Produção in vitro de embriões de mini vacas / In vitro embryo production of miniature cattle

With a focus on productivity, Brazil has advanced research and application of reproductive biotechnologies. However, much remains to be explored, such as in vitro embryo production and transfer in miniature breeds. Thus, the objective of the present work is to report the in vitro production of miniature bovine embryos and transfer to conventional sized cows located in Palmas/To. Nine miniature Jersey-Holstein cross breed donors were aspirated to obtain approximately 13 viable oocytes per female. The oocytes were matured, fertilized and cultured the in vitro, where the production of viable embryos was 23.2%. On day 7 of development (D7) the embryos were transferred to 17 crossbred dairy recipients and after 30 days of innovulation by ultrasound examination a conception rate of 76.4% was found. Thus, the technique of in vitro production and embryo transfer is viable for the reproduction of mini dairy cows, despite the lack of data regarding the work performed on small animals that serve as comparative parameters for the results achieved in the study.(AU)

Produção in vitro de embriões de mini vacas / In vitro embryo production of miniature cattle

With a focus on productivity, Brazil has advanced research and application of reproductive biotechnologies. However, much remains to be explored, such as in vitro embryo production and transfer in miniature breeds. Thus, the objective of the present work is to report the in vitro production of miniature bovine embryos and transfer to conventional sized cows located in Palmas/To. Nine miniature Jersey-Holstein cross breed donors were aspirated to obtain approximately 13 viable oocytes per female. The oocytes were matured, fertilized and cultured the in vitro, where the production of viable embryos was 23.2%. On day 7 of development (D7) the embryos were transferred to 17 crossbred dairy recipients and after 30 days of innovulation by ultrasound examination a conception rate of 76.4% was found. Thus, the technique of in vitro production and embryo transfer is viable for the reproduction of mini dairy cows, despite the lack of data regarding the work performed on small animals that serve as comparative parameters for the results achieved in the study.

Biotécnicas reprodutivas na produção de animais geneticamente modificados para pesquisa biomédica / Reproductive technologies in biomedical research with the use of genetic modified laboratory animals

Tecnologias reprodutivas têm uma participação fundamental na pesquisa biomédica. Por meio delas,novos modelos animais são produzidos e preservados de forma segura. A produção de animais geneticamentemodificados envolve diretamente o uso de embriões, sua manipulação e transferência para fêmeas receptoras.Uma vez produzida uma nova linhagem geneticamente modificada, a melhor forma de preservá-la é por meio dacriopreservação de espermatozoides ou embriões. Uma nova tecnologia para manipulação e modificação dogenoma do camundongo foi desenvolvida nos últimos anos. O sistema CRISPR/Cas9 tornou a produção destesanimais mais rápida, efetiva e com um custo mais baixo que as técnicas tradicionais. Esta tecnologiaimpulsionará o projeto mundial de produção de animais mutantes para cada um dos cerca de 25 mil genespresentes no camundongo. Ainda permitirá a produção de modelos com mutação dupla ou tripla e em animaiscom fundo genético distintos. Esta apresentação abordará a importância das técnicas reprodutivas neste processofundamental da pesquisa biomédica.(AU)

Reproductive technologies have a fundamental role in biomedical research. Through them, new animal modelsare produced and preserved safely. The production of genetically modified animals directly involves the use ofembryos, their manipulation and transference to recipient females. Once a new genetically modified lineage hasbeen produced, the best way to preserve it is by cryopreservation of sperm or embryos. A new technology formanipulating and modifying the mouse genome has been developed in recent years. The CRISPR / Cas9 systemmade the production of these animals faster, more effective and at a lower cost than traditional techniques. Thistechnology will boost the worldwide mutant animal production project for each of the approximately 25,000genes present in the mouse. It will also allow the production of models with double or triple mutation and inanimals with different genetic backgrounds. This presentation will address the importance of reproductivetechniques in this fundamental process of biomedical research.(AU)

Biotécnicas reprodutivas na produção de animais geneticamente modificados para pesquisa biomédica / Reproductive technologies in biomedical research with the use of genetic modified laboratory animals

Tecnologias reprodutivas têm uma participação fundamental na pesquisa biomédica. Por meio delas,novos modelos animais são produzidos e preservados de forma segura. A produção de animais geneticamentemodificados envolve diretamente o uso de embriões, sua manipulação e transferência para fêmeas receptoras.Uma vez produzida uma nova linhagem geneticamente modificada, a melhor forma de preservá-la é por meio dacriopreservação de espermatozoides ou embriões. Uma nova tecnologia para manipulação e modificação dogenoma do camundongo foi desenvolvida nos últimos anos. O sistema CRISPR/Cas9 tornou a produção destesanimais mais rápida, efetiva e com um custo mais baixo que as técnicas tradicionais. Esta tecnologiaimpulsionará o projeto mundial de produção de animais mutantes para cada um dos cerca de 25 mil genespresentes no camundongo. Ainda permitirá a produção de modelos com mutação dupla ou tripla e em animaiscom fundo genético distintos. Esta apresentação abordará a importância das técnicas reprodutivas neste processofundamental da pesquisa biomédica.

Reproductive technologies have a fundamental role in biomedical research. Through them, new animal modelsare produced and preserved safely. The production of genetically modified animals directly involves the use ofembryos, their manipulation and transference to recipient females. Once a new genetically modified lineage hasbeen produced, the best way to preserve it is by cryopreservation of sperm or embryos. A new technology formanipulating and modifying the mouse genome has been developed in recent years. The CRISPR / Cas9 systemmade the production of these animals faster, more effective and at a lower cost than traditional techniques. Thistechnology will boost the worldwide mutant animal production project for each of the approximately 25,000genes present in the mouse. It will also allow the production of models with double or triple mutation and inanimals with different genetic backgrounds. This presentation will address the importance of reproductivetechniques in this fundamental process of biomedical research.

Caracterização das proteínas caveolinas -1 e -2 na placenta de conceptos bovinos clonados transgênicos / Caracterization of caveolin -1 and -2 proteins in cloned and transgenic placenta of cattle

A utilização da transgenia com a proteína fluorescente verde (GFP) como marcador de células de origem fetal nas placentas de clones bovinos servirá de modelo inédito para estudo morfofisiológico e imunológico da interação materno-fetal, visto que possibilitará o seu mapeamento, diferenciando as células fetais das maternas. Tal modelo terá aplicação direta, principalmente porque estes são animais que apresentam problemas em relação ao seu desenvolvimento. Com o auxílio deste modelo, pretende-se verificar o transporte de substâncias entre a mãe e o feto via endocitose, pela imunolocalização das proteínas chamadas de caveolinas. Para tanto foram utilizados 06 bovinos clonados e 30 bovinos de inseminação artificial (IA) com idade até 90 dias de gestação, os quais tiveram seu desenvolvimento interrompido mediante abate humanitário das receptoras e ovariosalpingohisterectomia, com posterior recuperação do útero gestante. Foram coletados os placentônios e o cório. Uma parte das amostras foi recortada e fixada, por imersão, em solução de parafolmaldeído a 4% ou formoldeído a 10% em tampão fosfato de sódio (PBS) a 0,1M pH 7.4, solução de Zamboni (4% de paraformoldeído, 15% de ácido pícrico, em tampão fosfato de sódio a 0,1M pH 7.4), metacarn (60% de metanol, 30% de clorofórmio, e 10% de ácido acético glacial), para verificação da morfologia e realização de imuno-histoquímica para as proteínas caveolinas -1 e -2 (CAV -1 e CAV-2). As caveolinas -1 foram localizadas nos vilos fetais e maternos, mas sua marcação mais forte foi observada no estroma endometrial. As caveolinas -2 tiveram marcação positiva no trofoblasto e membrana córioalantoide, e, especificamente em célula trofoblástica gigante binucleada. Sendo assim, os resultados mostram que a proteína CAV-1 teve uma maior expressão em relação ...(AU)

The transgenic application of green fluorescent protein (GFP) as fetal cell marker on cattle cloned placenta could provide an exclusive model for studying the morphologic and immunologic maternal-fetal interactions, providing information about its mapping, distinguishing the fetal from maternal cells. This model will have direct application, mainly because these animals present problems during its development. With this model's support, we intend to verify the substances transport between mother and fetus during endocytosis, through the immunolocalization of protein named caveolae. For these, we used 06 cloned bovine and 30 cattle samples of artificial insemination (AI) with 90 days of pregnancy, which had been their development interrupted by humanitarian slaughter of the recipient and recovery of the pregnant uterus. We collected the placentome and the chorion. A part of the samples was cut and fixed, by immersion, on a solution containing 4% of parafomaldehyde or 10% of formaldehyde on a sodium phosphate buffer (PBS), at 0,1M pH 7.4, Zamboni solution (4% of paraformaldehyde, 15% of picric acid, on sodium phosphate buffer 0,1M pH 7.4), metacarn (60% of metanol, 30% of chloroform, and 10% glacial acetic acid), for morphologic and immunohistochemistry verification for caveolinas proteins -1 and -2 (CAV -1 and CAV- 2). The caveolins -1 were found in fetal and maternal villi, but its strongest staining was observed in the endometrial stroma. The caveolins -2 had positive staining in trophoblast and chorioallantoic membrane, and specifically in giant trophoblastic binucleated cell. Therefore the results were compared between cloned cattle and from AI or natural mating, for assisting on detection of the reason of many placental alterations, embryonic losses, spontaneous abortion, post-natal mortality and large offspring syndrome on laboratory-manipulated animals. The result suggests that the proteins caveolins -1 and -2 (CAV-1 and CAV-2) are part of the caveolae...(AU)

Caracterização das proteínas caveolinas -1 e -2 na placenta de conceptos bovinos clonados transgênicos / Caracterization of caveolin -1 and -2 proteins in cloned and transgenic placenta of cattle

A utilização da transgenia com a proteína fluorescente verde (GFP) como marcador de células de origem fetal nas placentas de clones bovinos servirá de modelo inédito para estudo morfofisiológico e imunológico da interação materno-fetal, visto que possibilitará o seu mapeamento, diferenciando as células fetais das maternas. Tal modelo terá aplicação direta, principalmente porque estes são animais que apresentam problemas em relação ao seu desenvolvimento. Com o auxílio deste modelo, pretende-se verificar o transporte de substâncias entre a mãe e o feto via endocitose, pela imunolocalização das proteínas chamadas de caveolinas. Para tanto foram utilizados 06 bovinos clonados e 30 bovinos de inseminação artificial (IA) com idade até 90 dias de gestação, os quais tiveram seu desenvolvimento interrompido mediante abate humanitário das receptoras e ovariosalpingohisterectomia, com posterior recuperação do útero gestante. Foram coletados os placentônios e o cório. Uma parte das amostras foi recortada e fixada, por imersão, em solução de parafolmaldeído a 4% ou formoldeído a 10% em tampão fosfato de sódio (PBS) a 0,1M pH 7.4, solução de Zamboni (4% de paraformoldeído, 15% de ácido pícrico, em tampão fosfato de sódio a 0,1M pH 7.4), metacarn (60% de metanol, 30% de clorofórmio, e 10% de ácido acético glacial), para verificação da morfologia e realização de imuno-histoquímica para as proteínas caveolinas -1 e -2 (CAV -1 e CAV-2)...

The transgenic application of green fluorescent protein (GFP) as fetal cell marker on cattle cloned placenta could provide an exclusive model for studying the morphologic and immunologic maternal-fetal interactions, providing information about its mapping, distinguishing the fetal from maternal cells. This model will have direct application, mainly because these animals present problems during its development. With this model's support, we intend to verify the substances transport between mother and fetus during endocytosis, through the immunolocalization of protein named caveolae. For these, we used 06 cloned bovine and 30 cattle samples of artificial insemination (AI) with 90 days of pregnancy, which had been their development interrupted by humanitarian slaughter of the recipient and recovery of the pregnant uterus. We collected the placentome and the chorion. A part of the samples was cut and fixed, by immersion, on a solution containing 4% of parafomaldehyde or 10% of formaldehyde on a sodium phosphate buffer (PBS), at 0,1M pH 7.4, Zamboni solution (4% of paraformaldehyde, 15% of picric acid, on sodium phosphate buffer 0,1M pH 7.4), metacarn (60% of metanol, 30% of chloroform, and 10% glacial acetic acid), for morphologic and immunohistochemistry verification for caveolinas proteins -1 and -2 (CAV -1 and CAV- 2). The caveolins -1 were found in fetal and maternal villi, but its strongest staining was observed in the endometrial stroma. The caveolins -2 had positive staining in trophoblast and chorioallantoic membrane, and specifically in giant trophoblastic binucleated cell. Therefore the results were compared between cloned cattle and from AI or natural mating, for assisting on detection of the reason of many placental alterations, embryonic losses, spontaneous abortion, post-natal mortality and large offspring syndrome on laboratory-manipulated animals. The result suggests that the proteins caveolins -1 and -2 (CAV-1 and CAV-2)...

GFP labelling and epigenetic enzyme expression of bone marrow-derived mesenchymal stem cells from bovine foetuses.

Mesenchymal stem cells (MSC) are multipotent progenitor cells defined by their ability to self-renew and give rise to differentiated progeny. Since MSC from adult tissues represent a promising source of cells for a wide range of cellular therapies, there is high scientific interest in better understanding the potential for genetic modification and the mechanism underlying differentiation. The main objective of this study was to evaluate the potential for gene delivery using a GFP vector and lipofectamine, and to quantify the expression of epigenetic enzymes during foetal bMSC multilineage differentiation. Proportion of GFP-positive cells achieved (15.7% ± 3.5) indicated moderately low transfection efficiency. Analysis of DNA methyltransferase expression during MSC multilineage differentiation suggested no association with osteogenic and chondrogenic differentiation. However, up-regulation of KDM6B expression during osteogenic differentiation was associated with adoption of osteogenic lineage. Furthermore, increase in epigenetic enzyme expression suggested an intense epigenetic regulation during adipogenic differentiation.

Modificação de células-tronco espermatogoniais para produção de bovinos transgênicos / Modification of spermatogonial stem cells to produce transgenic bovine

A espermatogênese em mamíferos é um processo sustentado pela auto-renovação e diferenciação de células-tronco espermatogoniais (SSCs). O estudo destas células oferece um excelente modelo para o melhor entendimento da biologia das células-tronco adultas e dos mecanismos que controlam as funções das SSCs. Além do potencial biomédico para estudos sobre infertilidade em diferentes espécies, as SSC possuem uma aplicação promissora na biotecnologia para a produção de animais transgênicos. Assim, o objetivo deste trabalho foi responder à pergunta: "SSCs bovinas LacZ+ podem integrar-se aos túbulos seminíferos de bezerros pré-púberes da raça Nelore após transplante autólogo?" Para isso, bezerros Nelore de 5 meses de idade (n=16) foram submetidos a uma orquiectomia unilateral para o isolamento de células espermatogoniais por digestão enzimática. Após o plaqueamento diferencial, as células foram transduzidas com um vetor lentiviral contendo a sequencia do gene marcador LacZ. Para isso, os animais foram aleatoriamente alocados em um dos quatro grupos experimentais: LacZ+/PKH26+, LacZ+/PKH26-, LacZ-/PKH26+, LacZ-/PKH26-. Após 60 h do início do cultivo in vitro, as células espermatogoniais foram transplantadas autologamente para o mediastino do testículo remanescente por injeção guiada por ultrassonografia. O testículo transplantado foi removido cirurgicamente após 45 dias e amostras de tecido foram submetidas a reação com x-gal para verificação da integração de células espermatogoniais transgênicas aos túbulos seminíferos. Células espermatogoniais foram isoladas e cultivadas in vitro com sucesso. Contudo, não foi possível obter uma população pura de SSCs por plaqueamento diferencial. Embora tenha sido eleito o transplante de células espermatogoniais e não de SSCs somente, sabe-se que também foram transplantadas SSCs, pois a caracterização das células isoladas demonstrou a expressão dos marcadores de SSCs ITGA6, GFRa-1, PGP 9.5 e afinidade pela lectina DBA. Crioseções de amostras de tecido testicular coradas com x-gal permitiram a observação de células transgênicas em 8 de 8 animais que receberam células LacZ+. Contudo, todas as células transgênicas observadas estavam situadas no interstício. Concluindo, não foi possível observar a integração das células transgênicas transplantadas aos túbulos seminíferos do testículo receptor após 45 dias do transplante autólogo utilizando a técnica de injeção intratesticular de células espermatogoniais LacZ+ no mediastino de bezerros pré-púberes da raça Nelore

Mammalian spermatogenesis is sustained by self renewal and differentiation of spermatogonial stem cells (SSCs). The study of these cells provides a model to better understand adult stem cell biology and the mechanisms that control SSC functions. Besides the biomedical potential to perform studies of infertility in many species, SSCs hold a promising biotechnological application at animal transgenesis. In this manner, the goal of this study was to answer the question: "Can LacZ+ bovine SSCs be integrated into seminiferous tubule of prepubertal Nelore bulls subjected to autologous transplantation?" Hence, 5 months old bulls (n=16) were hemicastrated and spermatogonial cells were isolated by a two step enzymatic digestion procedure. After differential plating, cells were transduced with a lentivirus vector carrying the LacZ reporter gene sequence. Animals were randomly allocated in four experimental groups: LacZ+/PKH26+, LacZ+/PKH26-, LacZ-/PKH26+, LacZ-/PKH26-. After 60 h of the onset of in vitro culture, spermatogonial cells were autologously transplanted to the remaining testes by an ultrasound guided needle injection at the testis mediastinum. The transplanted testes were surgically removed after 45 days and testicular tissue samples were subjected to x-gal staining to assess the integration of transgenic spermatogonial cells to seminiferous tubule. Spermatogonial cells were successfully isolated and in vitro cultured. However, it was not possible to obtain a SSC enriched population of cells by differential plating. Although it was decided by the transplant of spermatogonial cells instead of pure SSCs only, it was detected the expression of SSC marker genes ITGA6, PGP9.5, GFR-1 and the affinity for DBA by the isolated cells. Cryosections of x-gal stained testicular tissue samples allowed the observation of transgenic cells in 8 out of 8 animals that received LacZ+ cells. However, all transgenic cells observed were located at the interstitial space. In conclusion, it was not possible to observe the integration of the transplanted transgenic cells into seminiferous tubule of prepubertal Nelore bulls subjected to autologous transplantation using an ultrasound guided needle injection at the testis mediastinum, after 45 days of transplant

Efficiency of sperm-mediated gene transfer in the ovine by laparoscopic insemination, in vitro fertilization and ICSI.

Transgenesis constitutes an important tool for pharmacological protein production and livestock improvement. We evaluated the potential of laparoscopic insemination (LI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to produce egfp-expressing ovine embryos, using spermatozoa previously exposed to pCX-EGFP plasmid in two different sperm/DNA incubation treatments: "Long Incubation" (2 h at 17 C) and "Short Incubation" (5 min at 5 C). For LI, Merino sheep were superovulated and inseminated with treated fresh semen from Merino rams. The embryos were recovered by flushing the uterine horns. For IVF and ICSI, slaughterhouse oocytes were fertilized with DNA-treated frozen/thawed sperm. All recovered embryos were exposed to blue light (488 nm) to determine green fluorescent morulae and blastocysts rates. High cleavage and morulae/blastocysts rates accompanied the LI and IVF procedures, but no egfp-expressing embryos resulted. In contrast, regardless of the sperm/plasmid incubation treatment, egfp-expressing morulae and blastocysts were always obtained by ICSI, and the highest transgenesis rate (91.6%) was achieved with Short Incubation. In addition, following the incubation of labeled plasmid DNA, after Long or Short exposure treatments, with fresh or frozen/thawed spermatozoa, only non-motile fresh spermatozoa could maintain an attached plasmid after washing procedures. No amplification product could be detected following PCR treatment of LI embryos whose zonae pellucidae (ZP) had been removed. In order to establish conditions for transgenic ICSI in the ovine, we compared three different activation treatments, and over 60% of the obtained blastocysts expressed the transgene. For ICSI embryos, FISH analysis found possible signals compatible with integration events. In conclusion, our results show that in the ovine, under the conditions studied, ICSI is the only method capable of producing exogenous gene-expressing embryos using spermatozoa as vectors.

A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species.

Transgenesis is an essential tool in many biotechnological applications. Intracytoplasmic sperm injection (ICSI)-mediated gene transfer is a powerful technique to obtain transgenic pups; however, most domestic animal embryos do not develop properly after ICSI. An additional step in the protocol, namely assistance by haploid chemical activation, permits the use of ICSI-mediated gene transfer to generate transgenic preimplantation embryos in a wide range of domestic species, including ovine, porcine, feline, equine and bovine. In the present study, spermatozoa from five species were coincubated with pCX-EGFP plasmid and injected into metaphase II oocytes. The chemical activation protocol consisted of ionomycin plus 6-dimethylaminopurine. We detected high proportions of fluorescent EGFP embryos for all five species (23-60%), but with a high frequency of mosaic expression (range 60-85%). To our knowledge, this is the first study to produce exogenous DNA expression in feline and equine embryos. Chemical activation reduces the lag phase of egfp expression in ovine embryos. Our results show that this unique method could be used to obtain ovine, porcine, feline, bovine and equine transgenic preimplantation embryos.

Production of transgenic goat (Capra hircus) with human Granulocyte Colony Stimulating Factor (hG-CSF) gene in Brazil.

In order to produce transgenic goats with hG-CSF, a total of 24 adult Saanen and 48 adult undefined breed goats were used as donors and recipients, respectively. Donors were estrus-synchronized with vaginal sponges and superovulated by a treatment with 200 mg FSH given twice daily in decreasing doses over 3 days starting 48 h before sponge removal. Ovulation was induced by injecting 100 microg GnRH 36 h after sponge removal. The recipients also received an estrus synchronization treatment. Donors were mated with fertile Saanen bucks and, approximately 72 h after sponge removal, zygotes were recovered surgically by flushing oviducts. The recovered zygotes were briefly centrifuged to a reliable visualization of the pronuclei. The DNA construct containing hG-CSF gene flanked by goat and bovine alphas1-casein sequences was injected into pronuclei of 129 zygotes. The microinjected embryos (3-6 per female) were transferred to 27 recipients. Ten recipients became pregnant and 12 kids were born. One transgenic male founder was identified in the group of kids. This is the first report of a birth of a transgenic goat in Latin America.

Production of transgenic goat (Capra hircus) with human Granulocyte Colony Stimulating Factor (hG-CSF) gene in Brazil

In order to produce transgenic goats with hG-CSF, a total of 24 adult Saanen and 48 adult undefined breed goats were used as donors and recipients, respectively. Donors were estrus-synchronized with vaginal sponges and superovulated by a treatment with 200 mg FSH given twice daily in decreasing doses over 3 days starting 48 h before sponge removal. Ovulation was induced by injecting 100µg GnRH 36 h after sponge removal. The recipients also received an estrus synchronization treatment. Donors were mated with fertile Saanen bucks and, approximately 72 h after sponge removal, zygotes were recovered surgically by flushing oviducts. The recovered zygotes were briefly centrifuged to a reliable visualization of the pronuclei. The DNA construct containing hG-CSF gene flanked by goat and bovine alphas1-casein sequences was injected into pronuclei of 129 zygotes. The microinjected embryos (3-6 per female) were transferred to 27 recipients. Ten recipients became pregnant and 12 kids were born. One transgenic male founder was identified in the group of kids. This is the first report of a birth of a transgenic goat in Latin America.

A fim de produzir caprinos transgênicos para o hG-CSF, utilizou-se 24 cabras Saanen adultas e 48 cabras sem raça definida adultas como doadoras e receptoras, respectivamente. As doadoras tiveram o estro sincronizado por esponjas vaginais e foram superovuladas com 200 mg de FSH em doses decrescentes, duas vezes ao dia e iniciando 48 h antes da retirada da esponja. A ovulação foi induzida pela injeção de 100 µg de GnRH às 36 h após a retirada da esponja. As receptoras também receberam um tratamento de sincronização do estro. As doadoras foram cobertas por bodes Saanen férteis e, aproximadamente 72 h após a retirada da esponja, os zigotos foram colhidos cirurgicamente por lavagem dos ovidutos. Os zigotos colhidos foram rapidamente centrifugados para uma melhor visualização dos pró-núcleos. A construção de DNA, contendo o gene do hG-CSF flanqueado pelos genes caprino e bovino da alfas1-caseína, foi injetada em 129 embriões. Os embriões microinjetados (3 a 6 por receptora) foram transferidos para 27 receptoras que responderam ao tratamento. Dez receptoras ficaram gestantes e 12 crias foram produzidas. Um macho transgênico fundador foi identificado no grupo de crias nascidas. Este é o primeiro relato do nascimento de um caprino transgênico na América Latina.

High level expression of bioactive recombinant human growth hormone in the milk of a cloned transgenic cow.

Transgenic farm animals have been proposed as an alternative to current bioreactors for large scale production of biopharmaceuticals. However, the efficiency of both methods in the production of the same protein has not yet been established. Here we report the production of recombinant human growth hormone (hGH) in the milk of a cloned transgenic cow at levels of up to 5 g l(-1). The hormone is identical to that currently produced by expression in E. coli. In addition, the hematological and somatometric parameters of the cloned transgenic cow are within the normal range for the breed and it is fertile and capable of producing normal offspring. These results demonstrate that transgenic cattle can be used as a cost-effective alternative for the production of this hormone.

Isolation of transfected fibroblast clones for use in nuclear transfer and transgene detection in cattle embryos.

Transgenesis in cattle has provided numerous opportunities for livestock production. The development of nuclear transfer (NT) technology has improved the production of transgenic livestock. However, the isolation of pure colonies from a single transfection event remains laborious and can be a constraint in the production of transgenic livestock. We used 96-well cell culture plates to isolate cell lineages obtained from a single fibroblast transfected with the pCi-Neo plasmid. Since single mammalian cells do not grow well in fresh medium, we evaluated the use of conditioned medium. The neomycin phosphotransferase gene was detected in isolated colonies and NT embryos were produced from these cells. Multiplex-PCR assays were performed to detect the transfected fragment as well as autosomal satellite DNA in single NT embryos. This approach provided a reliable method for isolating transfected mammalian cells and for diagnosing the incorporation of desirable vectors in NT embryos. This method can reduce the time and cost of transgenic livestock production.

Isolation of transfected fibroblast clones for use in nuclear transfer and transgene detection in cattle embryos

Transgenesis in cattle has provided numerous opportunities for livestock production. The development of nuclear transfer (NT) technology has improved the production of transgenic livestock. However, the isolation of pure colonies from a single transfection event remains laborious and can be a constraint in the production of transgenic livestock. We used 96-well cell culture plates to isolate cell lineages obtained from a single fibroblast transfected with the pCi-Neo plasmid. Since single mammalian cells do not grow well in fresh medium, we evaluated the use of conditioned medium. The neomycin phosphotransferase gene was detected in isolated colonies and NT embryos were produced from these cells. Multiplex-PCR assays were performed to detect the transfected fragment as well as autosomal satellite DNA in single NT embryos. This approach provided a reliable method for isolating transfected mammalian cells and for diagnosing the incorporation of desirable vectors in NT embryos. This method can reduce the time and cost of transgenic livestock production.

Birth of normal kids after microinjection of pronuclear embryos in a transgenic goat (Capra hircus) production program in Brazil

This pilot project was designed to determine if normal kids could be produced after microinjection in pronuclear embryos and subsequent transfer to recipients in a transgenic goat program in Brazil. Twelve donors of the Saanen breed and 17 recipients of an undefined breed were used. The estrus of both donors and recipients was synchronized by a standard progestagen treatment and superovulation obtained by six pFSH injections. Donors in estrus were mated with fertile Saanen bucks. Zygotes were recovered surgically by flushing oviducts. The recovered zygotes with visible pronuclei were microinjected with 500 to 1000 copies of the human G-CSF gene. Two or four embryos were surgically transferred into the oviducts of recipients. One recipient became pregnant and two kids were born. No transgenic goat was identified after PCR analysis. Even though transgenic goats were not obtained, this experiment establishes the basis of a synchronization and superovulation regimen for use in goats raised in Brazil, for the purpose of collecting and manipulating the pronuclear embryos. This project also showed that microinjected one-cell goat embryos can survive to produce live young following surgical transfer

Birth of normal kids after microinjection of pronuclear embryos in a transgenic goat (Capra hircus) production program in Brazil.

This pilot project was designed to determine if normal kids could be produced after microinjection in pronuclear embryos and subsequent transfer to recipients in a transgenic goat program in Brazil. Twelve donors of the Saanen breed and 17 recipients of an undefined breed were used. The estrus of both donors and recipients was synchronized by a standard progestagen treatment and superovulation obtained by six pFSH injections. Donors in estrus were mated with fertile Saanen bucks. Zygotes were recovered surgically by flushing oviducts. The recovered zygotes with visible pronuclei were microinjected with 500 to 1000 copies of the human G-CSF gene. Two or four embryos were surgically transferred into the oviducts of recipients. One recipient became pregnant and two kids were born. No transgenic goat was identified after PCR analysis. Even though transgenic goats were not obtained, this experiment establishes the basis of a synchronization and superovulation regimen for use in goats raised in Brazil, for the purpose of collecting and manipulating the pronuclear embryos. This project also showed that microinjected one-cell goat embryos can survive to produce live young following surgical transfer.