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1.
Proc Natl Acad Sci U S A ; 120(29): e2301250120, 2023 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-37428903

RESUMO

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease caused by mutations in the DMD gene, leading to complete absence of dystrophin and progressive degeneration of skeletal musculature and myocardium. In DMD patients and in a corresponding pig model with a deletion of DMD exon 52 (DMDΔ52), expression of an internally shortened dystrophin can be achieved by skipping of DMD exon 51 to reframe the transcript. To predict the best possible outcome of this strategy, we generated DMDΔ51-52 pigs, additionally representing a model for Becker muscular dystrophy (BMD). DMDΔ51-52 skeletal muscle and myocardium samples stained positive for dystrophin and did not show the characteristic dystrophic alterations observed in DMDΔ52 pigs. Western blot analysis confirmed the presence of dystrophin in the skeletal muscle and myocardium of DMDΔ51-52 pigs and its absence in DMDΔ52 pigs. The proteome profile of skeletal muscle, which showed a large number of abundance alterations in DMDΔ52 vs. wild-type (WT) samples, was normalized in DMDΔ51-52 samples. Cardiac function at age 3.5 mo was significantly reduced in DMDΔ52 pigs (mean left ventricular ejection fraction 58.8% vs. 70.3% in WT) but completely rescued in DMDΔ51-52 pigs (72.3%), in line with normalization of the myocardial proteome profile. Our findings indicate that ubiquitous deletion of DMD exon 51 in DMDΔ52 pigs largely rescues the rapidly progressing, severe muscular dystrophy and the reduced cardiac function of this model. Long-term follow-up studies of DMDΔ51-52 pigs will show if they develop symptoms of the milder BMD.


Assuntos
Distrofia Muscular de Duchenne , Animais , Suínos , Distrofia Muscular de Duchenne/metabolismo , Distrofina/genética , Distrofina/metabolismo , Proteoma/metabolismo , Volume Sistólico , Função Ventricular Esquerda , Músculo Esquelético/metabolismo , Éxons/genética
3.
Xenotransplantation ; 31(2): e12858, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38646921

RESUMO

One of the prerequisites for successful organ xenotransplantation is a reasonable size match between the porcine organ and the recipient's organ to be replaced. Therefore, the selection of a suitable genetic background of source pigs is important. In this study, we investigated body and organ growth, cardiac function, and genetic diversity of a colony of Auckland Island pigs established at the Center for Innovative Medical Models (CiMM), LMU Munich. Male and female Auckland Island pig kidney cells (selected to be free of porcine endogenous retrovirus C) were imported from New Zealand, and founder animals were established by somatic cell nuclear transfer (SCNT). Morphologically, Auckland Island pigs have smaller body stature compared to many domestic pig breeds, rendering their organ dimensions well-suited for human transplantation. Furthermore, echocardiography assessments of Auckland Island pig hearts indicated normal structure and functioning across various age groups throughout the study. Single nucleotide polymorphism (SNP) analysis revealed higher runs of homozygosity (ROH) in Auckland Island pigs compared to other domestic pig breeds and demonstrated that the entire locus coding the swine leukocyte antigens (SLAs) was homozygous. Based on these findings, Auckland Island pigs represent a promising genetic background for organ xenotransplantation.


Assuntos
Variação Genética , Suínos , Transplante Heterólogo , Nova Zelândia , Suínos/genética , Animais , Masculino , Feminino , Humanos , Coração/anatomia & histologia , Coração/diagnóstico por imagem , Ecocardiografia , Genótipo , Homozigoto
4.
Nature ; 564(7736): 430-433, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30518863

RESUMO

Heart transplantation is the only cure for patients with terminal cardiac failure, but the supply of allogeneic donor organs falls far short of the clinical need1-3. Xenotransplantation of genetically modified pig hearts has been discussed as a potential alternative4. Genetically multi-modified pig hearts that lack galactose-α1,3-galactose epitopes (α1,3-galactosyltransferase knockout) and express a human membrane cofactor protein (CD46) and human thrombomodulin have survived for up to 945 days after heterotopic abdominal transplantation in baboons5. This model demonstrated long-term acceptance of discordant xenografts with safe immunosuppression but did not predict their life-supporting function. Despite 25 years of extensive research, the maximum survival of a baboon after heart replacement with a porcine xenograft was only 57 days and this was achieved, to our knowledge, only once6. Here we show that α1,3-galactosyltransferase-knockout pig hearts that express human CD46 and thrombomodulin require non-ischaemic preservation with continuous perfusion and control of post-transplantation growth to ensure long-term orthotopic function of the xenograft in baboons, the most stringent preclinical xenotransplantation model. Consistent life-supporting function of xenografted hearts for up to 195 days is a milestone on the way to clinical cardiac xenotransplantation7.


Assuntos
Transplante de Coração , Xenoenxertos/transplante , Papio , Suínos , Transplante Heterólogo , Animais , Anticorpos/análise , Anticorpos/sangue , Proteínas do Sistema Complemento/análise , Enzimas/sangue , Fibrina/análise , Galactosiltransferases/deficiência , Galactosiltransferases/genética , Xenoenxertos/patologia , Humanos , Fígado/enzimologia , Masculino , Proteína Cofatora de Membrana/genética , Proteína Cofatora de Membrana/metabolismo , Miocárdio/enzimologia , Necrose , Perfusão , Contagem de Plaquetas , Tempo de Protrombina , Trombomodulina/genética , Trombomodulina/metabolismo , Fatores de Tempo
5.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33658378

RESUMO

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons.


Assuntos
Animais Geneticamente Modificados/genética , Sistemas CRISPR-Cas , Galinhas/genética , Edição de Genes , Gado/genética , Suínos/genética , Animais
6.
Proc Natl Acad Sci U S A ; 118(37)2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34508004

RESUMO

ß cells produce, store, and secrete insulin upon elevated blood glucose levels. Insulin secretion is a highly regulated process. The probability for insulin secretory granules to undergo fusion with the plasma membrane or being degraded is correlated with their age. However, the molecular features and stimuli connected to this behavior have not yet been fully understood. Furthermore, our understanding of ß cell function is mostly derived from studies of ex vivo isolated islets in rodent models. To overcome this translational gap and study insulin secretory granule turnover in vivo, we have generated a transgenic pig model with the SNAP-tag fused to insulin. We demonstrate the correct targeting and processing of the tagged insulin and normal glycemic control of the pig model. Furthermore, we show specific single- and dual-color granular labeling of in vivo-labeled pig pancreas. This model may provide unprecedented insights into the in vivo insulin secretory granule behavior in an animal close to humans.


Assuntos
Animais Geneticamente Modificados/metabolismo , Membrana Celular/metabolismo , Corantes Fluorescentes/química , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas SNARE/metabolismo , Vesículas Secretórias/metabolismo , Animais , Exocitose , Glucose/metabolismo , Secreção de Insulina , Masculino , Suínos
7.
FASEB J ; 36(6): e22337, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35486003

RESUMO

The mammalian blastocyst undergoes two lineage segregations, that is, formation of the trophectoderm and subsequently differentiation of the hypoblast (HB) from the inner cell mass, leaving the epiblast (EPI) as the remaining pluripotent lineage. To clarify the expression patterns of markers specific for these lineages in bovine embryos, we analyzed day 7, 9, and 12 blastocysts completely produced in vivo by staining for OCT4, NANOG, SOX2 (EPI), and GATA6, SOX17 (HB) and identified genes specific for these developmental stages in a global transcriptomics approach. To study the role of OCT4, we generated OCT4-deficient (OCT4 KO) embryos via somatic cell nuclear transfer or in vitro fertilization. OCT4 KO embryos reached the expanded blastocyst stage by day 8 but lost NANOG and SOX17 expression, while SOX2 and GATA6 were unaffected. Blastocysts transferred to recipient cows from day 6 to 9 expanded, but the OCT4 KO phenotype was not rescued by the uterine environment. Exposure of OCT4 KO embryos to exogenous FGF4 or chimeric complementation with OCT4 intact embryos did not restore NANOG or SOX17 in OCT4-deficient cells. Our data show that OCT4 is required cell autonomously for the maintenance of pluripotency of the EPI and differentiation of the HB in bovine embryos.


Assuntos
Blastocisto , Regulação da Expressão Gênica no Desenvolvimento , Animais , Blastocisto/metabolismo , Bovinos , Diferenciação Celular/genética , Feminino , Genes Homeobox , Camadas Germinativas , Mamíferos/genética
8.
Xenotransplantation ; 29(1): e12719, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34935207

RESUMO

BACKGROUND: Islet xenotransplantation is a promising concept for beta-cell replacement therapy. Reporter genes for noninvasive monitoring of islet engraftment, graft mass changes, long-term survival, and graft failure support the optimization of transplantation strategies. Near-infrared fluorescent protein (iRFP) is ideal for fluorescence imaging (FI) in tissue, but also for multispectral optoacoustic tomography (MSOT) with an even higher imaging depth. Therefore, we generated reporter pigs ubiquitously expressing iRFP. METHODS: CAG-iRPF720 transgenic reporter pigs were generated by somatic cell nuclear transfer from FACS-selected stable transfected donor cells. Neonatal pig islets (NPIs) were transplanted into streptozotocin-diabetic immunodeficient NOD-scid IL2Rgnull (NSG) mice. FI and MSOT were performed to visualize different numbers of NPIs and to evaluate associations between signal intensity and glycemia. MSOT was also tested in a large animal model. RESULTS: CAG-iRFP transgenic NPIs were functionally equivalent with wild-type NPIs. Four weeks after transplantation under the kidney capsule, FI revealed a twofold higher signal for 4000-NPI compared to 1000-NPI grafts. Ten weeks after transplantation, the fluorescence intensity of the 4000-NPI graft was inversely correlated with glycemia. After intramuscular transplantation into diabetic NSG mice, MSOT revealed clear dose-dependent signals for grafts of 750, 1500, and 3000 NPIs. Dose-dependent MSOT signals were also revealed in a pig model, with stronger signals after subcutaneous (depth ∼6 mm) than after submuscular (depth ∼15 mm) placement of the NPIs. CONCLUSIONS: Islets from CAG-iRFP transgenic pigs are fully functional and accessible to long-term monitoring by state-of-the-art imaging modalities. The novel reporter pigs will support the development and preclinical testing of novel matrices and engraftment strategies for porcine xeno-islets.


Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Animais , Animais Geneticamente Modificados , Glicemia , Xenoenxertos , Transplante das Ilhotas Pancreáticas/métodos , Camundongos , Camundongos Endogâmicos NOD , Proteína Estafilocócica A , Suínos , Transplante Heterólogo/métodos
9.
BMC Genomics ; 22(1): 139, 2021 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-33639836

RESUMO

BACKGROUND: The transcriptional changes around the time of embryonic genome activation in pre-implantation embryos indicate that this process is highly dynamic. In vitro produced porcine blastocysts are known to be less competent than in vivo developed blastocysts. To understand the conditions that compromise developmental competence of in vitro embryos, it is crucial to evaluate the transcriptional profile of porcine embryos during pre-implantation stages. In this study, we investigated the transcriptome dynamics in in vivo developed and in vitro produced 4-cell embryos, morulae and hatched blastocysts. RESULTS: In vivo developed and in vitro produced embryos displayed largely similar transcriptome profiles during development. Enriched canonical pathways from the 4-cell to the morula transition that were shared between in vivo developed and in vitro produced embryos included oxidative phosphorylation and EIF2 signaling. The shared canonical pathways from the morula to the hatched blastocyst transition were 14-3-3-mediated signaling, xenobiotic metabolism general signaling pathway, and NRF2-mediated oxidative stress response. The in vivo developed and in vitro produced hatched blastocysts further were compared to identify molecular signaling pathways indicative of lower developmental competence of in vitro produced hatched blastocysts. A higher metabolic rate and expression of the arginine transporter SLC7A1 were found in in vitro produced hatched blastocysts. CONCLUSIONS: Our findings suggest that embryos with compromised developmental potential are arrested at an early stage of development, while embryos developing to the hatched blastocyst stage display largely similar transcriptome profiles, irrespective of the embryo source. The hatched blastocysts derived from the in vitro fertilization-pipeline showed an enrichment in molecular signaling pathways associated with lower developmental competence, compared to the in vivo developed embryos.


Assuntos
Blastocisto , Transcriptoma , Animais , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Fertilização in vitro , Mórula , Suínos
10.
Xenotransplantation ; 28(2): e12664, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33241624

RESUMO

BACKGROUND: Many genetically multi-modified donor lines for xenotransplantation have a background of domestic pigs with rapid body and organ growth. The intrinsic growth potential of porcine xeno-organs may impair their long-term function after orthotopic transplantation in non-human primate models. Since growth hormone is a major stimulator of postnatal growth, we deleted its receptor (GHR-KO) to reduce the size of donor pigs in one step. METHODS: Heart weight and proteome profile of myocardium were investigated in GHR-KO and control pigs. GHR-KO mutations were introduced using CRISPR/Cas9 in an α1,3-galactosyltransferase (GGTA1)-deficient background expressing the human cluster of differentiation (hCD46) and human thrombomodulin (hTHBD) to generate quadruple-modified (4GM) pigs. RESULTS: At age 6 months, GHR-KO pigs had a 61% reduced body weight and a 63% reduced heart weight compared with controls. The mean minimal diameter of cardiomyocytes was 28% reduced. A holistic proteome study of myocardium samples from the two groups did not reveal prominent differences. Two 4GM founder sows had low serum insulin-like growth factor 1 (IGF1) levels (24 ± 1 ng/mL) and reached body weights of 70.3 and 73.4 kg at 9 months. Control pigs with IGF1 levels of 228 ± 24 ng/mL reached this weight range three months earlier. The 4GM sows showed normal sexual development and were mated with genetically multi-modified boars. Offspring revealed the expected Mendelian transmission of the genetic modifications and consistent expression of the transgenes. CONCLUSION: GHR-KO donor pigs can be used at an age beyond the steepest phase of their growth curve, potentially reducing the problem of xeno-organ overgrowth in preclinical studies.


Assuntos
Galactosiltransferases , Receptores da Somatotropina , Animais , Animais Geneticamente Modificados , Feminino , Técnicas de Inativação de Genes , Xenoenxertos , Masculino , Primatas , Receptores da Somatotropina/genética , Sus scrofa , Suínos , Transplante Heterólogo
11.
Proc Natl Acad Sci U S A ; 115(4): 708-713, 2018 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-29311328

RESUMO

Genetically engineered pigs play an indispensable role in the study of rare monogenic diseases. Pigs harboring a gene responsible for a specific disease can be efficiently generated via somatic cell cloning. The generation of somatic cell-cloned pigs from male cells with mutation(s) in an X chromosomal gene is a reliable and straightforward method for reproducing X-linked genetic diseases (XLGDs) in pigs. However, the severe symptoms of XLGDs are often accompanied by impaired growth and reproductive disorders, which hinder the reproduction of these valuable model animals. Here, we generated unique chimeric boars composed of mutant cells harboring a lethal XLGD and normal cells. The chimeric boars exhibited the cured phenotype with fertility while carrying and transmitting the genotype of the XLGD. This unique reproduction system permits routine production of XLGD model pigs through the male-based breeding, thereby opening an avenue for translational research using disease model pigs.


Assuntos
Técnicas de Cultura Embrionária/métodos , Doenças Genéticas Ligadas ao Cromossomo X/genética , Reprodução/genética , Animais , Animais Geneticamente Modificados/genética , Cruzamento , Quimera , Clonagem de Organismos/métodos , Modelos Animais de Doenças , Fertilidade , Técnicas de Inativação de Genes/métodos , Engenharia Genética/métodos , Masculino , Técnicas de Transferência Nuclear , Suínos/genética
12.
Xenotransplantation ; 27(5): e12585, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32056300

RESUMO

The activation of the endothelial surface in xenografts is still a poorly understood process and the consequences are unpredictable. The role of Ca2+ -messaging during the activation of endothelial cells is well recognized and routinely measured by synthetic Ca2+ -sensitive fluorophors. However, these compounds require fresh loading immediately before each experiment and in particular when grown in state-of-the-art 3D cell culture systems, endothelial cells are difficult to access with such sensors. Therefore, we developed transgenic pigs expressing a Ca2+ -sensitive protein and examined its principal characteristics. Primary transgenic endothelial cells stimulated by ATP showed a definite and short influx of Ca2+ into the cytosol, whereas exposure to human serum resulted in a more intense and sustained response. Surprisingly, not all endothelial cells reacted identically to a stimulus, rather activation took place in adjacent cells in a timely decelerated way and with distinct intensities. This effect was again more pronounced when cells were stimulated with human serum. Finally, we show clear evidence that antibody binding alone significantly activated endothelial cells, whereas antibody depletion dramatically reduced the stimulatory potential of serum. Transgenic porcine endothelial cells expressing a Ca2+ -sensor represent an interesting tool to dissect factors inducing activation of porcine endothelial cells after exposure to human blood or serum.


Assuntos
Sinalização do Cálcio , Células Endoteliais , Soro , Animais , Animais Geneticamente Modificados , Cálcio , Células Cultivadas , Células Endoteliais/citologia , Humanos , Suínos , Transplante Heterólogo
13.
Xenotransplantation ; 27(1): e12560, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31591751

RESUMO

BACKGROUND: Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are α-1,3 Gal (Galactose-alpha-1,3-galactose) causing hyperacute rejection, also Neu5Gc (N-glycolylneuraminic acid) and Sd(a) blood group antigens both of which are likely to elicit acute vascular rejection given the known human immune status. Porcine cells with knockouts of the three genes responsible, GGTA1, CMAH and B4GALNT2, revealed minimal xenoreactive antibody binding after incubation with human serum. However, human leucocyte antigen (HLA) antibodies cross-reacted with swine leucocyte antigen class I (SLA-I). We previously demonstrated efficient generation of pigs with multiple xeno-transgenes placed at a single genomic locus. Here we wished to assess whether key xenoreactive antigen genes can be simultaneously inactivated and if combination with the multi-transgenic background further reduces antibody deposition and complement activation. METHODS: Multiplex CRISPR/Cas9 gene editing and somatic cell nuclear transfer were used to generate pigs carrying functional knockouts of GGTA1, CMAH, B4GALNT2 and SLA class I. Fibroblasts derived from one- to four-fold knockout animals, and from multi-transgenic cells (human CD46, CD55, CD59, HO1 and A20) with the four-fold knockout were used to examine the effects on human IgG and IgM binding or complement activation in vitro. RESULTS: Pigs were generated carrying four-fold knockouts of important xenoreactive genes. In vitro assays revealed that combination of all four gene knockouts reduced human IgG and IgM binding to porcine kidney cells more effectively than single or double knockouts. The multi-transgenic background combined with GGTA1 knockout alone reduced C3b/c and C4b/c complement activation to such an extent that further knockouts had no significant additional effect. CONCLUSION: We showed that pigs carrying several xenoprotective transgenes and knockouts of xenoreactive antigens can be readily generated and these modifications will have significant effects on xenograft survival.


Assuntos
Galactosiltransferases/genética , Rejeição de Enxerto/imunologia , Transplante de Rim , Oxigenases de Função Mista/genética , N-Acetilgalactosaminiltransferases/genética , Animais , Anticorpos Heterófilos/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Proteínas do Sistema Complemento/metabolismo , Antígenos HLA/imunologia , Xenoenxertos/imunologia , Antígenos de Histocompatibilidade Classe I , Humanos , Suínos , Transplante Heterólogo
14.
Reprod Fertil Dev ; 31(4): 820-826, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30384878

RESUMO

Immunosurgical isolation of the inner cell mass (ICM) from blastocysts is based on complement-mediated lysis of antibody-coated trophectoderm (TE) cells. Conventionally, anti-species antisera, containing antibodies against multiple undefined TE-cell epitopes, have been used as the antibody source. We previously generated α-1,3-galactosyltransferase deficient (GTKO) pigs to prevent hyperacute rejection of pig-to-primate xenotransplants. Since GTKO pigs lack galactosyl-α-1,3-galactose (αGal) but are exposed to this antigen (e.g. αGal on gut bacteria), they produce anti-αGal antibodies. In this study, we examined whether serum from GTKO pigs could be used as a novel antibody source for multi-species embryo immunosurgery. Mouse, rabbit, pig and cattle blastocysts were used for the experiment. Expression of αGal epitopes on the surface of TE cells was detected in blastocysts of all species tested. GTKO pig serum contained sufficient anti-αGal antibodies to induce complement-mediated lysis of TE cells in blastocysts from all species investigated. Intact ICMs could be successfully recovered and the majority showed the desired level of purity. Our study demonstrates that GTKO pig serum is a reliable and effective source of antibodies targeting the αGal epitopes of TE cells for multi-species embryo immunosurgery.


Assuntos
Blastocisto/imunologia , Epitopos , Galactose/imunologia , Animais , Bovinos , Camundongos , Coelhos , Suínos
15.
Xenotransplantation ; 25(4): e12449, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30264883

RESUMO

For clinical xenotransplantation, transplants must be free of porcine cytomegalovirus (PCMV). Piglets become infected primarily in the perinatal period by the mother sow. While individual donor animals can be protected from infection by isolation husbandry, success is not guaranteed and this strategy poses the risk of undetected infections and raises animal welfare questions. Here, we present the establishment of a completely PCMV-negative pig herd for breeding donor animals for xenotransplantation. Eleven pregnant DanAvl Basic hybrid sows were purchased from a designated pathogen-free (DPF), PCMV-positive colony and transferred to a new pig facility at the Centre for Innovative Medical Models (CiMM) 4 weeks prior to farrowing. At the age of 24 hours, piglets were early-weaned and transferred to a commercially available Rescue Deck system dedicated to motherless rearing of piglets. Sows were removed from the facility. The PCMV status of F1-generation animals was determined at regular intervals over a period of 14 months by a sensitive real-time PCR-based detection method testing blood, nasal swabs and cultured peripheral blood mononuclear cells (PBMCs). F1 sows were used as recipients of genetically modified embryos to generate a xenotransplant donor herd. Offspring were tested for PCMV accordingly. All offspring have remained PCMV negative over the whole observation period of 14 months. A completely PCMV-negative pig herd for xenotransplantation has thus been successfully established.


Assuntos
Infecções por Citomegalovirus , Citomegalovirus/genética , Leucócitos Mononucleares/virologia , Transplante Heterólogo , Animais , Animais Geneticamente Modificados , Citomegalovirus/isolamento & purificação , Xenoenxertos/virologia , Suínos , Doadores de Tecidos , Desmame
16.
Xenotransplantation ; 25(2): e12382, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29359453

RESUMO

BACKGROUND: Multiple xenoprotective transgenes are best grouped at a single locus to avoid segregation during breeding and simplify production of donor animals. METHODS: We used transgene stacking to place a human CD55 transgene adjacent to a human heme oxygenase 1 construct at the porcine ROSA26 locus. A transgenic pig was analyzed by PCR, RT-PCR, droplet digital PCR, immunohistochemistry, immunofluorescence, and flow cytometry. Resistance to complement-mediated cell lysis and caspase 3/7 activation were determined in vitro. RESULTS: The ROSA26 locus was retargeted efficiently, and animals were generated by nuclear transfer. RNA and protein analyses revealed abundant expression in all organs analyzed, including pancreatic beta cells. Transgenic porcine kidney fibroblasts were almost completely protected against complement-mediated lysis and showed reduced caspase 3/7 activation. CONCLUSION: Step-by-step placement enables highly expressed single-copy xenoprotective transgenes to be grouped at porcine ROSA26.


Assuntos
Células Secretoras de Insulina/citologia , Transplante Heterólogo , Animais , Animais Geneticamente Modificados/genética , Antígenos CD55/genética , Antígenos CD59/genética , Fibroblastos/citologia , Loci Gênicos , Heme Oxigenase-1/genética , Humanos , Regiões Promotoras Genéticas/genética , Suínos , Transgenes/genética , Transplante Heterólogo/métodos
17.
Transgenic Res ; 26(2): 309-318, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27943082

RESUMO

Due to a rising demand of porcine models with complex genetic modifications for biomedical research, the approaches for their generation need to be adapted. In this study we describe the direct introduction of a gene construct into the pronucleus (PN)-like structure of cloned embryos as a novel strategy for the generation of genetically modified pigs, termed "nuclear injection". To evaluate the reliability of this new strategy, the developmental ability of embryos in vitro and in vivo as well as the integration and expression efficiency of a transgene carrying green fluorescence protein (GFP) were examined. Eighty percent of the cloned pig embryos (633/787) exhibited a PN-like structure, which met the prerequisite to technically perform the new method. GFP fluorescence was observed in about half of the total blastocysts (21/40, 52.5%), which was comparable to classical zygote PN injection (28/41, 68.3%). In total, 478 cloned embryos injected with the GFP construct were transferred into 4 recipients and from one recipient 4 fetuses (day 68) were collected. In one of the fetuses which showed normal development, the integration of the transgene was confirmed by PCR in different tissues and organs from all three primary germ layers and placenta. The integration pattern of the transgene was mosaic (48 out of 84 single-cell colonies established from a kidney were positive for GFP DNA by PCR). Direct GFP fluorescence was observed macro- and microscopically in the fetus. Our novel strategy could be useful particularly for the generation of pigs with complex genetic modifications.


Assuntos
Animais Geneticamente Modificados/genética , Núcleo Celular/genética , Transgenes/genética , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Clonagem de Organismos/métodos , DNA/genética , Transferência Embrionária/métodos , Proteínas de Fluorescência Verde/genética , Técnicas de Transferência Nuclear , Suínos , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismo
18.
Hum Mol Genet ; 22(21): 4368-82, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23784375

RESUMO

Duchenne muscular dystrophy (DMD) is caused by mutations in the X-linked dystrophin (DMD) gene. The absence of dystrophin protein leads to progressive muscle weakness and wasting, disability and death. To establish a tailored large animal model of DMD, we deleted DMD exon 52 in male pig cells by gene targeting and generated offspring by nuclear transfer. DMD pigs exhibit absence of dystrophin in skeletal muscles, increased serum creatine kinase levels, progressive dystrophic changes of skeletal muscles, impaired mobility, muscle weakness and a maximum life span of 3 months due to respiratory impairment. Unlike human DMD patients, some DMD pigs die shortly after birth. To address the accelerated development of muscular dystrophy in DMD pigs when compared with human patients, we performed a genome-wide transcriptome study of biceps femoris muscle specimens from 2-day-old and 3-month-old DMD and age-matched wild-type pigs. The transcriptome changes in 3-month-old DMD pigs were in good concordance with gene expression profiles in human DMD, reflecting the processes of degeneration, regeneration, inflammation, fibrosis and impaired metabolic activity. In contrast, the transcriptome profile of 2-day-old DMD pigs showed similarities with transcriptome changes induced by acute exercise muscle injury. Our studies provide new insights into early changes associated with dystrophin deficiency in a clinically severe animal model of DMD.


Assuntos
Distrofina/genética , Distrofina/metabolismo , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/fisiopatologia , Distrofia Muscular de Duchenne/fisiopatologia , Envelhecimento , Animais , Peso ao Nascer , Distrofina/deficiência , Éxons , Feminino , Marcação de Genes , Humanos , Masculino , Músculo Esquelético/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Técnicas de Transferência Nuclear , Fenótipo , Deleção de Sequência , Estresse Mecânico , Suínos , Transcriptoma
19.
Transgenic Res ; 24(3): 509-17, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25716163

RESUMO

Oncogenic mutations of KRAS play a major role in human carcinogenesis. Here we describe viable gene-targeted pigs carrying a latent KRAS (G12D) mutant allele that can be activated by Cre recombination. These have been produced as part of a program to model human cancers in pigs by replicating genetic lesions known to initiate and drive human disease. Cre-activated KRAS (G12D) animals add to a growing set of gene-targeted pigs that includes a Cre-activated oncogenic mutant TP53, a Cre-responsive dual fluorescent reporter and two truncating mutations of APC (adenomatous polyposis coli). These alleles can be combined and activated in various tissues to produce new models for cancer research.


Assuntos
Marcação de Genes/métodos , Mutação , Proteínas Proto-Oncogênicas/genética , Sus scrofa/genética , Proteínas ras/genética , Animais , Feminino , Integrases/genética , Células-Tronco Mesenquimais/fisiologia , Técnicas de Transferência Nuclear
20.
Gastroenterology ; 143(5): 1173-1175.e7, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22864254

RESUMO

We created gene-targeted pigs with mutations in the adenomatous polyposis coli (APC) gene (APC) that are orthologous to those responsible for human familial adenomatous polyposis (FAP). One-year-old pigs with the APC(1311) mutation (orthologous to human APC(1309)) have aberrant crypt foci and low- and high-grade dysplastic adenomas in the large intestine, similar to the precancerous lesions that develop in patients with FAP. Dysplastic adenomas accumulate ß-catenin and lose heterozygosity of APC. This large-animal, genetic model of FAP will be useful in the development of diagnostics and therapeutics for colorectal cancer. DNA sequence data: NCBI accession number GU951771.


Assuntos
Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Modelos Animais de Doenças , Genes APC , Polipose Adenomatosa do Colo/metabolismo , Animais , Heterozigoto , Humanos , Masculino , Mutação , Suínos , beta Catenina/metabolismo
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