the nuclear and plasma membrane integrity, organelle disintegration, cytoskeletal damage, a reduced thickness of the zona pellucida and a formation of fractures in the zona pellucida. Further studies are required to understand and decrease the harmful effects of cryopreservation.

Cell-mediated transgenesis in rabbits: chimeric and nuclear transfer animals.

The ability to perform precise genetic engineering such as gene targeting in rabbits would benefit biomedical research by enabling, for example, the generation of genetically defined rabbit models of human diseases. This has so far not been possible because of the lack of functional rabbit embryonic stem cells and the high fetal and perinatal mortality associated with rabbit somatic cell nuclear transfer. We examined cultured pluripotent and multipotent cells for their ability to support the production of viable animals. Rabbit putative embryonic stem (ES) cells were derived and shown capable of in vitro and in vivo pluripotent differentiation. We report the first live born ES-derived rabbit chimera. Rabbit mesenchymal stem cells (MSCs) were derived from bone marrow, and multipotent differentiation was demonstrated in vitro. Nuclear transfer was carried out with both cell types, and embryo development was assessed in vitro and in vivo. Rabbit MSCs were markedly more successful than ES cells as nuclear donors. MSCs were transfected with fluorescent reporter gene constructs and assessed for nuclear transfer competence. Transfected MSCs supported development with similar efficiency as normal MSCs and resulted in the first live cloned rabbits from genetically manipulated MSCs. Reactivation of fluorescence reporter gene expression in reconstructed embryos was investigated as a means of identifying viable embryos in vitro but was not a reliable predictor. We also examined serial nuclear transfer as a means of rescuing dead animals.

Nucleolar proteins and ultrastructure in bovine in vivo developed, in vitro produced, and parthenogenetic cleavage-stage embryos.

In the present study, ribosomal RNA (rRNA) gene activation, monitored through nucleolus development, was studied by autoradiography following (3)H-uridine incubation, transmission electron microscopy, and immunofluorescence confocal laser scanning microscopy of key nucleolar proteins involved in rRNA transcription (topoisomerase I, upstream binding factor, and RNA polymerase I) and processing (fibrillarin, nucleolin, and nucleophosmin) in in vivo developed, in vitro produced, and parthenogenetic bovine embryos. In general, in vivo developed embryos displayed formation of fibrillo-granular nucleoli during the 4th post-fertilization cell cycle. During the previous stages of development, nucleolus precursor bodies (NPBs) were observed. However, on some occasions the initial steps of nucleolus formation were observed already at the 2- and 4-cell stage in cases where such embryos were collected from superovulated animals together with later embryonic stages presenting nucleolar development and autoradiographic labeling. The in vitro produced embryos displayed very synchronous formation of fibrillo-granular nucleoli and autoradiographic labeling during the 4th cell cycle. In vivo developed and in vitro produced embryos displayed allocation of nucleolar proteins to fibrillar and granular compartments of the developing nucleoli during the 4th cell cycle. The parthenogenetic embryos typically displayed formation of fibrillo- granular nucleoli during the 5th cell cycle and autoradiographic labeling was not observed until the morula stage. Moreover, the 1-, 2-, and 4-cell parthenogenetic embryos practically lacked NPBs. On the other hand, parthenogenetic embryos displayed allocation of nucleoar proteins to nuclear entities during the 4th cell cycle. In conclusion, both in vivo developed and in vitro produced bovine embryos displayed activation of transcription and nucleolar development during the 4th cell cycle. However, in vivo developed embryos flushed together with later developmental stages displayed premature activation of these processes. Parthenogenetic bovine embryos, on the other hand, displayed a delayed activation.

Effect of donor cell age on the efficiency of nuclear transfer in rabbits.

The ability of rabbit fibroblasts of different ages to be reprogrammed following nuclear transfer (NT) to aged recipient oocytes was evaluated. The rate of NT blastocysts reconstructed with presumptive G1 stage morula cells or fetal fibroblasts was significantly higher (41.5% and 51.4%) than was those of cloned embryos reconstructed with fibroblasts from young (4-month-old) or aged (5-year-old) animals (16.7% and 7.1%, respectively, P < 0.025). Serum starvation significantly increased the development of NT embryos to the morula-blastocyst stage (67.6% versus 22.9%, P < 0.025). Transfer of 168 NT embryos derived from nuclei of morula cells and 106 control embryos into 21 recipients resulted in 10 pregnancies, 2 NT and 18 control pups, respectively. In the first experiment, transfer of 142 cleaved NT embryos reconstructed with fetal fibroblasts and 86 control embryos into eight recipient does resulted in five pregnancies and the birth of 20 control pups. In the second experiment, after transfer of 112 NT embryos derived from fetal fibroblasts into six recipients, 10 (8.9%) sites of implantation were revealed in two does (33.3%) on day 14 of gestation. This study provides evidence that nuclei of morula cells and fetal and adult fibroblasts differ in their ability to be reprogrammed by recipient cytoplasm following nuclear transfer.

Effects of high concentrations of hyaluronan in culture medium on development and survival rates of fresh and frozen-thawed bovine embryos produced in vitro.

Hyaluronic acid (HA) is the main glycosaminoglycan present in follicular, oviductal and uterine fluids. The main functions of HA include dynamic processes that are mediated through interaction with extracellular matrix components, regulation of gene expression, cell proliferation and cell differentiation. HA increases the viscosity of solutions and also has several physiological functions, including regulation of water distribution and water-binding capacity. The addition of 6 mg HA ml(-1) to synthetic oviduct fluid (SOF; SOF-HA) culture medium on day 5 (IVF = day 0) significantly (P < 0.001) increased the viscosity of the medium in comparison with SOF culture medium containing BSA (SOF-BSA). On day 8, rate of blastocyst development in SOF-HA culture medium was significantly (P < 0.05) higher than in SOF-BSA culture medium (38.2 versus 29.3%). The number of trophectoderm cells and the total number of cells of expanded blastocysts cultured in the presence of HA were significantly (P < 0.01) higher in comparison with expanded blastocysts cultured in the presence of BSA (88.9 +/- 7.3 versus 67.6 +/- 3.0 and 130.1 +/- 10.9 versus 104.8 +/- 2.5, respectively). After freezing and thawing, the percentage of day 8 embryos that re-expanded and hatched when cultured with SOF-HA was greater than that of embryos cultured with SOF-BSA (11.3 and 10.5% versus 75.5 and 36.8%, respectively). After thawing, the ATP contents of in vivo-derived, SOF-HA and SOF-BSA expanded blastocysts were similar. The embryos cultured with HA showed less ultrastructural deviation and de-differentiation after freezing and thawing than the embryos cultured with BSA. This study demonstrates that HA improves the developmental capacity of bovine embryos under in vitro conditions and is warranted as a supplement for in vitro production of bovine embryos, particularly if they are to be cryopreserved.

Nucleolar protein allocation and ultrastructure in bovine embryos produced by nuclear transfer from granulosa cells.

In the present study immunofluorescence confocal laser scanning microscopy, autoradiography following (3)H-uridine incubation and transmission electron microscopy were used to evaluate the nucleolar protein localization, transcriptional activity, and nucleolar ultrastructure during genomic re-programming in bovine embryos reconstructed by nuclear transfer from granulosa cells into non-activated cytoplasts followed by activation. During the 1st cell cycle (1-cell embryos), no autoradiographic labelling was detected. Ultrastructurally, nucleoli devoid of a granular component were observed. During the 2nd cell cycle (2-cell embryos) autoradiographic labelling was also lacking and the embryos displayed varying degrees of nucleolar inactivation. During both the 3rd (4-cell embryos) and 4th (tentative 8-cell embryos), cell cycles autoradiographic labelling was lacking in some embryos, while others displayed labelling and associated formation of fibrillo-granular nucleoli. During the 5th cell cycle (tentative 16-cell embryos), all embryos displayed autoradiographic labelling and fibrillo-granular nucleoli. In some blastomeres, however, deviant nucleolar ultrastructure was observed. During the first cell cycle labelling of RNA polymerase I, fibrillarin, upstream binding factor (UBF) and nucleolin (C23) was localized to nuclear entities. During the 2nd cell cycle, only labelling of RNA polymerase I and fibrillarin persisted. During the 3rd and 4th cell cycle labelling of fibrillarin persisted, labelling of nucleophosmin (B23) appeared and that of nucleolin re-appeared. During the 5th cell cycle almost all embryos showed complete labelling of all proteins except for UBF, which lacked in more than half of the embryos. In conclusion, bovine granulosa cell nuclear transfer embryos showed re-modelling of the nucleoli to an inactive form followed by re-formation of fibrillo-granular nucleoli. The re-formation of fibrillo-granular nucleoli was initiated already during the 3rd cell cycle, which is one cell cycle earlier than in in vivo- and in vitro-derived bovine embryos. Moreover, in more than half of the embryos, UBF could not be immunocytochemically localized to the nucleolar compartment during the 5th cell cycle indicating lack of developmental potentials.

Mammalian oocyte activation: lessons from the sperm and implications for nuclear transfer.

Events after fertilisation have been carefully studied in the last decades. However, there are still several questions to be clarified in relation to the signalling pathway initiated by the sperm, the identification of proteins or factors involved in the activation of the arrested oocyte, and the inactivation of specific molecules involved in the meiotic arrest. The present state of knowledge in mammalian fertilisation allows the development of activation protocols that closely mimic the events initiated by the sperm according to certain major factors (MPF activity and MAPk activity). These protocols are successfully used for the activation of oocytes after NT giving rise to offspring. Few cloned animals have yet been produced. However, the pregnancy and the survival rates after birth are not significantly different when different activation protocols are compared. This fact argues fora major reason forthe low success in the efficiency of NT. Eventually, factors related to the recipient oocyte, the donor cell or the culture conditions are part of these major problems that the reconstructed embryo has to overcome to develop into a normal offspring. Nonetheless, the development of activation protocols that closely imitate the mechanism of activation initiated by the sperm are of special interest to improve the developmental potential of cloned embryos.

Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos.

Mouse embryos undergo genome-wide methylation reprogramming by demethylation in early preimplantation development, followed by remethylation thereafter. Here we show that genome-wide reprogramming is conserved in several mammalian species and ask whether it also occurs in embryos cloned with the use of highly methylated somatic donor nuclei. Normal bovine, rat, and pig zygotes showed a demethylated paternal genome, suggesting active demethylation. In bovine embryos methylation was further reduced during cleavage up to the eight-cell stage, and this reduction in methylation was followed by de novo methylation by the 16-cell stage. In cloned one-cell embryos there was a reduction in methylation consistent with active demethylation, but no further demethylation occurred subsequently. Instead, de novo methylation and nuclear reorganization of methylation patterns resembling those of differentiated cells occurred precociously in many cloned embryos. Cloned, but not normal, morulae had highly methylated nuclei in all blastomeres that resembled those of the fibroblast donor cells. Our study shows that epigenetic reprogramming occurs aberrantly in most cloned embryos; incomplete reprogramming may contribute to the low efficiency of cloning.

Nuclear transfer in cattle with non-transfected and transfected fetal or cloned transgenic fetal and postnatal fibroblasts.

The efficiency of nuclear transfer (NT) using two primary cultures of fetal fibroblasts (FF1 and FF2) was compared vs. the same cultures transfected with an expression vector in which the bovine prochymosin coding sequence is placed under the control of the bovine alpha(S1)-casein promoter (TFF1 and TFF2). In addition, fibroblasts of a cloned transgenic fetus (TRFF1) derived from TFF1 and ear skin fibroblasts of a 1-month-old cloned transgenic calf (TRCF1) derived from TRFF1 were used as nuclear donors. Embryos reconstructed from FF1 (44%) and FF2 (52%) developed to the blastocyst stage at a significantly (P < 0.05) higher rate than those derived from TFF1 (24%) and TFF2 (27%). The proportions of cleaved embryos and blastocysts were significantly (P < 0.05) higher with TRFF1 than with TRCF1 used as nuclear donors (75 vs. 66% and 33 vs. 16%, respectively). Transfer of NT embryos derived from FF2 and TFF2 to recipients resulted in similar pregnancy rates on day 30 (52 and 48%, respectively). However, with TFF2 embryos, the majority of pregnancies (8/11; 73%) was lost in the first and second trimesters of gestation, whereas 4/11 (36%) pregnancies with FF2 embryos were lost during the full period of in vivo development. Of 11 FF2 and 6 TFF2 born calves (25 and 13% of transferred embryos, respectively), 6 and 3 survived including one oversized FF2 calf. After transfer of TRFF1 and TRCF1 NT embryos to recipients, initial pregnancy rate was as a tendency higher in the TRFF1 (49%) than in the TRCF1 group (30%). The majority (14/17) of TRFF1 pregnancies and all TRCF1 pregnancies were lost in the first and second trimester. A high proportion of TRFF1 calves (5/8) showed increased body weights, and only two calves which were also large survived. These findings demonstrate that (i) extended culture associated with transfection and selection procedures may induce changes of donor cells which markedly decrease the efficiency of nuclear transfer and (ii) these changes are not reversed by recloning.

Quantitation of retroviral-mediated transfer using luciferase in living and lysed cells.

We have developed a murine retroviral vector containing an improved luciferase gene for the study of retroviral gene transfer and expression in living or lysed cells. We used a cytosolic form of luciferase gene (luc+) with transcriptional enhancements that yielded greater expression levels. The luc+ gene was subcloned into the retroviral plasmids pDON-AI, in which almost the entire U3 region has been replaced with the heterologous human cytomegalovirus immediate-early promoter A stable ecotropic and amphotropic retrovirus-producing cell line was generated with a titer 1 x 10(6) cfu/mL. NIH/3T3(tk-) cells transduced with ecotropic luciferase retrovirus demonstrated a high level of luminescence on the third day. Lysed NIH/3T3(tk-) cells demonstrated a 10-fold increase in activity as compared to living cultures. The creation of a new retroviral system allowed a substantial decrease to 5 days from the 10-14 days previously needed to evaluate viral transfer using the standard neomycin method. Our assay also provides a quantitative assessment in contrast to the beta-galactosidase detection method, which also takes 5-6 days but lacks quantitative evaluation. Thus, the expression of an integrated luc+ gene in eukaryotic cells provides a powerful tool for the study of retroviral gene transfer and will greatly facilitate functional studies in both living and lysed cells.

Remodeling of donor nuclei, DNA-synthesis, and ploidy of bovine cumulus cell nuclear transfer embryos: effect of activation protocol.

The purpose of this study was to investigate the effects of two activation protocols on nuclear remodeling, DNA synthesis during the first cell cycle, chromosome segregation after first mitosis and development to blastocyst of embryos produced by somatic nuclear transfer. Pronuclear formation was significantly higher when activation lasted 5 hr compared to 3 hr for both ethanol-cycloheximide and ionomycin-bohemine treatment. However, the presence of a single nucleus was significantly higher in embryos activated for 3 hr in bohemine. Initiation of DNA synthesis was delayed in ethanol-cycloheximide group, however, after 12 hr labeling 100% of embryos synthesized DNA in both groups. Embryos activated with ethanol-cycloheximide developed to blastocysts at a significantly higher rate than those activated with ionomycin-bohemine. Analysis of 2-cell embryos with DNA probes for chromosome 6, 7, and 15 by fluorescence in situ hybridization showed that at least 50% of NT embryos were of normal ploidy independent of the activation stimulus. The results presented in this study show differences between the protocols compared on the nuclear events during the first cell cycle and on the development to blastocyst. Mol. Reprod. Dev. 59: 371-379, 2001.

Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: correlation with morphological criteria and developmental capacity after in vitro fertilization and culture.

In this study, we evaluated mitochondrial distribution and ATP content of individual bovine oocytes before and after in vitro maturation (IVM). Cumulus-oocyte complexes were classified according to morphological criteria: category 1, homogeneous oocyte cytoplasm, compact multilayered cumulus oophorus; category 2, cytoplasm with small inhomogeneous areas, more than five layers of compact cumulus; category 3, heterogeneous/vacuolated cytoplasm, three to five layers of cumulus including small areas of denuded zona pellucida; category 4, heterogeneous cytoplasm, completely or in great part denuded. In immature oocytes, staining with MitoTracker green revealed mitochondrial clumps in the periphery of the cytoplasm, with a strong homogenous signal in category 1 oocytes, a weaker staining in category 2 oocytes, allocation of mitochondria around vacuoles in category 3 oocytes, and poor staining of mitochondria in category 4 oocytes. After IVM, mitochondrial clumps were allocated more toward the center, became larger, and stained more intensive in category 1 and 2 oocytes. This was also true for category 3 oocytes; however, mitochondria maintained their perivacuolar distribution. No mitochondrial reorganization was seen for category 4 oocytes. Before IVM, the average ATP content of category 1 oocytes (1.8 pmol) tended to be higher than that of category 2 oocytes (1.6 pmol) and was significantly (P < 0.01) higher than in category 3 (1.4 pmol) and 4 oocytes (0.9 pmol). The IVM resulted in a significant (P < 0.01) increase in the average ATP content of all oocyte categories, with no difference between oocytes extruding versus nonextruding a polar body. After in vitro fertilization (IVF) and culture, significantly (P < 0.05) more category 1 and 2 than category 3 and 4 oocytes developed to the morula or blastocyst stage (determined 168 h after IVF). Total cell numbers of expanded blastocysts derived from category 1 and 2 oocytes were significantly (P < 0.05) higher than of those originating from category 3 and 4 oocytes. These data indicate that mitochondrial reorganization and ATP levels are different between morphologically good and poor oocytes and may be responsible for their different developmental capacity after IVF.

Nucleolar protein allocation and ultrastructure in bovine embryos produced by nuclear transfer from embryonic cells.

In the present study, immunofluorescence confocal laser scanning microscopy, autoradiography following (3)H-uridine incubation, and transmission electron microscopy were used to evaluate the nucleolar protein localization, transcriptional activity, and nucleolar ultrastructure during genomic reprogramming in bovine embryos reconstructed by nuclear transfer from in vitro-produced bovine morulae to activated cytoplasts. During the first cell cycle (one-cell embryos), no autoradiographic labelling was detected. Ultrastructurally, whorls consisting of densely packed fibrillar material were observed instead of nucleoli. During the second, third, and fourth cell cycle (two-, four-, and tentative eight-cell embryos), autoradiographically unlabelled nuclei contained vacuolated bodies consisting of densely packed fibrillar material. Also, during the fourth cell cycle, the first nucleoplasmic autoradiographic labelling was observed, but still without formation of fibrillo-granular nucleoli. During the fifth cell cycle (tentative 16-cell embryos), the nuclei displayed autoradiographic labelling over both nucleoplasm and presumptive nucleoli, and the formation of fibrillo-granular nucleoli was observed. In a certain proportion of blastomeres, however, abnormal patterns of nucleolar formation and apoptosis were noted. During the first two cell cycles, labelling of RNA polymerase I, fibrillarin, upstream binding factor (UBF), nucleolin (C23), and nucleophosmin (B23) was localized to nuclear entities. During the third cell cycle, labelling of topoisomerase I was observed in addition. During the fourth and fifth cell cycles, a substantial portion of the embryos presented blastomeres that lacked labelling of several of these nucleolar proteins. In conclusion, the nuclear transfer procedure was associated with remodelling of the nucleoli to an inactive form, followed by reformation of fibrillo-granular nucleoli during the fifth cell cycle. Moreover, a certain proportion of blastomeres failed to form functional nucleoli with respect to both ultrastructural organization and protein allocation.

Intracytoplasmic sperm injection in bovine: effects of oocyte activation, sperm pretreatment and injection technique.

Intracytoplasmic sperm injection (ICSI) is a very important technique for treating male subfertility and for basic research. The efficiency of ICSI in bovine is very limited because of the necessity for additional oocyte activation before or after the ICSI procedure. In this study, we compared the effects of seven different protocols on activation and fertilization rates of bovine oocytes after ICSI and on their subsequent development under in vitro conditions. The protocols include 1) different chemical activation of oocytes, 2) pretreated or nonpretreated sperm, and 3) conventional or Piezo-driven injection techniques. In all three groups, ICSI, sham-injected, and noninjected, the highest activation rates were obtained after treatment of oocytes with ionomycin followed by 6-dimethylaminopurine (6-DMAP). Using this treatment for oocyte activation, 59% of oocytes were activated and 31% of oocytes were fertilized using dithiothreitol (DTT) pretreated spermatozoa and Piezo-driven injection. Using the protocols with the same oocyte activation or activation with calcium ionophore (Ca-I) and cycloheximide (CHX), nonpretreated sperm, and conventional injection technique, early cleavage rate (79.6% and 77.6%, respectively) were significantly (P <0.01) higher when compared with all other protocols. The latter protocol resulted in 8% blastocyst and 90% of the obtained blastocysts were found to be diploid. Our results demonstrate that activation of oocytes, sperm treatment, and injection technique separately or together could improve the success of bovine ICSI.

Behavior of M-phase synchronized blastomeres after nuclear transfer in cattle.

M-phase synchronized bovine blastomeres were used to study the effect of nuclear-cytoplasmic synchronization on the developmental potential after nuclear transfer (NT). The capacity of nocodazole and benomyl to reversibly synchronize blastomeres from embryos in M-phase was evaluated. Nocodazole reversibly arrested bovine embryos at the studied stages and induced high rates of M-phases in morulae and compact morulae. In contrast, benomyl was less efficient than nocodazole to synchronize in M-phase. After transfer of an M-phase blastomere, premature chromatin condensation was the prevalent finding 1 hr post-fusion (hpf). Condensed chromosomes non-arranged in the equatorial plate (1-3 hpf) that acquired an organized structure over time (3-7 hpf) were subsequently observed. Anaphase-telophase structures were predominantly recorded at 4-9 hpf. About 50% of the embryos activated at both 3-4 and 6-7 hpf extruded a polar body-like structure 5 hr after activation, but this was not observed in embryos activated immediately after fusion. A significantly lower activation rate was observed for oocytes activated 3-4 hpf compared to those activated 6-7 hpf. However, the ability to undergo first cleavage was significantly lower in the latter group. Reconstructed embryos activated immediately after fusion showed no difference in the rate of activation compared to those activated 6-7 hpf, although the cleavage rate was higher. DNA synthesis was observed at a significantly higher rate in embryos activated both immediately and 3-4 hpf that did not extrude a PB-like structure than in those activated 3-4 hpf that extruded a polar body-like structure. Under the conditions tested M-phase donor cells cannot be properly remodeled after NT in cattle to trigger normal embryonic development. Our observations of chromatin structures together with DNA synthesis suggest that the failure in the development may be due to improper chromatin remodeling of mitotic nuclei after NT, which may result in chromosomal abnormalities incompatible with normal embryo development.

Mitochondrial DNA heteroplasmy in cloned cattle produced by fetal and adult cell cloning.

Mammals have been cloned from adult donor cells. Here we report the first cases of mitochondrial DNA (mtDNA) heteroplasmy in adult mammalian clones generated from fetal and adult donor cells. The heteroplasmic clones included a healthy cattle equivalent of the sheep Dolly, for which a lack of heteroplasmy was reported.

Activation of ribosomal RNA genes in preimplantation cattle and swine embryos.

Transcription of ribosomal RNA (rRNA) genes occurs in the nucleolus resulting in ribosome synthesis. In cattle and swine embryos, functional ribosome-synthesizing nucleoli become structurally recognizable towards the end of the fourth and third post-fertilization cell cycle, respectively. In cattle, a range of important nucleolar proteins become localized to the nucleolar anlage over several cell cycles and this localization is apparently completed towards the end of the fourth cell cycle. In swine, the localization of these proteins to the anlage is more synchronous and occurs towards the end of the third cell cycle and is apparently completed at the onset of the fourth. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus, serve to evaluate the developmental potential of embryos originating from different embryo technological procedures. By this approach, we have demonstrated that in vitro produced porcine embryos display a lack of localization of nucleolar proteins to the nucleolar anlage as compared with in vivo developed counterparts. Similarly, bovine embryos produced by nuclear transfer from morulae display such deviations as compared with in vitro produced counterparts. Collectively, this information may help to explain the appearance of abnormalities seen in a certain proportion of offspring derived from in vitro produced embryos and after cloning.

Activation of bovine oocytes by specific inhibition of cyclin-dependent kinases.

Activation of bovine oocytes by experimental procedures that closely mimic normal fertilization and allow to obtain haploid oocytes is essential both for intracytoplasmic sperm injection (ICSI) and for nuclear transfer. Therefore, with the goal of producing haploid activated oocytes, this study evaluated whether bohemine, either alone or in combination with ionomycin, is able to activate young matured bovine oocytes. Furthermore, the effect of bohemine on the patterns of DNA synthesis after pronuclear formation as well as changes in histone H1 kinase and MAP kinase activities during the process of activation were studied. Our results with bohemine show that the specific inhibition of CDKs in metaphase II bovine oocytes induces parthenogenetic activation in a dose-dependent manner (25, 50, and 100 microM, respectively), either alone (3%, 30%, and 50%) or in combination with ionomycin (30%, 70%, and 87.5%). A single pronucleus and extrusion of the second polar body was observed (97%) when Ca(2+) influx was stimulated in the presence of bohemine, although pronuclear formation without polar body extrusion was observed when bohemine was used alone. Bohemine-activated oocytes started to synthesize DNA in the first hour (37%) after their removal from bohemine-supplemented medium (6-7 hr post-activation; hpa). A high synchrony in the S-phase was registered with more than 85% of parthenotes actively synthesizing DNA 8 hpa. By contrast, DNA synthesis was absent in oocytes cultured for 4, 6, and 8 hpa in the presence of bohemine and a low rate was observed by those cultured for 18 hr (30%) in bohemine-supplemented medium. This confirms the ability of the inhibitor to arrest the cell cycle in the G1/S boundary for at least 8 hr. A drop in histone H1 kinase activity was observed in bohemine-activated oocytes. The activity of MBP kinase decreased later than histone H1 kinase and even 4 hr after inomycin-bohemine treatment at least half of this activity was still detectable. Then, the MBP kinase activity decreased and the lowest level could be seen 6-8 hpa. In summary, our study shows that in vitro matured bovine oocytes can be successfully activated by a synthetic inhibitor of CDKs. This effect can be improved by combination with ionomycin. The targeting of CDKs in the way to activate bovine oocytes can be an approach to improve the efficiency of mammalian oocyte activation.

Transgenic technology in farm animals--progress and perspectives.

Current applications of gene transfer in farm animals include the improvement of product quality and quantity, disease resistance, the production of valuable proteins in the mammary gland or other organs, the genetic modification of pigs for xenotransplantation and the generation of new animal models in cases where rodent models are not sufficient for studying the problem under evaluation. Although DNA microinjection into pronuclei of zygotes from various farm animal species has happened since 1985, the efficiency of this method is low. Further drawbacks are related to the random integration process which may cause mosaicism, insertional mutations and varying expression due to position effects. Sperm-mediated gene transfer is not routinely established yet, although the mechanisms of binding and internalisation of DNA by sperm cells is becoming increasingly clearer. New protocols for the use of retroviral vectors to infect metaphase II oocytes which are subsequently fertilised resulted in efficient production of transgenic cattle. In spite of extensive efforts to establish pluripotent stem cells from farm animal species, no germ-line competent cells have been reported in mammalian species other than mouse so far. However, recent success in cloning sheep, cattle, goats and pigs from cultured cells provides an alternative route for efficient and targeted genetic modifications of farm animals.