Perspectives of gene editing for cattle farming in tropical and subtropical regions

Cattle productivity in tropical and subtropical regions can be severely affected by the environment. Reproductive performance, milk and meat production are compromised by the heat stress imposed by the elevated temperature and humidity. The resulting low productivity contributes to reduce the farmer's income and to increase the methane emissions per unit of animal protein produced and the pressure on land usage. The introduction of highly productive European cattle breeds as well as crossbreeding with local breeds have been adopted as strategies to increase productivity but the positive effects have been limited by the low adaptation of European animals to hot climates and by the reduction of the heterosis effect in the following generations. Gene editing tools allow precise modifications in the animal genome and can be an ally to the cattle industry in tropical and subtropical regions. Alleles associated with production or heat tolerance can be shifted between breeds without the need of crossbreeding. Alongside assisted reproductive biotechnologies and genome selection, gene editing can accelerate the genetic gain of indigenous breeds such as zebu cattle. This review focuses on some of the potential applications of gene editing for cattle farming in tropical and subtropical regions, bringing aspects related to heat stress, milk yield, bull reproduction and methane emissions.(AU)

Light up the embryos: knock-in reporter generation for mouse developmental biology

Developmental biology seeks to understand the sophisticated regulated process through which a single cell – a fertilized egg – generates a highly organized organism. The most effective way to reveal the nature of these processes is to follow single cells and cell lineages in real-time. Recent advances in imaging equipment, fluorescent tags and computational tools have made long term multi-color imaging of cells and embryos possible. However, there is still one major challenging for achieving live imaging of mammalian embryos- the generation of embryos carrying reporters that recapitulate the endogenous expression pattern of marker genes. Recent developments of genome editing technology played important roles in enabling efficient generation of reporter mouse models. This mini review discusses recent developments of technologies for efficiently generate knock-in reporter mice and the application of these models in live imaging development. With these developments, we are starting to realize the long-sought promises of realtime analysis of mammalian development.

Light up the embryos: knock-in reporter generation for mouse developmental biology

Developmental biology seeks to understand the sophisticated regulated process through which a single cell a fertilized egg generates a highly organized organism. The most effective way to reveal the nature of these processes is to follow single cells and cell lineages in real-time. Recent advances in imaging equipment, fluorescent tags and computational tools have made long term multi-color imaging of cells and embryos possible. However, there is still one major challenging for achieving live imaging of mammalian embryos- the generation of embryos carrying reporters that recapitulate the endogenous expression pattern of marker genes. Recent developments of genome editing technology played important roles in enabling efficient generation of reporter mouse models. This mini review discusses recent developments of technologies for efficiently generate knock-in reporter mice and the application of these models in live imaging development. With these developments, we are starting to realize the long-sought promises of realtime analysis of mammalian development.(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).[...](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).[...]

THE ROLE OF THE CD46 RECEPTOR IN THE PATHOGENICITY OF THE BOVINE VIRAL DIARRHEA VIRUS INFECTION IN BOVINE CULTURED CELLS

O vírus da Diarreia Viral Bovina (BVDV) é um dos principais patógenos virais que causam importantes doenças clínicas em bovinos. A presença deste vírus em rebanhos representa um grande risco para a produtividade por perdas econômicas significativas. O uso da tecnologia de edição gênica, como o sistema CRISPR/Cas9, em animais de produção pode contribuir para o desenvolvimento de modelos de resistência a doenças, como a BVD. Mesmo que os mecanismos de infecção, entrada e liberação do BVDV não sejam totalmente compreendidos, a molécula CD46 é considerada o principal receptor celular para o BVDV em bovinos. O mapeamento do local de adesão ao BVDV demonstrou que dois peptídeos, localizados no módulo mais distal da proteína 1 de controle do complemento (CCP1), fornecem a plataforma de adesão ao BVDV. Os objetivos deste estudo foram (i) caracterizar o CD46 em linhagens celulares bovinas; (ii) modificar a plataforma de adesão do receptor por edição gênica; e (iii) investigar a permissibilidade do BVDV à infecção celular em células editadas geneticamente para o gene CD46. Para caracterizar o receptor, foi sequenciado o DNA complementar do gene CD46 em amostras de fibroblastos bovinos e em linhagens celulares de rim bovino Madin-Darby suscetíveis (MDBK) ou resistentes (CRIB) ao BVDV. Posteriormente, por edição gênica pelo sistema CRISPR/Cas9, criaram-se indels no éxon 1 do gene CD46, removendo oligopeptídeos que conformam a plataforma de adesão do receptor CD46 ao BVDV. Para realizar a edição genômica, duas sequências de RNA-guia (gRNA) foram selecionadas para a deleção do éxon 1 do CD46 em células MDBK. Posteriormente, dois plasmídeos px458, específicos para cada umas das gRNAs, e com expressão da proteína fluorescente verde (GFP), foram co-transfectados em células MDBK. O isolamento de populações de células clonais com modificações específicas das células transfectadas foi realizado por citometria de fluxo, seguido de um período de expansão para estabelecer sub-populações de células clonais. O DNA genômico foi isolado e amplificado por PCR. O produto do PCR foi ligado em plasmídeos pCR-TOPO, transformado em células TOP10 E. coli e sequenciado. Como resultados, identificou-se que as células CRIB codificam uma proteína CD46 semelhante às MDBK e aos fibroblastos, sem nenhuma mutação na região de interesse, com o receptor CD46 não apresentando aparente importância na resistência destas células à infecção por BVDV. Além disso, obtivemos três linhagens celulares MDBK com deleção em segmentos específicos na região codificante para o CCP1 referente a plataforma de adesão ao BVDV. Uma das linhagens apresentou a deleção bialélica, com a edição gênica sendo diferente em cada alelo; outra linhagem apresentou uma edição bialélica, com um dos alelos não apresentando a deleção da plataforma de adesão; e a última linhagem apresentou uma deleção bialélica homozigota. O estudo da susceptibilidade destas células mutantes à infecção pelo BVDV encontra-se em andamento. Com as três linhagens celulares bovinas modificadas geneticamente para a plataforma de adesão do receptor CD46 ao BVDV, tem-se a expectativa que estas modificações genéticas possivelmente levarão a uma diminuição na infecção pelo BVDV, o que permitirá obtermos mais informações sobre o mecanismo de entrada e infecção do vírus em células bovinas. Tal estratégia mostra-se viável para a futura geração de bovinos resistentes ao BVDV por biotécnicas avançadas da reprodução, como pela clonagem por transferência nuclear de células somáticas (TNCS).

The Bovine Viral Diarrhea virus (BVDV) is one of the major viral pathogens that cause important clinical diseases in cattle. The presence of the virus in herds represents a major risk for productivity due to significant economic losses. The use of gene-editing technology, such as the CRISPR/Cas9 system, in livestock animals can contribute to the development of disease resistance models, such as BVD. Even though the mechanisms of infection, entry and release of BVDV are not yet fully understood, the CD46 molecule is considered the major cellular receptor for BVDV in cattle. The mapping of the adhesion site to the BVDV revealed that two peptides, located in the most distal domain of the complement control protein 1 (CCP1), provide the adhesion platform for the virus. The aims of this study were (i) to characterize CD46 in bovine cell lines; (ii) to modify the platform of adhesion of the CD46 receptor by gene edition; and (iii) to investigate the permissibility of BVDV to cellular infection in CD46 gene-edited cells. To characterize the receptor, the complementary DNAof the gene CD46 was sequenced on samples of fibroblast cells and in Madin-Darby bovine kidney cell lines (MDBK) or MDBK susceptible cells and on cells resistant to BVDV infection (CRIB cells). Afterwards, by gene editing using the CRISPR/Cas9 system, indels were created in the exon 1 of the CD46 gene, removing oligopeptides that form the CD46 receptor adhesion platform to BVDV. To achieve the genomic edition, two sequences of guiding RNA (gRNAs) were selected for exon 1 deletion of CD46 in MDBK cells. Subsequently, two px458 plasmids, each encoding one of the gRNAs, and expressing the green fluorescent protein (GFP), were co-transfected into MDBK cells. Isolation of clonal cell populations with specific modifications of the transfected cells was done through Fluorescence Activated Cell Sorting, followed by an expansion period to establish new clonal cell lines. Genomic DNA was isolated and amplified by PCR. The PCR amplified products were ligated into pCR-TOPO plasmid and transformed into TOP10 E. coli cells, and DNA-sequenced. Our results indicated that CRIB cells express a CD46 protein similar to MDBK and fibroblast cells, without any mutation in the region of interest, with the CD46 receptor in CRIB cells showing an apparent no importance in the resistance to BVDV infection. Furthermore, we obtained three MDBK cell lines with deletion in specific segments in the coding region for CCP1 referring to the BVDV adhesion platform. One of the lines presented a biallelic deletion of the adhesion platform, with gene edition being different in each allele; another line presented a biallelic edition, with one of the alleles not containing the adhesion platform deletion; and the last line presented a biallelic homozygous deletion. However, the study of the susceptibility of such mutant cells to BVDV infection is still ongoing. With the three genetically modified bovine cell lines to the CD46 receptor adhesion platform on BVDV, such genetic modification will possibly lead to a decrease in BVDV infection, which may allow us to obtain more pieces of information regarding mechanisms of virus entry and infection in bovine cells. Such a strategy proves to be feasible for the future generation of BVDV resistant animals by the use of advanced reproductive technologies, such as cloning by somatic cell nuclear transfer (SCNT).