RESUMO
Chickens genetically resistant to avian influenza could prevent future outbreaks. In chickens, influenza A virus (IAV) relies on host protein ANP32A. Here we use CRISPR/Cas9 to generate homozygous gene edited (GE) chickens containing two ANP32A amino acid substitutions that prevent viral polymerase interaction. After IAV challenge, 9/10 edited chickens remain uninfected. Challenge with a higher dose, however, led to breakthrough infections. Breakthrough IAV virus contained IAV polymerase gene mutations that conferred adaptation to the edited chicken ANP32A. Unexpectedly, this virus also replicated in chicken embryos edited to remove the entire ANP32A gene and instead co-opted alternative ANP32 protein family members, chicken ANP32B and ANP32E. Additional genome editing for removal of ANP32B and ANP32E eliminated all viral growth in chicken cells. Our data illustrate a first proof of concept step to generate IAV-resistant chickens and show that multiple genetic modifications will be required to curtail viral escape.
Assuntos
Vírus da Influenza A , Influenza Aviária , Embrião de Galinha , Animais , Influenza Aviária/genética , Edição de Genes , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Galinhas/genética , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismoRESUMO
Chickens are an important resource for smallholder farmers who raise locally adapted, genetically distinct breeds for eggs and meat. The development of efficient reproductive technologies to conserve and regenerate chicken breeds safeguards existing biodiversity and secures poultry genetic resources for climate resilience, biosecurity, and future food production. The majority of the over 1600 breeds of chicken are raised in low and lower to middle income countries under resource-limited, small-scale production systems, which necessitates a low-tech, cost-effective means of conserving diversity is needed. Here, we validate a simple biobanking technique using cryopreserved embryonic chicken gonads. The gonads are quickly isolated, visually sexed, pooled by sex, and cryopreserved. Subsequently, the stored material is thawed and dissociated before injection into sterile host chicken embryos. By using pooled GFP and RFP-labelled donor gonadal cells and Sire Dam Surrogate mating, we demonstrate that chicks deriving entirely from male and female donor germ cells are hatched. This technology will enable ongoing efforts to conserve chicken genetic diversity for both commercial and smallholder farmers, and to preserve existing genetic resources at poultry research facilities.
Assuntos
Cruzamento/métodos , Galinhas/genética , Criopreservação/veterinária , Células Germinativas/citologia , Infertilidade/veterinária , Animais , Bancos de Espécimes Biológicos , Galinhas/fisiologia , Análise Custo-Benefício , Feminino , Variação Genética , MasculinoRESUMO
In birds, males are the homogametic sex (ZZ) and females are the heterogametic sex (ZW). Here, we investigate the role of chromosomal sex and germ cell competition on avian germ cell differentiation. We recently developed genetically sterile layer cockerels and hens for use as surrogate hosts for primordial germ cell (PGC) transplantation. Using in vitro propagated and cryopreserved PGCs from a pedigree Silkie broiler breed, we now demonstrate that sterile surrogate layer hosts injected with same sex PGCs have normal fertility and produced pure breed Silkie broiler offspring when directly mated to each other in Sire Dam Surrogate mating. We found that female sterile hosts carrying chromosomally male (ZZ) PGCs formed functional oocytes and eggs, which gave rise to 100% male offspring after fertilization. Unexpectedly, we also observed that chromosomally female (ZW) PGCs carried by male sterile hosts formed functional spermatozoa and produced viable offspring. These findings demonstrate that avian PGCs are not sexually restricted for functional gamete formation and provide new insights for the cryopreservation of poultry and other bird species using diploid stage germ cells.
RESUMO
In macrolecithal species, cryopreservation of the oocyte and zygote is not possible due to the large size and quantity of lipid deposited within the egg. For birds, this signifies that cryopreserving and regenerating a species from frozen cellular material are currently technically unfeasible. Diploid primordial germ cells (PGCs) are a potential means to freeze down the entire genome and reconstitute an avian species from frozen material. Here, we examine the use of genetically engineered (GE) sterile female layer chicken as surrogate hosts for the transplantation of cryopreserved avian PGCs from rare heritage breeds of chicken. We first amplified PGC numbers in culture before cryopreservation and subsequent transplantation into host GE embryos. We found that all hatched offspring from the chimera GE hens were derived from the donor rare heritage breed broiler PGCs, and using cryopreserved semen, we were able to produce pure offspring. Measurement of the mutation rate of PGCs in culture revealed that 2.7 × 10-10 de novo single-nucleotide variants (SNVs) were generated per cell division, which is comparable with other stem cell lineages. We also found that endogenous avian leukosis virus (ALV) retroviral insertions were not mobilized during in vitro propagation. Taken together, these results show that mutation rates are no higher than normal stem cells, essential if we are to conserve avian breeds. Thus, GE sterile avian surrogate hosts provide a viable platform to conserve and regenerate avian species using cryopreserved PGCs.
Assuntos
Animais Geneticamente Modificados/genética , Cruzamento/métodos , Galinhas/genética , Células Germinativas/citologia , Infertilidade/veterinária , Animais , Animais Geneticamente Modificados/fisiologia , Galinhas/fisiologia , Criopreservação , Diploide , Transferência Embrionária , Feminino , Edição de Genes , Engenharia Genética , MasculinoRESUMO
BACKGROUND: The global market for protein drugs has the highest compound annual growth rate of any pharmaceutical class but their availability, especially outside of the US market, is compromised by the high cost of manufacture and validation compared to traditional chemical drugs. Improvements in transgenic technologies allow valuable proteins to be produced by genetically-modified animals; several therapeutic proteins from such animal bioreactors are already on the market after successful clinical trials and regulatory approval. Chickens have lagged behind mammals in bioreactor development, despite a number of potential advantages, due to the historic difficulty in producing transgenic birds, but the production of therapeutic proteins in egg white of transgenic chickens would substantially lower costs across the entire production cycle compared to traditional cell culture-based production systems. This could lead to more affordable treatments and wider markets, including in developing countries and for animal health applications. RESULTS: Here we report the efficient generation of new transgenic chicken lines to optimize protein production in eggs. As proof-of-concept, we describe the expression, purification and functional characterization of three pharmaceutical proteins, the human cytokine interferon α2a and two species-specific Fc fusions of the cytokine CSF1. CONCLUSION: Our work optimizes and validates a transgenic chicken system for the cost-effective production of pure, high quality, biologically active protein for therapeutics and other applications.
Assuntos
Animais Geneticamente Modificados/genética , Biotecnologia/métodos , Galinhas/genética , Citocinas/genética , Animais , Animais Geneticamente Modificados/metabolismo , Reatores Biológicos/economia , Biotecnologia/economia , Galinhas/metabolismo , Citocinas/economia , Citocinas/metabolismo , Humanos , Interferon-alfa/economia , Interferon-alfa/genética , Interferon-alfa/metabolismo , Fator Estimulador de Colônias de Macrófagos/economia , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Proteínas Recombinantes/economia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
In this work we use TALE nucleases (TALENs) to target a reporter construct to the DDX4 (vasa) locus in chicken primordial germ cells (PGCs). Vasa is a key germ cell determinant in many animal species and is posited to control avian germ cell formation. We show that TALENs mediate homology-directed repair of the DDX4 locus on the Z sex chromosome at high (8.1%) efficiencies. Large genetic deletions of 30â kb encompassing the entire DDX4 locus were also created using a single TALEN pair. The targeted PGCs were germline competent and were used to produce DDX4 null offspring. In DDX4 knockout chickens, PGCs are initially formed but are lost during meiosis in the developing ovary, leading to adult female sterility. TALEN-mediated gene targeting in avian PGCs is therefore an efficient process.
Assuntos
RNA Helicases DEAD-box/metabolismo , Marcação de Genes , Células Germinativas/citologia , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Alelos , Animais , Animais Geneticamente Modificados , Galinhas/genética , Cruzamentos Genéticos , Feminino , Deleção de Genes , Técnicas de Inativação de Genes , Proteínas de Fluorescência Verde/metabolismo , Masculino , Meiose , TransgenesRESUMO
Precise self-renewal of the germ cell lineage is fundamental to fertility and reproductive success. The early precursors for the germ lineage, primordial germ cells (PGCs), survive and proliferate in several embryonic locations during their migration to the embryonic gonad. By elucidating the active signaling pathways in migratory PGCs in vivo, we were able to create culture conditions that recapitulate this embryonic germ cell environment. In defined medium conditions without feeder cells, the growth factors FGF2, insulin, and Activin A, signaling through their cognate-signaling pathways, were sufficient for self-renewal of germline-competent PGCs. Forced expression of constitutively active MEK1, AKT, and SMAD3 proteins could replace their respective upstream growth factors. Unexpectedly, we found that BMP4 could replace Activin A in non-clonal growth conditions. These defined medium conditions identify the key molecular pathways required for PGC self-renewal and will facilitate efforts in biobanking of chicken genetic resources and genome editing.
Assuntos
Embrião de Galinha/citologia , Células Germinativas Embrionárias/citologia , Fatores de Crescimento de Fibroblastos/metabolismo , Insulina/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Ativinas/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Proliferação de Células , Células Cultivadas , Embrião de Galinha/metabolismo , Galinhas , Células Germinativas Embrionárias/metabolismo , Feminino , MasculinoRESUMO
BACKGROUND: Macrophages have many functions in development and homeostasis as well as innate immunity. Recent studies in mammals suggest that cells arising in the yolk sac give rise to self-renewing macrophage populations that persist in adult tissues. Macrophage proliferation and differentiation is controlled by macrophage colony-stimulating factor (CSF1) and interleukin 34 (IL34), both agonists of the CSF1 receptor (CSF1R). In the current manuscript we describe the origin, function and regulation of macrophages, and the role of CSF1R signaling during embryonic development, using the chick as a model. RESULTS: Based upon RNA-sequencing comparison to bone marrow-derived macrophages grown in CSF1, we show that embryonic macrophages contribute around 2% of the total embryo RNA in day 7 chick embryos, and have similar gene expression profiles to bone marrow-derived macrophages. To explore the origins of embryonic and adult macrophages, we injected Hamburger-Hamilton stage 16 to 17 chick embryos with either yolk sac-derived blood cells, or bone marrow cells from EGFP+ donors. In both cases, the transferred cells gave rise to large numbers of EGFP+ tissue macrophages in the embryo. In the case of the yolk sac, these cells were not retained in hatched birds. Conversely, bone marrow EGFP+ cells gave rise to tissue macrophages in all organs of adult birds, and regenerated CSF1-responsive marrow macrophage progenitors. Surprisingly, they did not contribute to any other hematopoietic lineage. To explore the role of CSF1 further, we injected embryonic or hatchling CSF1R-reporter transgenic birds with a novel chicken CSF1-Fc conjugate. In both cases, the treatment produced a large increase in macrophage numbers in all tissues examined. There were no apparent adverse effects of chicken CSF1-Fc on embryonic or post-hatch development, but there was an unexpected increase in bone density in the treated hatchlings. CONCLUSIONS: The data indicate that the yolk sac is not the major source of macrophages in adult birds, and that there is a macrophage-restricted, self-renewing progenitor cell in bone marrow. CSF1R is demonstrated to be limiting for macrophage development during development in ovo and post-hatch. The chicken provides a novel and tractable model to study the development of the mononuclear phagocyte system and CSF1R signaling.
Assuntos
Galinhas/imunologia , Sistema Fagocitário Mononuclear/embriologia , Sistema Fagocitário Mononuclear/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Transdução de Sinais , Animais , Células Sanguíneas/efeitos dos fármacos , Células Sanguíneas/metabolismo , Densidade Óssea/efeitos dos fármacos , Células da Medula Óssea , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Embrião de Galinha , Galinhas/genética , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Sistema Fagocitário Mononuclear/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacos , Saco Vitelino/citologiaRESUMO
We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Macrófagos/citologia , Animais , Animais Geneticamente Modificados , Sequência de Bases , Aves , Linhagem da Célula , Galinhas , Células Dendríticas/citologia , Genes Reporter , Técnicas Genéticas , Sistema Imunitário , Íntrons , Dados de Sequência Molecular , Fagocitose , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Transgenes , Saco Vitelino/fisiologiaRESUMO
In this work, we describe a single piggyBac transposon system containing both a tet-activator and a doxycycline-inducible expression cassette. We demonstrate that a gene product can be conditionally expressed from the integrated transposon and a second gene can be simultaneously targeted by a short hairpin RNA contained within the transposon, both in vivo and in mammalian and avian cell lines. We applied this system to stably modify chicken primordial germ cell (PGC) lines in vitro and induce a reporter gene at specific developmental stages after injection of the transposon-modified germ cells into chicken embryos. We used this vector to express a constitutively-active AKT molecule during PGC migration to the forming gonad. We found that PGC migration was retarded and cells could not colonise the forming gonad. Correct levels of AKT activation are thus essential for germ cell migration during early embryonic development.
Assuntos
Movimento Celular , Elementos de DNA Transponíveis , Células Germinativas/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ativação Transcricional , Células-Tronco Adultas/metabolismo , Animais , Linhagem Celular , Embrião de Galinha , Galinhas , Clonagem Molecular , Células-Tronco Embrionárias/metabolismo , Fibroblastos/metabolismo , Expressão Gênica , Engenharia Genética , Células Germinativas/transplante , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos , Camundongos Transgênicos , Sêmen/citologia , Transdução de SinaisRESUMO
The derivation of germ-line competent avian primordial germ cells establishes a cell-based model system for the investigation of germ cell differentiation and the production of genetically modified animals. Current methods to modify primordial germ cells using DNA or retroviral vectors are inefficient and prone to epigenetic silencing. Here, we validate the use of transposable elements for the genetic manipulation of primordial germ cells. We demonstrate that chicken primordial germ cells can be modified in vitro using transposable elements. Both piggyBac and Tol2 transposons efficiently transpose primordial germ cells. Tol2 transposon integration sites were spread throughout both the macro- and microchromosomes of the chicken genome and were more prevalent in gene transcriptional units and intronic regions, consistent with transposon integrations observed in other species. We determined that the presence of insulator elements was not required for reporter gene expression from the integrated transposon. We further demonstrate that a gene-trap cassette carried in the Tol2 transposon can trap and mutate endogenous transcripts in primordial germ cells. Finally, we observed that modified primordial germ cells form functional gametes as demonstrated by the generation of transgenic offspring that correctly expressed a reporter gene carried in the transposon. Transposable elements are therefore efficient vectors for the genetic manipulation of primordial germ cells and the chicken genome.
Assuntos
Animais Geneticamente Modificados/genética , Elementos de DNA Transponíveis/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Células Germinativas/metabolismo , Animais , Southern Blotting , Técnicas de Cultura de Células , Embrião de Galinha , Primers do DNA/genética , Genes Reporter/genética , Reação em Cadeia da Polimerase/métodosRESUMO
The proposal that birds descended from theropod dinosaurs with digits 2, 3 and 4 was recently given support by short-term fate maps, suggesting that the chick wing polarizing region-a group that Sonic hedgehog-expressing cells-gives rise to digit 4. Here we show using long-term fate maps that Green fluorescent protein-expressing chick wing polarizing region grafts contribute only to soft tissues along the posterior margin of digit 4, supporting fossil data that birds descended from theropods that had digits 1, 2 and 3. In contrast, digit IV of the chick leg with four digits (I-IV) arises from the polarizing region. To determine how digit identity is specified over time, we inhibited Sonic hedgehog signalling. Fate maps show that polarizing region and adjacent cells are specified in parallel through a series of anterior to posterior digit fates-a process of digit specification that we suggest is involved in patterning all vertebrate limbs with more than three digits.
Assuntos
Evolução Biológica , Aves/crescimento & desenvolvimento , Padronização Corporal , Asas de Animais/crescimento & desenvolvimento , Animais , Aves/anatomia & histologia , Aves/classificação , Aves/genética , Expressão Gênica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Asas de Animais/anatomia & histologia , Asas de Animais/metabolismoRESUMO
Point mutations in the intronic ZRS region of Lmbr1, a limb specific cis-regulatory element of Sonic hedgehog (Shh), are associated with polydactyly in humans, cats, and mice. We and others have recently mapped the dominant preaxial polydactyly (Po) locus in Silkie chickens to a single nucleotide polymorphism (SNP) in the ZRS region. Using polymorphisms in the chicken Shh sequence, we confirm that the ZRS region directly regulates Shh expression in the developing limb causing ectopic Shh expression in the anterior leg, prolonged Shh expression in the posterior limb, and allelic imbalance between wt and Slk Shh alleles in heterozygote limbs. Using Silkie legs, we have explored the consequences of increased Shh expression in the posterior leg on the patterning of the toes, and the induction of preaxial polydactyly.
Assuntos
Extremidades/embriologia , Proteínas Hedgehog/metabolismo , Animais , Gatos , Embrião de Galinha , Galinhas , Genótipo , Proteínas Hedgehog/genética , Hibridização In Situ , Camundongos , Polidactilia , Polimorfismo de Fragmento de Restrição/genética , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genéticaRESUMO
Hoxd13, Tbx2, Tbx3, Sall1 and Sall3 genes are candidates for encoding antero-posterior positional values in the developing chick wing and specifying digit identity. In order to build up a detailed profile of gene expression patterns in cell lineages that give rise to each of the digits over time, we compared 3 dimensional (3D) expression patterns of these genes during wing development and related them to digit fate maps. 3D gene expression data at stages 21, 24 and 27 spanning early bud to digital plate formation, captured from in situ hybridisation whole mounts using Optical Projection Tomography (OPT) were mapped to reference wing bud models. Grafts of wing bud tissue from GFP chicken embryos were used to fate map regions of the wing bud giving rise to each digit; 3D images of the grafts were captured using OPT and mapped on to the same models. Computational analysis of the combined computerised data revealed that Tbx2 and Tbx3 are expressed in digit 3 and 4 progenitors at all stages, consistent with encoding stable antero-posterior positional values established in the early bud; Hoxd13 and Sall1 expression is more dynamic, being associated with posterior digit 3 and 4 progenitors in the early bud but later becoming associated with anterior digit 2 progenitors in the digital plate. Sox9 expression in digit condensations lies within domains of digit progenitors defined by fate mapping; digit 3 condensations express Hoxd13 and Sall1, digit 4 condensations Hoxd13, Tbx3 and to a lesser extent Tbx2. Sall3 is only transiently expressed in digit 3 progenitors at stage 24 together with Sall1 and Hoxd13; then becomes excluded from the digital plate. These dynamic patterns of expression suggest that these genes may play different roles in digit identity either together or in combination at different stages including the digit condensation stage.
Assuntos
Padronização Corporal/genética , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Imageamento Tridimensional , Fatores de Transcrição/genética , Asas de Animais/embriologia , Asas de Animais/metabolismo , Animais , Desenvolvimento Ósseo/genética , Linhagem da Célula/genética , Embrião de Galinha , Biologia Computacional , Proteínas de Fluorescência Verde/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Infection of chickens with avian influenza virus poses a global threat to both poultry production and human health that is not adequately controlled by vaccination or by biosecurity measures. A novel alternative strategy is to develop chickens that are genetically resistant to infection. We generated transgenic chickens expressing a short-hairpin RNA designed to function as a decoy that inhibits and blocks influenza virus polymerase and hence interferes with virus propagation. Susceptibility to primary challenge with highly pathogenic avian influenza virus and onward transmission dynamics were determined. Although the transgenic birds succumbed to the initial experimental challenge, onward transmission to both transgenic and nontransgenic birds was prevented.
Assuntos
Animais Geneticamente Modificados , Galinhas/genética , Virus da Influenza A Subtipo H5N1/fisiologia , Influenza Aviária/prevenção & controle , Influenza Aviária/transmissão , RNA Interferente Pequeno/genética , Animais , Linhagem Celular , Galinhas/virologia , Cloaca/virologia , Virus da Influenza A Subtipo H5N1/enzimologia , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/virologia , Orofaringe/virologia , RNA Interferente Pequeno/metabolismo , RNA Viral/análise , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Transfecção , Replicação Viral , Eliminação de Partículas ViraisRESUMO
BACKGROUND: Regulatory elements that control expression of specific genes during development have been shown in many cases to contain functionally-conserved modules that can be transferred between species and direct gene expression in a comparable developmental pattern. An example of such a module has been identified at the rat myosin light chain (MLC) 1/3 locus, which has been well characterised in transgenic mouse studies. This locus contains two promoters encoding two alternatively spliced isoforms of alkali myosin light chain. These promoters are differentially regulated during development through the activity of two enhancer elements. The MLC3 promoter alone has been shown to confer expression of a reporter gene in skeletal and cardiac muscle in transgenic mice and the addition of the downstream MLC enhancer increased expression levels in skeletal muscle. We asked whether this regulatory module, sufficient for striated muscle gene expression in the mouse, would drive expression in similar domains in the chicken. RESULTS: We have observed that a conserved downstream MLC enhancer is present in the chicken MLC locus. We found that the rat MLC1/3 regulatory elements were transcriptionally active in chick skeletal muscle primary cultures. We observed that a single copy lentiviral insert containing this regulatory cassette was able to drive expression of a lacZ reporter gene in the fast-fibres of skeletal muscle in chicken in three independent transgenic chicken lines in a pattern similar to the endogenous MLC locus. Reporter gene expression in cardiac muscle tissues was not observed for any of these lines. CONCLUSIONS: From these results we conclude that skeletal expression from this regulatory module is conserved in a genomic context between rodents and chickens. This transgenic module will be useful in future investigations of muscle development in avian species.
Assuntos
Regulação da Expressão Gênica , Músculo Esquelético/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Células Cultivadas , Galinhas , Elementos Facilitadores Genéticos , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Cadeias Leves de Miosina/genética , Regiões Promotoras Genéticas , Ratos , Alinhamento de SequênciaRESUMO
The outgrowth of the vertebrate tail is thought to involve the proliferation of regionalised stem/progenitor cell populations formed during gastrulation. To follow these populations over extended periods, we used cells from GFP-positive transgenic chick embryos as a source for donor tissue in grafting experiments. We determined that resident progenitor cell populations are localised in the chicken tail bud. One population, which is located in the chordoneural hinge (CNH), contributes descendants to the paraxial mesoderm, notochord and neural tube, and is serially transplantable between embryos. A second population of mesodermal progenitor cells is located in a separate dorsoposterior region of the tail bud, and a corresponding population is present in the mouse tail bud. Using heterotopic transplantations, we show that the fate of CNH cells depends on their environment within the tail bud. Furthermore, we show that the anteroposterior identity of tail bud progenitor cells can be reset by heterochronic transplantation to the node region of gastrula-stage chicken embryos.
Assuntos
Neurônios/metabolismo , Células-Tronco/metabolismo , Cauda/embriologia , Cauda/metabolismo , Animais , Animais Geneticamente Modificados , Contagem de Células , Diferenciação Celular , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Neurônios/citologia , Transplante de Células-Tronco , Células-Tronco/citologia , Cauda/citologiaRESUMO
The potential of using transgenic animals for the synthesis of therapeutic proteins was suggested over twenty years ago. Considerable progress has been made in developing methods for the production of transgenic animals and specifically in the expression of therapeutic proteins in the mammary glands of cows, sheep and goats. Development of transgenic hens for protein production in eggs has lagged behind these systems. The positive features associated with the use of the chicken in terms of cost, speed of development of a production flock and potentially appropriate glycosylation of target proteins have led to significant advances in transgenic chicken models in the past few years.
Assuntos
Animais Geneticamente Modificados , Reatores Biológicos , Galinhas , Proteínas/metabolismo , Animais , Galinhas/genética , Óvulo/metabolismo , Proteínas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
An effective method for genetic modification of chickens has yet to be developed. An efficient technology, enabling production of transgenic birds at high frequency and with reliable expression of transgenes, will have many applications, both in basic research and in biotechnology. We investigated the efficiency with which lentiviral vectors could transduce the chicken germ line and examined the expression of introduced reporter transgenes. Ten founder cockerels transmitted the vector to between 4% and 45% of their offspring and stable transmission to the G2 generation was demonstrated. Analysis of expression of reporter gene constructs in several transgenic lines showed a conserved expression profile between individuals that was maintained after transmission through the germ line. These data demonstrate that lentiviral vectors can be used to generate transgenic lines with an efficiency in the order of 100-fold higher than any previously published method, with no detectable silencing of transgene expression between generations.