Production of recombinant therapeutic proteins in genetically engineered animals: the dawn of a new era / Produção de proteínas terapêuticas recombinantes em animais geneticamente modificados: o surgimento de uma nova era

Background: The production of transgenic animals has been envisioned as a viable strategy to improve food quality, animal yield, and for the production of bioproducts that can be used for the benefit of the human and animal population. Transgenic animals have been used to improve production traits, to add value to animal products, to minimize the impact on the environment, to promote disease resistance, and most notably, to produce recombinant proteins in natural fluids, such as milk, that can be collected, purified and used as biomedical products (biopharming). This review aims to discuss past and recent technological advances in animal transgenesis, and the perspective for biopharming in Brazil.Review: Since the production of recombinant human insulin from Escherichia coli in the 1970s, continuous development of new platforms has allowed a significant expansion in the biopharmaceutical market. The animal platform has been shown to be highly competitive by adding value as low cost implementation, production and scale up, as well as high productivity of synthesized proteins. The expression of recombinant proteins in milk represents the most developed system for production of biopharmaceutical drugs in animals, with two approved biopharmaceuticals for human use: Atryn®, a recombinant antithrombin produced in the milk of goats, approved in 2006 by European Medicines Agency (EMA) and in 2009 by US Food and Drug Administration (FDA), and more recently, Ruconest®, a recombinant human C1 esterase inhibitor protein (C1INH) produced in the milk of rabbits, first approved by EMA in 2012, followed by the FDA approval in 2014. Transgenic animals have been produced by many strategies that have gradually evolved over the decades, including the use of embryo microinjection, viral vectors and transposable elements, sperm-mediated gene transfer, and cloning by somatic cell nuclear transfer (SCNT).[...](AU)

Production of recombinant therapeutic proteins in genetically engineered animals: the dawn of a new era / Produção de proteínas terapêuticas recombinantes em animais geneticamente modificados: o surgimento de uma nova era

Background: The production of transgenic animals has been envisioned as a viable strategy to improve food quality, animal yield, and for the production of bioproducts that can be used for the benefit of the human and animal population. Transgenic animals have been used to improve production traits, to add value to animal products, to minimize the impact on the environment, to promote disease resistance, and most notably, to produce recombinant proteins in natural fluids, such as milk, that can be collected, purified and used as biomedical products (biopharming). This review aims to discuss past and recent technological advances in animal transgenesis, and the perspective for biopharming in Brazil.Review: Since the production of recombinant human insulin from Escherichia coli in the 1970s, continuous development of new platforms has allowed a significant expansion in the biopharmaceutical market. The animal platform has been shown to be highly competitive by adding value as low cost implementation, production and scale up, as well as high productivity of synthesized proteins. The expression of recombinant proteins in milk represents the most developed system for production of biopharmaceutical drugs in animals, with two approved biopharmaceuticals for human use: Atryn®, a recombinant antithrombin produced in the milk of goats, approved in 2006 by European Medicines Agency (EMA) and in 2009 by US Food and Drug Administration (FDA), and more recently, Ruconest®, a recombinant human C1 esterase inhibitor protein (C1INH) produced in the milk of rabbits, first approved by EMA in 2012, followed by the FDA approval in 2014. Transgenic animals have been produced by many strategies that have gradually evolved over the decades, including the use of embryo microinjection, viral vectors and transposable elements, sperm-mediated gene transfer, and cloning by somatic cell nuclear transfer (SCNT).[...]

Clonagem animal: a sobrevivência dos mais aptos / Animal cloning: survival of the fittest

A clonagem animal por transferência nuclear de célula somática (TNCS) apresenta inúmeras aplicações científicas e comerciais, incluindo a produção de animais transgênicos, a preservação de animais de genética desejável, rara ou em extinção, ou mesmo a aplicação para o estudo de aspectos básicos em biologia molecular, celular e do desenvolvimento. Não obstante, a clonagem por TNCS ainda é ineficiente, com menos de 5% dos embriões clones produzidos resultando em animais nascidos vivos. O sucesso na clonagem exige o exímio domínio técnico e científico de várias disciplinas e áreas de conhecimento, havendo pelo menos cinco etapas críticas no processo associadas a falhas de desenvolvimento, desde a produção in vitro dos embriões até o nascimento de um animal viável. A identificação de fatores associados às falhas em cada etapa, em especial aqueles relacionados ao oócito receptor (citoplasto), à célula doadora (carioplasto) e aos procedimentos técnicos per se de produção de embriões clones, além da observação cuidadosa dos sinais de anormalidades subsequentes à transferência dos embriões para fêmeas receptoras, é essencial para a optimização de todos os procedimentos para a obtenção, em seu final, de um animal clonado viável e que sobreviva até a vida adulta. Esta revisão visa descrever alguns eventos técnicos e biológicos associados ao sucesso e/ou insucesso da clonagem animal.

Animal cloning by somatic cell nuclear transfer (SCNT) has numerous scientific and commercial applications, including the production of transgenic animals, preservation of animals from desirable or rare gene pools, and animals in risk of extinction, or even for the study of basic aspects in molecular, cell and developmental biology. Nevertheless, cloning by SCNT is still inefficient, with less than 5% of cloned embryos resulting in liveborn animals. The cloning success depends on a proficient technical and scientific know-how of a number of disciplines and areas of knowledge, with at least five critical steps in the process associated with developmental failures, from the in vitro production of cloned embryos through the birth of a viable animal. The identification of factors associated with failures in each step, in special to those related to the recipient oocyte (cytoplast), to the nucleus donor cell (karyoplast), and to the technical procedures for the production of cloned embryos per se, along with the careful observation of signs of abnormalities following the transfer of embryos to recipient females, is essential for the optimization of procedures that, ultimately, may result in a cloned animal that survives to adulthood. This review aims to discuss some technical and biological events associated with success and/or failure in animal cloning.

Clonagem animal: a sobrevivência dos mais aptos / Animal cloning: survival of the fittest

A clonagem animal por transferência nuclear de célula somática (TNCS) apresenta inúmeras aplicações científicas e comerciais, incluindo a produção de animais transgênicos, a preservação de animais de genética desejável, rara ou em extinção, ou mesmo a aplicação para o estudo de aspectos básicos em biologia molecular, celular e do desenvolvimento. Não obstante, a clonagem por TNCS ainda é ineficiente, com menos de 5% dos embriões clones produzidos resultando em animais nascidos vivos. O sucesso na clonagem exige o exímio domínio técnico e científico de várias disciplinas e áreas de conhecimento, havendo pelo menos cinco etapas críticas no processo associadas a falhas de desenvolvimento, desde a produção in vitro dos embriões até o nascimento de um animal viável. A identificação de fatores associados às falhas em cada etapa, em especial aqueles relacionados ao oócito receptor (citoplasto), à célula doadora (carioplasto) e aos procedimentos técnicos per se de produção de embriões clones, além da observação cuidadosa dos sinais de anormalidades subsequentes à transferência dos embriões para fêmeas receptoras, é essencial para a optimização de todos os procedimentos para a obtenção, em seu final, de um animal clonado viável e que sobreviva até a vida adulta. Esta revisão visa descrever alguns eventos técnicos e biológicos associados ao sucesso e/ou insucesso da clonagem animal.(AU)

Animal cloning by somatic cell nuclear transfer (SCNT) has numerous scientific and commercial applications, including the production of transgenic animals, preservation of animals from desirable or rare gene pools, and animals in risk of extinction, or even for the study of basic aspects in molecular, cell and developmental biology. Nevertheless, cloning by SCNT is still inefficient, with less than 5% of cloned embryos resulting in liveborn animals. The cloning success depends on a proficient technical and scientific know-how of a number of disciplines and areas of knowledge, with at least five critical steps in the process associated with developmental failures, from the in vitro production of cloned embryos through the birth of a viable animal. The identification of factors associated with failures in each step, in special to those related to the recipient oocyte (cytoplast), to the nucleus donor cell (karyoplast), and to the technical procedures for the production of cloned embryos per se, along with the careful observation of signs of abnormalities following the transfer of embryos to recipient females, is essential for the optimization of procedures that, ultimately, may result in a cloned animal that survives to adulthood. This review aims to discuss some technical and biological events associated with success and/or failure in animal cloning.(AU)

In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre-and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the first third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may influence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells. Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the x² or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student's test (P < 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were similar between one another. Cloned GFP embryos had lower in vitro development to the blastocyst stage than WT embryos, which had more TCN than parthenote or aggregated chimeric WT/GFP embryos. Aggregated GFP embryos had fewer cells than the other embryo groups. Discussion: The in vitro development of GFP cloned embryos was lower than WT embryos, with no effects on cell allocation in resulting blastocysts. Differences in blastocyst rate between groups were likely due to lower GFP-expressing cell viability, as GFP donor cells were at high population cell doublings when used for cloning. On a per embryo basis, embryo aggregation on Day 1 resulted in blastocyst development similar to non-aggregated embryos on Day 7, with no differences in cell proportion between groups. The use of GFP-expressing cells was proven a promising strategy for the study of cell allocation during embryo development, which may assist in the elucidation of mechanisms of abnormalities after in vitro embryo manipulations, leading to the development of improved protocols for the in vitro production (IVP) of bovine embryos.

In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos / In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos

Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student´s test (P 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were simi

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the fi rst third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may infl uence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells.Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then

Microbiological and functional evaluation of an alternative device (OB®) for estrous synchronization in ewes

The use of synthetic progestagens released by vaginal devices is an important tool to overcome the reproductive seasonality in sheep, but cost and/or subsequent vaginitis are limiting factors for their use. To identify economic, simple and innocuous alternative vaginal devices for estrous synchronization/induction protocols in sheep, this study aimed to evaluate the microbiological and functional viability of the human vaginal tampons (OB®) impregnated with medroxyprogesterone acetate (MAP) on reproductive performance of ewes. The study compared them with commercial vaginal inserts (CIDR®) and polyurethane sponges impregnated with MAP. In Experiment 1, the device loss rate, the degree of vaginitis during the device removal, the count and identification of bacterial colonies at the device insertion and removal, and efficiency in estrous synchronization and estrus temporal distribution were evaluated. Pubertal ewes at the beginning of the breeding season were randomly allocated to three experimental groups: CIDR®, PSP (polyurethane sponge) and OB®. No device losses occurred in any group, but the use of OB® caused milder signs of vaginitis than polyurethane sponges, with a similar vaginal bacterial growth and microbiota than the CIDR group. The estrus distribution was more disperse in the CIDR than PSP or OB groups. In Experiment 2, pregnancy rates using CIDR® or OB® devices were compared, with estrus manifestation (85.4% and 89.8%) and pregnancy rates (58.3% and 49.0%) being similar between groups (P>0.05), respectively. In conclusion, the use of human intra-vaginal tampons (OB®) impregnated with MAP was proven highly hygienic, practical and effective as a low-cost alternative for estrous synchronization and AI in sheep.

O uso de progestágeno sintético liberado por pessários vaginais é uma importante ferramenta para suplantar a sazonalidade reprodutiva em ovelhas. Todavia, seu uso é limitado pelo custo ou pelas subsequentes vaginites. Na busca de uma alternativa simples e de baixo custo para sincronizar estro em ovelhas, este estudo avaliou o tampão vaginal humano (OB®) impregnado com MAP, na performance reprodutiva de ovelhas, comparando com o CIDR® e as esponjas de poliuretano, estas também impregnadas com MAP. No experimento 1 foram avaliados a taxa de perdas; o grau das vaginites no momento da remoção do pessário; a contagem e identificação das colônias bacterianas; bem como a eficiência da sincronização e a distribuição temporal dos cios. As ovelhas foram aleatoriamente distribuídas em um de três grupos experimentais: CIDR, Esponjas e OB, no inicio da estação reprodutiva. Não ocorreram perdas de pessários em qualquer grupo, porém o OB causou menor grau de vaginite em relação às esponjas, com um crescimento bacteriano e microbiota similares ao grupo CIDR. A distribuição dos cios foi mais dispersa no grupo CIDR do que nos grupos Esponja ou OB. No experimento 2, foram comparados o CIDR e OB em relação à manifestação de cio (85,4% e 89,8%) e taxa de prenhez (58,3% e 49,0%), que foram similares (P 0,05). Conclui-se que o pessário OB impregnado com MAP é higiênico, de baixo custo, prático e efetivo como para a sincronização de cios e IA em ovelhas.

Microbiological and functional evaluation of an alternative device (OB®) for estrous synchronization in ewes

The use of synthetic progestagens released by vaginal devices is an important tool to overcome the reproductive seasonality in sheep, but cost and/or subsequent vaginitis are limiting factors for their use. To identify economic, simple and innocuous alternative vaginal devices for estrous synchronization/induction protocols in sheep, this study aimed to evaluate the microbiological and functional viability of the human vaginal tampons (OB®) impregnated with medroxyprogesterone acetate (MAP) on reproductive performance of ewes. The study compared them with commercial vaginal inserts (CIDR®) and polyurethane sponges impregnated with MAP. In Experiment 1, the device loss rate, the degree of vaginitis during the device removal, the count and identification of bacterial colonies at the device insertion and removal, and efficiency in estrous synchronization and estrus temporal distribution were evaluated. Pubertal ewes at the beginning of the breeding season were randomly allocated to three experimental groups: CIDR®, PSP (polyurethane sponge) and OB®. No device losses occurred in any group, but the use of OB® caused milder signs of vaginitis than polyurethane sponges, with a similar vaginal bacterial growth and microbiota than the CIDR group. The estrus distribution was more disperse in the CIDR than PSP or OB groups. In Experiment 2, pregnancy rates using CIDR® or OB® devices were compared, with estrus manifestation (85.4% and 89.8%) and pregnancy rates (58.3% and 49.0%) being similar between groups (P>0.05), respectively. In conclusion, the use of human intra-vaginal tampons (OB®) impregnated with MAP was proven highly hygienic, practical and effective as a low-cost alternative for estrous synchronization and AI in sheep.

O uso de progestágeno sintético liberado por pessários vaginais é uma importante ferramenta para suplantar a sazonalidade reprodutiva em ovelhas. Todavia, seu uso é limitado pelo custo ou pelas subsequentes vaginites. Na busca de uma alternativa simples e de baixo custo para sincronizar estro em ovelhas, este estudo avaliou o tampão vaginal humano (OB®) impregnado com MAP, na performance reprodutiva de ovelhas, comparando com o CIDR® e as esponjas de poliuretano, estas também impregnadas com MAP. No experimento 1 foram avaliados a taxa de perdas; o grau das vaginites no momento da remoção do pessário; a contagem e identificação das colônias bacterianas; bem como a eficiência da sincronização e a distribuição temporal dos cios. As ovelhas foram aleatoriamente distribuídas em um de três grupos experimentais: CIDR, Esponjas e OB, no inicio da estação reprodutiva. Não ocorreram perdas de pessários em qualquer grupo, porém o OB causou menor grau de vaginite em relação às esponjas, com um crescimento bacteriano e microbiota similares ao grupo CIDR. A distribuição dos cios foi mais dispersa no grupo CIDR do que nos grupos Esponja ou OB. No experimento 2, foram comparados o CIDR e OB em relação à manifestação de cio (85,4% e 89,8%) e taxa de prenhez (58,3% e 49,0%), que foram similares (P 0,05). Conclui-se que o pessário OB impregnado com MAP é higiênico, de baixo custo, prático e efetivo como para a sincronização de cios e IA em ovelhas.

In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos / In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos

Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student´s test (P 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were simi

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the fi rst third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may infl uence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells.Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then