Manual handling of biological cells is routine in most laboratories. This is gradually changing with the development of robotic cell handling systems, and micro-scale lab-on-chip devices. Attempts were made to develop devices that automate or assist cell handling in the context of a bovine nuclear transfer (NT) system. The system, a zona-free bovine NT cloning system, formed a baseline reference for tool design and performance evaluation. Bovine NT can, as other cell handling procedures, be improved by rapid and precise cell positioning. Improvements in cell handling can increase the quantity of cells processed, and the uniformity of conditions the cells are subject to during processing. Tools were developed for two areas of cell handling: cell fusion and cell transportation. Designs suitable for implementation in microscale lab-on-chip systems were evaluated. Tool development was predominantly experimental, assisted by numerical modelling. The experimental investigation concerned device fabrication and operational performance. A number of cell handling tool designs were built and tested. Coplanar electrodes are not commonly used in bovine NT and reports on their efficacy were not available. These electrodes, which are simple to fabricate, were tested to determine fusion rates achievable in comparison with those of the baseline procedure. A novel fusion device, the micropit, was designed to assist bovine cell pairing and electrofusion. It was initially uncertain whether this device was capable of achieving cell fusion. Tests were conducted; and cell fusion and micro-positioning were demonstrated, as was an increase in biological cell processing throughput. Many miniaturised lab-on-chip systems rely on cell transportation. One illustration in the baseline procedure is the on-chip transport of cells to the cell fusion device. Potential cell transport mechanisms for a miniature cloning system were evaluated by prototype construction and testing. These mechanisms included travelling wave dielectrophoresis and capillary fluid actuation. To facilitate automation of on-chip cell transportation, a low cost electrically isolated cell detection system was developed based on a DVD pick-up unit. Various obstacles that were encountered during the course of device construction are noted, as are the fabrication methods employed.

Somatic cells in semen are a potential source of nuclei for cloning animals bysomatic cell nuclear transfer. Culture of the cells from frozen semen, if possible, wouldbe extremely valuable for preservation or restoration of endangered, exotic, and extinctanimals when other ways of obtaining somatic cells are unavailable. In the present study,somatic cells isolated from ovine and bovine semen samples were characterized, culturesystems were evaluated for attachment and proliferation of these cells, and usefulness ofthese cells for somatic cell nuclear transfer was determined.Semen samples were collected from eight rams representing three breeds:Dorper, Suffolk, and Hampshire and nine bulls representing three breeds: Charolais,Brahman, and a crossbred Brahman. Somatic cells were isolated immediately postcollection by centrifuging through percoll columns and the epithelial cells wereidentified by immunofluorescence analysis. Culture systems were evaluated for theirability to support attachment and proliferation of the cells. A supplemented mediumcomposed of DMEM/F12, 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 30 g/ml bovine pituitary extract, 5 g/ml insulin, 10 ng/ml cholera toxin, and 50 g/mlgentamicin significantly improved cell proliferation over sheep fetal fibroblastconditionedmedium, 3T3 cell-conditioned medium, and basic medium (p<0.05). Cellproliferation and attachment were further improved when Matrigel-coated culturesurfaces were used (p<0.05). However, the system was not adequate for obtaining cellgrowth from frozen semen.To check the chromosome anomalies, metaphase chromosomal complements ofthe cells cultured from 4 rams were evaluated. The predominant chromosome number ofcells from three of the rams (Dorper 18-month-old; Suffolk 17-month-old; Suffolk 18-month-old) was 2n = 54, which is the normal modal number for sheep. However, thenumbers of chromosomes of cells cultured from the fourth ram (Hampshire, 18-monthold)were near-triploid. These results indicate the need for chromosome analysis of cellsbefore using them for cloning experiments. In our attempts to clone animals, blastocyststage embryos were successfully produced using epithelial cells cultured from semen ofthree different bulls. However, no compact morulae or blastocysts were obtained whensomatic cells isolated from frozen semen but not cultured were used as donor cells.

Somatic cells in semen are a potential source of nuclei for cloning animals bysomatic cell nuclear transfer. Culture of the cells from frozen semen, if possible, wouldbe extremely valuable for preservation or restoration of endangered, exotic, and extinctanimals when other ways of obtaining somatic cells are unavailable. In the present study,somatic cells isolated from ovine and bovine semen samples were characterized, culturesystems were evaluated for attachment and proliferation of these cells, and usefulness ofthese cells for somatic cell nuclear transfer was determined.Semen samples were collected from eight rams representing three breeds:Dorper, Suffolk, and Hampshire and nine bulls representing three breeds: Charolais,Brahman, and a crossbred Brahman. Somatic cells were isolated immediately postcollection by centrifuging through percoll columns and the epithelial cells wereidentified by immunofluorescence analysis. Culture systems were evaluated for theirability to support attachment and proliferation of the cells. A supplemented mediumcomposed of DMEM/F12, 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 30 g/ml bovine pituitary extract, 5 g/ml insulin, 10 ng/ml cholera toxin, and 50 g/mlgentamicin significantly improved cell proliferation over sheep fetal fibroblastconditionedmedium, 3T3 cell-conditioned medium, and basic medium (p<0.05). Cellproliferation and attachment were further improved when Matrigel-coated culturesurfaces were used (p<0.05). However, the system was not adequate for obtaining cellgrowth from frozen semen.To check the chromosome anomalies, metaphase chromosomal complements ofthe cells cultured from 4 rams were evaluated. The predominant chromosome number ofcells from three of the rams (Dorper 18-month-old; Suffolk 17-month-old; Suffolk 18-month-old) was 2n = 54, which is the normal modal number for sheep. However, thenumbers of chromosomes of cells cultured from the fourth ram (Hampshire, 18-monthold)were near-triploid. These results indicate the need for chromosome analysis of cellsbefore using them for cloning experiments. In our attempts to clone animals, blastocyststage embryos were successfully produced using epithelial cells cultured from semen ofthree different bulls. However, no compact morulae or blastocysts were obtained whensomatic cells isolated from frozen semen but not cultured were used as donor cells.

The advent of somatic cell nuclear transfer in cattle has provided the opportunity for researchers to generate genetically identical animals as well as animals that possess precise genetic modifications for agriculture and biomedical purposes. However, in spite of the revolutionary impact this technology presents, problems remain which hinder the production of healthy animals on a consistent basis. Research on cloned mice implicates improper reprogramming of epigenetic modifications and genomic imprinting for the low pregnancy rates and high incidence of abnormalities that are manifested in cloned animals; however, a systematic and comprehensive analysis of nuclear reprogramming in cloned cattle remains undone. The purpose of this research is to assess and characterize the patterns of genomic imprinting in normal and nuclear transfer derived bovine fetuses. To facilitate the identification of imprinted genes in the bovine, a Bos gaurus/Bos taurus interspecies model has been incorporated to maximize the genetic heterozygosity that exists between the alleles of putative imprinted genes for allelic discrimination and parental inheritance. The sequence of twenty-six genes, previously reported as imprinted in mice and humans, was analyzed in Bos gaurus (Gaur) and Bos taurus (Angus) cattle for the presence of single nucleotide polymorphisms (SNP). SNPs were detected in the Gene trap locus 2 (GTL2), Insulin like growth factor 2 (IGF2), Wilms tumor 1 (WT1) and the X chromosome inactivation specific transcript (XIST). Allelic expression analysis in interspecies hybrids indicated maternal genomic imprinting at the IGF2 and XIST loci, paternal genomic imprinting at the GTL2 locus and no imprinting at the WT1 locus. Analysis in cloned hybrids indicated fidelity of allelic expression at the IGF2 and GTL2 loci, however disruption of imprinting was observed at the XIST locus in the placenta of clones. These results are the largest identification of imprinted genes in the bovine and the first identification of the disruption of an imprinted gene in an animal derived from somatic cell nuclear transfer.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / This thesis encompasses work that aimed to further understand genomic reprogramming, an event crucial in obtaining development in cloned embryos produced by somatic cell nuclear transfer (SCNT). An increasing number of different mammalian species have been cloned using nuclear transfer technology since Dolly the cloned sheep was first successfully produced. However, the biological mechanisms involved in the process of nuclear reprogramming are yet to be fully described. At the centre of this study was an intergeneric SCNT model, which was implemented to determine whether reprogramming factors are conserved across genera. The interaction between donor nucleus and recipient ooplasm was characterised with regard to developmental potential, timing of genome activation, nucleolus formation, and expression of significant proteins. In initial studies, fusion parameters of the intergeneric SCNT procedure were optimised for the ovine cytoplast and porcine donor granulosa cell. Cell fusion and lysis percentages were determined over a range of electrical pulse voltage, duration and repetition. The optimal electrofusion settings were a single DC pulse of 1.5 kV/cm for 20 usec following a 2 sec 400 kHz alignment pulse. In addition, it was demonstrated that ovine oocytes were sensitive to electric stimulation to the extreme that oocyte activation would occur no matter how low the voltage. The practical significance was that it would not be possible to implement a fusion before activation protocol. The ability of the ooplasm of one species to replicate chromosomes and support early embryo cleavage was determined in a preliminary experiment where intergeneric embryos were produced by SCNT using bovine and ovine foetal fibroblasts, and ovine ooplasm. After their construction, the embryos were allowed to develop for 7 days in vitro and the developmental stage determined by Hoechst staining and nuclei counting. In addition, chromosome spreads of the ovine and bovine somatic foetal fibroblast cell lines used in SCNT, as well as the intra- and intergeneric SCNT embryos were prepared to determine whether the ovine ooplasm was replicating the chromosomes according to the karyotype of the donor nucleus. The somatic cells were karyotyped with 54 and 60 chromosomes counted for ovine and bovine cells respectively. Bovine-ovine embryos were characterised as having a bovine karyotype as distinct from an ovine karyotype, due to the presence of only two metacentric chromosomes as compared with six that are found in the latter. These preliminary results indicated that bovine nuclei obtained from foetal fibroblast cells could initiate early pre-implantation embryo development with the support of ovine oocyte cytoplasm. The development of a proportion (33%) of ovine-ovine intrageneric SCNT embryos beyond the 16-cell stage indicated that an extensive characterisation of an intergeneric model could be performed satisfactorily. It was hypothesised that the ovine ooplasm would possess the ability to direct in vitro preimplantation embryo development after nuclear transfer using donor nuclei from a different genus, as has been demonstrated in studies using bovine and rabbit ooplasm. In this study, intergeneric SCNT embryos were constructed by the separate fusion of porcine and bovine cells with ovine cytoplasts (bovine-ovine and porcine-ovine respectively), cultured in vitro and the developmental characteristics compared with ovine-ovine SeNT embryos as well as ovine in vitro produced (IVP) embryos. These four groups of embryos were sampled to determine embryo cell numbers at 24, 36, 48, 72, 96, 120 and 168 h post-activation to compare development over time. Despite cleaving normally and undergoing the first three cleavage divisions at a rate comparable with ovine-ovine SCNT embryos, a major block in development occurred in the intergeneric embryos at the 8-16 cell stage. Consequently, no blastocyst formation was obtained as observed for the IVP and ovine-ovine SCNT controls. These results indicate that unlike the rabbit and bovine ooplasm, the ovine ooplasm is not suitable for intergeneric reprogramming of somatic nuclei from another genus, at least of porcine or bovine origin. To determine the effect of a less differentiated donor nucleus on intergeneric developmental potential, embryonic cell nuclear transfer (ECNT) was conducted in a separate experiment by fusing pluripotent bovine and ovine donor cells (obtained from day-4 preimplantation embryos) to ovine cytoplasts. After 7 days of culture, the cell number of embryos was determined by Hoechst staining and fluorescent observation. Despite observing a single bovine-ovine blastocyst (4.8%), the developmental block remained at the 8-16 cell stage of development. This outcome indicates that a less differentiated nucleus does not increase intergeneric developmental capability. It is well documented that the ooplasm supplies a large amount of mRNA and protein to the newly formed embryo, crucial for normal development leading up to the major activation of the embryonic genome. However, the interaction between the ooplasm as compared with the donor nucleus in SCNT embryos during this developmental period is poorly understood. This intergeneric SCNT model provided an opportunity to determine the role of the ooplasm on nucleolus formation, which is a marker for genome activation. Ultrastructural evidence was obtained that indicates the ovine ooplasm directs the initial assembly of the nucleolus independent of the species of the nuclear donor. Intergeneric porcine-ovine SCNT and intrageneric ovine-ovine SCNT embryos were constructed and the nucleolus ultrastructure and nucleolus associated rRNA synthesis examined in 1-,2-,4-, early 8-, late 8-and 16-cell embryos using transmission electron microscopy (TEM) and light microscopical autoradiography. Intergeneric porcine-ovine SCNT embryos exhibited nucleolar precursor bodies (NPBs) of an ovine (ruminant) ultrastructure, but no active rRNA producing fibrillogranular nucleoli at any of the stages. Unusually, cytoplasmic organelles were located inside the nucleus of two porcineovine SCNT embryos. The ovine-ovine SCNT embryos, on the other hand, revealed fibrillogranular nucleoli in 16-cell embryos. In parallel, autoradiographic labelling over the nucleoplasm and, in particular, the nulcleoli was detected. Bovine-ovine SCNT embryos at the 8-cell stage were examined for nucleolar morphology and exhibited ruminant-type NPBs as well as structures that appeared to comprise of broken down fibrillar material, perhaps formerly of nucleolar origin from the donor cell. These observations indicate that factors within the ovine ooplasm are playing a role in the initial assembly of the embryonic nucleolus in intrageneric SCNT embryos. To further characterise nucleolus formation, immunocytochemical localisation by confocal microscopy of nucleolin, fibrillarin and RNA polymerase, three key proteins involved in processing rRNA transcripts, was performed on early 8-, late 8- and 16-cell embryos for ovineovine and porcine-ovine SCNT embryos. Nucleolin was localised throughout the nucleoplasm for all developmental stages examined in porcine-ovine and ovine-ovine SCNT embryos and, in particular, intensity around the presumptive nucleolar compartments in the later developmental stages. Fibrillarin and RNA polymerase I, on the other hand, were not detected in any ovineovine or porcine-ovine SCNT embryos or ovine IVP controls, although both proteins were detected in control bovine IVP blastocysts. This result indicates that the antifibrillarin and anti-RNA polymerase I were not compatible with the ovine form of these respective proteins. As nucleolin is not present in porcine in vivo embryos before the major activation of the embryonic genome, its presence in porcine-ovine SCNT embryo nucleus indicates that nucleolin is derived from the abundant protein and mRNA stored in the ovine ooplasm. The intergeneric SCNT model established in this thesis demonstrates that the ovine ooplasm lacks the ability to support embryonic development beyond the 16-cell stage. The TEM and autoradiographical studies, in combination with the protein immunocytochemistry study, confirmed that these embryos are unable to undergo the major activation of the embryonic genome, and that the ooplasm influences the initial nucleolar assembly in these embryos. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1167553 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture and Wine, 2005

In normal embryonic development, cells generated from a fertilised oocyte lose their pluripotent status and become restricted to a particular differentiation pathway. This production of functionally distinct cell lineages is thought to be mediated by epigenetic processes that help control gene expression both temporally and spatially without any changes to the DNA sequence. These epigenetic changes consist of posttranslational modifications of the N-terminal tails of histones and differential DNA methylation. Together these act by altering local chromatin structure, which in turn directs gene transcription by regulating the accessibility of the underlying DNA. To examine the potential developmental roles of these modifications, we determined the global cellular patterns of DNA methylation, as well as histone H3 lysine 9 (H3K9) and histone H4 lysine 20 (H4K20) methylation in the developing zebrafish embryo. These modifications are seen as hallmarks of heterochromatin, which consists of DNA that is tightly packaged, gene-poor and transcriptionally silent. Thus using immunostaining techniques, we confirmed the occurrence of genome-wide DNA methylation changes during zebrafish embryogenesis, as well as observing the unique localisation of this mark around the nuclear periphery in conjunction with pericentric heterochromatin. For mono-, di- and tri-methylated H3K9, it was observed by both immunostaining and immunoblotting that these marks became apparent after the onset of zygotic transcription. Ultimately their levels increased as development progressed, in a fashion similar to that of DNA methylation, consistent with a link between these epigenetic marks. Using the same methodology, the three methylation states of H4K20 were seen to vary differentially during zebrafish development, where in particular the levels of H4K20me1 decreased in concert with a potentially sumoylated form. In contrast, the levels of H4K20me2 increased progressively during embryogenesis, while those of H4K20me3 decreased rapidly after the mid-blastula transition. Together, these findings demonstrate that both DNA and histone lysine methylation take place in a highly dynamic manner, further supporting their roles in augmenting chromatin structure and directing cellular differentiation, while also providing a valuable comparison to the developmental epigenetics of other model organisms characterised to date. Preparatory work for somatic cell nuclear transfer in zebrafish was also undertaken. In future studies, the dynamics of these marks could be compared with those of cloned embryos, so that the specific epigenetic profiles necessary for development can be elucidated. Epigenetically, a homologous process occurs within pluripotent embryonic stem cells (ESCs), which can differentiate into any cell type or undergo indefinite self-renewal. Advantageously, we were able to derive zebrafish ESC-like clusters which were morphologically similar to those derived from mice. These clusters were alkaline phosphatase-positive and expressed key ESC markers as detected by RT-PCR and immunofluorescence. In pilot studies, GFP-expressing ESC-like clusters have so far also contributed to ectodermal tissues when transplanted into wild type zebrafish embryos. Subsequently, these ESC-like clusters were epigenetically profiled using immunofluorescence, which showed that they had a similar complement of modifications to ESCs derived from mice. The derivation and initial characterisation of these ESC-like clusters from zebrafish, in addition to the development of somatic cell nuclear transfer in this species, will help pave the way for future studies involving tissue repair and regeneration, as well as opening up the potential of targeted genetic manipulation in this valuable model organism.

A produção de animais transgênicos possui aplicações que envolvem desde a pesquisa básica à produção agropecuária. O recente progresso na clonagem animal por transferência nuclear (TN) possibilitou a produção de animais transgênicos utilizando linhagens de células doadoras de núcleo previamente modificadas geneticamente. A possibilidade de manipulação genética, estudo da expressão gênica e adequada seleção da célula doadora de núcleo na TN não somente pode garantir a presença da construção gênica em toda a prole, como também pode evitar a produção de animais portadores de modificações indesejáveis resultantes da inserção do inserto em regiões codificantes do genoma, em decorrência da inserção aleatória das técnicas de transferência gênica mais comuns. Este trabalho teve como objetivo geral produzir animais transgênicos a partir de transferência nuclear utilizando como células doadoras de núcleo fibroblastos modificados geneticamente por transdução lentiviral. Objetivos específicos foram a produção e caracterização de linhagens de fibroblasto fetal portadores do gene da Proteína Fluorescente Verde (eGFP) quanto à seleção da expressão do transgene, passagem celular e posição da inserção do transgene e sua utilização na técnica de transferência de núcleos para a análise da competência de desenvolvimento a blastocisto e estabelecimento de gestações. Para tal, fibroblastos fetais bovinos foram transduzidos pelo sistema lentiviral. Células expressando o gene da eGFP foram selecionadas por citometria de fluxo e utilizadas como doadoras de núcleo na TN. Foram analisados o efeito do período de cultivo dos fibroblastos, assim como o efeito da reclonagem na competência de desenvolvimento a blastocisto e estabelecimento de gestações. O cultivo celular submetido à reclonagem foi analisado quanto à posição de inserção do transgene, sendo constatado nos fetos produzidos neste experimento a presença de uma inserção única em região não transcrita do cromossomo 14. Não houve efeito neste experimento do tempo de cultivo na competência de desenvolvimento a blastocisto, mas houve efeito benéfico da reclonagem celular. Além disso, foram obtidos 4 fetos, sendo 3 transgênicos, dos embriões transferidos provenientes das TNs que utilizaram células transgênicas de inserção aleatória em baixas e altas passagens e 6 fetos dos 37 embriões transferidos provenientes das TN que utilizaram células reclonadas, sendo todos transgênicos. Conclui-se que a produção de células transgênicas mediante mecanismo de transdução lentiviral, pode resultar, após TNCS, em embriões geneticamente idênticos à células doadora capazes de sustentar o desenvolvimento in vitro a blastocisto e o estabelecimento de gestações. Finalmente, são discutidos fatores ligados ao processo de seleção e reclonagem que aumentam a eficiência da produção de gestações por TNCS. / Genetically modified animals have numerous applications ranging from basic research to agriculture production. Recent progress in animal cloning by nuclear transfer (NT) has made possible the production of transgenic animals using previously genetically modified cell lineages. The possibility of genetic manipulation, gene expression studies and adequate selection of the nuclei donor cell for NT not only can guarantee the presence of the gene construction in the offspring, but also can avoid the production of animals that carries undesirable characteristics, often as a result of the random insertion of transgenes in transcripted areas of the genome. General objective of this study was to produce transgenic animals by nuclear transfer using lentivirus-genetically modified nuclei donor fibroblasts. Specifically, objectives were the production and characterization of fetal fibroblasts lineages expressing eGFP (enhanced Green Fluorescent Protein, eGFP) gene in different cell passages regarding transgene insertion position and its use for the nuclear transfer procedure. The potential of blastocyst development and pregnancy establishment were analyzed in embryos reconstructed with late and early passages and with clonal or random insertion of transgenes (recloning). For that, bovine fetal fibroblasts were transduced with lentiviruses. eGFP expressing cells were selected by flow citometry sorting and used as nuclei donor cells for NT. Transgene integration site of the cell culture submitted to recloning was analised. It was observed that an unique insertion in a non-transcribed área of chromossome 14 was present in the fetuses recovered in this recloning experiment. No effect of culture time on development of blastocysts was observed, however, there was a beneficial effect of the cell recloning Besides, 4 fetuses (3 of them were transgenic) were obtained when 64 embryos reconstructed with random transgene position cells in late and early passages were transferred and 6 fetuses were obtained when 37 embryos reconstructed with recloned cells were transferred (all of them were transgenic). In conclusion, lentivirus transduction was able to produce transgenic cells with a stable expression of the transgene. These cells, when used for SCNT, can be reprogrammed and genetically identical embryos able to sustain in vitro culture, pregnancy establishment and recognition. Finally, recloning and cell selection procedures are discussed as a possible approach to increase pregnancy efficiency after TNCS.

Somatic cell nuclear transfer (SCNT) is a powerful tool for production of transgenic animals for various biomedical and agricultural applications. For instance, our group is using SCNT to produce transgenic goats to study the role of cardiac fibrosis in initiation and progression of atrial fibrillation. There is a possibility of cell transfer from a transgenic fetus to its non-transgenic surrogate mother, known as fetal microchimerism; from a transgenic mother to non-transgenic fetus, maternal microchimerism and from a transgenic twin to non-transgenic twin in utero. Initially, we have assessed the presence of fetal microchimerism in tissue samples from non-transgenic surrogates that delivered transgenic SCNT generated offspring. Then, the SCNT derived transgenic goats were naturally bred and non-transgenic offspring were used for the assessment of maternal microchimerism. Additionally, fetal-fetal microchimerism was evaluated using the tissue samples from non-transgenic twins of transgenic offspring. We investigated DNA from kidney, liver, lung, lymph node and spleen for the presence of neomycin resistant gene (Neo), which all transgenic SCNT generated females and their transgenic offspring tested positive for. We found no detectable maternal or fetal-fetal microchimerism, but fetal microchimerism was detected in lymph node of one of the surrogate dams that carried a SCNT pregnancy. The results of the study have direct implications on the use and disposal of non-transgenic surrogates and non-transgenic offspring.

A produção de animais transgênicos possui aplicações que envolvem desde a pesquisa básica à produção agropecuária. O recente progresso na clonagem animal por transferência nuclear (TN) possibilitou a produção de animais transgênicos utilizando linhagens de células doadoras de núcleo previamente modificadas geneticamente. A possibilidade de manipulação genética, estudo da expressão gênica e adequada seleção da célula doadora de núcleo na TN não somente pode garantir a presença da construção gênica em toda a prole, como também pode evitar a produção de animais portadores de modificações indesejáveis resultantes da inserção do inserto em regiões codificantes do genoma, em decorrência da inserção aleatória das técnicas de transferência gênica mais comuns. Este trabalho teve como objetivo geral produzir animais transgênicos a partir de transferência nuclear utilizando como células doadoras de núcleo fibroblastos modificados geneticamente por transdução lentiviral. Objetivos específicos foram a produção e caracterização de linhagens de fibroblasto fetal portadores do gene da Proteína Fluorescente Verde (eGFP) quanto à seleção da expressão do transgene, passagem celular e posição da inserção do transgene e sua utilização na técnica de transferência de núcleos para a análise da competência de desenvolvimento a blastocisto e estabelecimento de gestações. Para tal, fibroblastos fetais bovinos foram transduzidos pelo sistema lentiviral. Células expressando o gene da eGFP foram selecionadas por citometria de fluxo e utilizadas como doadoras de núcleo na TN. Foram analisados o efeito do período de cultivo dos fibroblastos, assim como o efeito da reclonagem na competência de desenvolvimento a blastocisto e estabelecimento de gestações. O cultivo celular submetido à reclonagem foi analisado quanto à posição de inserção do transgene, sendo constatado nos fetos produzidos neste experimento a presença de uma inserção única em região não transcrita do cromossomo 14. Não houve efeito neste experimento do tempo de cultivo na competência de desenvolvimento a blastocisto, mas houve efeito benéfico da reclonagem celular. Além disso, foram obtidos 4 fetos, sendo 3 transgênicos, dos embriões transferidos provenientes das TNs que utilizaram células transgênicas de inserção aleatória em baixas e altas passagens e 6 fetos dos 37 embriões transferidos provenientes das TN que utilizaram células reclonadas, sendo todos transgênicos. Conclui-se que a produção de células transgênicas mediante mecanismo de transdução lentiviral, pode resultar, após TNCS, em embriões geneticamente idênticos à células doadora capazes de sustentar o desenvolvimento in vitro a blastocisto e o estabelecimento de gestações. Finalmente, são discutidos fatores ligados ao processo de seleção e reclonagem que aumentam a eficiência da produção de gestações por TNCS. / Genetically modified animals have numerous applications ranging from basic research to agriculture production. Recent progress in animal cloning by nuclear transfer (NT) has made possible the production of transgenic animals using previously genetically modified cell lineages. The possibility of genetic manipulation, gene expression studies and adequate selection of the nuclei donor cell for NT not only can guarantee the presence of the gene construction in the offspring, but also can avoid the production of animals that carries undesirable characteristics, often as a result of the random insertion of transgenes in transcripted areas of the genome. General objective of this study was to produce transgenic animals by nuclear transfer using lentivirus-genetically modified nuclei donor fibroblasts. Specifically, objectives were the production and characterization of fetal fibroblasts lineages expressing eGFP (enhanced Green Fluorescent Protein, eGFP) gene in different cell passages regarding transgene insertion position and its use for the nuclear transfer procedure. The potential of blastocyst development and pregnancy establishment were analyzed in embryos reconstructed with late and early passages and with clonal or random insertion of transgenes (recloning). For that, bovine fetal fibroblasts were transduced with lentiviruses. eGFP expressing cells were selected by flow citometry sorting and used as nuclei donor cells for NT. Transgene integration site of the cell culture submitted to recloning was analised. It was observed that an unique insertion in a non-transcribed área of chromossome 14 was present in the fetuses recovered in this recloning experiment. No effect of culture time on development of blastocysts was observed, however, there was a beneficial effect of the cell recloning Besides, 4 fetuses (3 of them were transgenic) were obtained when 64 embryos reconstructed with random transgene position cells in late and early passages were transferred and 6 fetuses were obtained when 37 embryos reconstructed with recloned cells were transferred (all of them were transgenic). In conclusion, lentivirus transduction was able to produce transgenic cells with a stable expression of the transgene. These cells, when used for SCNT, can be reprogrammed and genetically identical embryos able to sustain in vitro culture, pregnancy establishment and recognition. Finally, recloning and cell selection procedures are discussed as a possible approach to increase pregnancy efficiency after TNCS.

Since the production of the first sheep by somatic cell nuclear transfer a great deal of effort has been made to improve efficiency and to understand nuclear reprogramming mechanisms. Unfortunately efficiency remains low, and nuclear reprogramming mechanisms remain uncharacterized. The objectives of this research were to identify factors associated with somatic cell nuclear transfer efficiency and to analyze the transcriptome of blastocyst-stage clone and control embryos and cotyledonary tissue in an effort to elucidate mechanisms responsible for the low developmental efficiency and high post-implantation losses. The experiments reported here identify factors including oocyte source and timing of activation following nuclear transfer that yield improved efficiencies. It was determined the use of cow oocytes for somatic cell nuclear transfer results in improved in vitro development and increased pregnancy rates. These data further indicate prolonged exposure of the donor nucleus to pre-activated oocyte cytoplasm results in increased nuclear fragmentation and reduced developmental efficiency in vitro. Several aberrantly expressed genes were identified in nuclear transfer blastocysts and cotyledons that could impact cloning efficiency. Major histocompatibility complex I and down-regulator of transcription 1 were overexpressed in nuclear transfer blastocysts, and retinol binding protein 1 was overexpressed in nuclear transfer cotyledons. The functions of these genes in immune response, transcriptional regulation, and retinol binding and transport make them attractive candidates for further nuclear transfer research. Expression levels of six developmentally important genes were analyzed in various stages of preimplantation nuclear transfer embryos by real-time polymerase chain reaction to determine the timing of nuclear reprogramming following nuclear transfer. Five of the six genes were aberrantly expressed multiple developmental stages, however by the blastocyst stage only one gene was aberrantly expressed. These data indicate reprogramming is delayed in nuclear transfer embryos resulting in over- or under-expression of developmentally important genes during early embryogenesis. These experiments report factors associated with improved nuclear transfer efficiency; provide insight into potential mechanisms for low developmental rates, abnormal placentation, and fetal loss of clones; and characterize the timing of nuclear reprogramming following somatic cell nuclear transfer.