Papers by Andrea Lucas-Hahn
Theriogenology, 1994
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The viral disease African swine fever (ASF) has spread in Eastern Europe over the last years. The... more The viral disease African swine fever (ASF) has spread in Eastern Europe over the last years. The virus causes up to 100 % mortality in domestic pigs and is a potential threat to the European pork industry. Efforts to eradicate or cure the disease with vaccines or antiviral therapy have been unsuccessful. Therefore, new approaches to treat the disease are frequently sought. Genome editing applying the CRISPR/Cas9 system has become a standardized tool in research and also qualifies to address ASF. By integrating Cas9 into the porcine genome; Cas9 may act upon infection and inhibit virus replication. Previous in vitro experiments in wild boar lung cells targeting the p30 gene showed resistance towards a European strain of ASF. p30 is expressed by CP204L and is critical for virus replication. However, due to variation in the CP204L sequence no resistance was found towards a Kenyan strain (Hübner et al., 2018). The study aims to further explore the feasibility to integrate Cas9 into the...
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Mit dem Klonschaf "Dolly" ist ein langjahriges Dogma der Biologie gefallen, nach dem ei... more Mit dem Klonschaf "Dolly" ist ein langjahriges Dogma der Biologie gefallen, nach dem eine differenzierte Zelle nicht mehr in einen "embryonalen" (pluripotenten) Zustand zuruckprogrammiert werden kann. Die mit dem erfolgreichen Klonen beim Saugetier verbundenen viefaltigen Anwendungsperspektiven in Landwirtschaft und Medizin haben zu einer breiten, haufig kontroversen offentlichen Diskussion gefuhrt. Der aktuelle Entwicklungsstand des Klonens von Nutztieren und die sich daraus abzeichnenden Anwendungsperspektiven sind Thema dieses Beitrags.
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Reproduction, Fertility and Development, 2022
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Scientific Reports, 2020
In modern livestock farming horned cattle pose an increased risk of injury for each other as well... more In modern livestock farming horned cattle pose an increased risk of injury for each other as well as for the farmers. Dehorning without anesthesia is associated with stress and pain for the calves and raises concerns regarding animal welfare. Naturally occurring structural variants causing polledness are known for most beef cattle but are rare within the dairy cattle population. The most common structural variant in beef cattle consists of a 202 base pair insertion-deletion (Polled Celtic variant). For the generation of polled offspring from a horned Holstein–Friesian bull, we isolated the Polled Celtic variant from the genome of an Angus cow and integrated it into the genome of fibroblasts taken from the horned bull using the CRISPR/Cas12a system (formerly Cpf1). Modified fibroblasts served as donor cells for somatic cell nuclear transfer and reconstructed embryos were transferred into synchronized recipients. One resulting pregnancy was terminated on day 90 of gestation for the ex...
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Cellular Reprogramming, 2020
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Zygote, 2019
SummaryBrilliant cresyl blue (BCB) vital labelling is a powerful method for analyzing the quality... more SummaryBrilliant cresyl blue (BCB) vital labelling is a powerful method for analyzing the quality of porcine cumulus–oocyte complexes. Our aim was to investigate the correlation between the selection of porcine oocytes using BCB labelling and selected intranuclear characteristics of porcine oocytes and parthenotes. Moreover, BCB labelling was correlated with the diameter of the oocyte and the developmental potential of the parthenotes. The following methods were used: BCB labelling, measurement of the diameter of the oocyte, parthenogenetic activation, immunocytochemistry, transmission electron microscopy, enucleation and relative protein concentration (RPC) analysis. We determined that the diameter of the oocytes in the BCB-positive (BCB+) group was significantly larger than in the BCB-negative (BCB−) group. Immediately after oocyte selection according to BCB labelling, we found significant difference in chromatin configuration between the analyzed groups. BCB+ oocytes were signifi...
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Xenotransplantation, 2019
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Xenotransplantation, 2018
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Zygote, 2018
SummaryThe present study examines the role of RNA polymerase I (RPI)-mediated transcription, mate... more SummaryThe present study examines the role of RNA polymerase I (RPI)-mediated transcription, maternally inherited rRNA and nucleolar proteins in the resumption of fibrillogranular nucleoli during embryonic genome activation (EGA) in porcine embryos. Late 4-cell embryos were incubated in the absence (control) or presence of actinomycin D (AD) (0.2 μg/ml for inhibition of RPI; 2.0 μg/ml for inhibition of total transcription) and late 2-cell embryos were cultured to the late 4-cell stage with 0.2 μg/ml AD to block EGA. Embryos were then processed for reverse-transcriptase polymerase chain reaction (RT-PCR), and for autoradiography (ARG), transmission electron microscopy (TEM), fluorescence in situ hybridization (FISH), silver staining and immunofluorescence (for RPI). Embryos in the control group displayed extranucleolar and intranucleolar ARG labelling, and exhibited de novo synthesis of rRNA and reticulated functional nucleoli. Nucleolar proteins were located in large foci. After RPI...
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Reproduction, Fertility and Development, 2018
Recently (Spate et al. 2017 Reprod. Fertil. Dev. 29, 150), a new medium [TCM-199 supplemented wit... more Recently (Spate et al. 2017 Reprod. Fertil. Dev. 29, 150), a new medium [TCM-199 supplemented with hCG 10 IU, pregnant mare serum gonadotropin (PMSG) 10 IU mL−1, fibroblast growth factor (FGF) 40 ng mL−1, leukemia inhibitory factor (LIF) 2000 U mL−1, IGF-1 20 ng mL−1, epidermal growth factor (EGF) 10 ng mL−1], termed FLI medium, was demonstrated to improve porcine oocyte maturation in vitro. The effects on embryo development and quality have not yet been investigated. The purpose of the present study was to compare the FLI medium in porcine in vitro embryo production (IVP) with our standard maturation medium (DMEM supplemented with 10 IU mL−1 PMSG and hCG, 50 ng mL−1 EGF, 100 ng mL−1 IGF1, and 5 ng mL−1 FGF). Briefly, gilt oocytes were collected via aspiration of follicles from abattoir ovaries and matured for 44 h in either FLI or standard DMEM medium at 39°C, 5% CO2 in humidified air. In vitro fertilization was performed with freshly ejaculated sperm (250,000 mL−1) of a multi-tran...
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Reproduction, Fertility and Development, 2016
Induced pluripotent stem cells (iPSCs) are promising for developing novel cell-based medical trea... more Induced pluripotent stem cells (iPSCs) are promising for developing novel cell-based medical treatments. One potential use of iPSCs is for xenotransplantation via production of chimeric organisms. The generation of organs originated from the iPSCs of one individual in a foreign organism could allow the production of immune compatible organ transplants. One of the major problems down this road is to define the different nutrient needs of chimeric embryos (i.e. iPSCs and the host embryos). Here, we evaluated different media for supporting development of chimeric embryos consisting of parthenogenetic porcine embryos and iPS cells, either from pig or nonhuman primate, to identify the optimal medium conditions for pig-monkey aggregation chimeras. First, we cultured 3-day-old porcine parthenogenetic embryos in porcine zygote medium (PZM), iPSC-medium, and mixtures of the two media to identify the most suitable culture conditions. Three-day-old parthenogenetic embryos developed poorly in p...
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Reproduction, Fertility and Development, 2012
The acute vascular rejection (AVR) remains the main hurdle for long-term survival of a porcine xe... more The acute vascular rejection (AVR) remains the main hurdle for long-term survival of a porcine xenograft after transplantation into primates. Immunological reactions and molecular incompatibilities can lead to endothelial activation and microvascular thrombosis. Knock-down of tissue factor (TF) could be a promising mechanism to prevent AVR. Tissue factor is a key molecule of the extrinsic coagulation pathway. It functions as cell surface receptor for coagulation factor VIIa and thereby initiates thrombin formation. Since TF knockout was lethal in the mouse model (Toomey et al. 1996 Blood 88, 1583–1587), we tested different small interfering (si) RNAs in their efficiency to knock down TF in a porcine cell line. Two siRNAs reduced the mRNA level of TF to <3% compared with wild type controls as determined by RT-PCR. Subsequently, porcine fetal fibroblasts (PFF) were co-transfected with constructs coding for one of the most promising siRNAs and a DsRed vector, which confers neomycin ...
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Cryobiology, 1988
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The vast majority of human embryos are aneuploid. Aneuploidy frequently arises during the early m... more The vast majority of human embryos are aneuploid. Aneuploidy frequently arises during the early mitotic divisions of the embryo, but the origin of this remains elusive. Using bovine embryos as a model for human embryos, we identify an error-prone mechanism of parental genome unification which often results in aneuploidy. Surprisingly, genome unification initiates hours before breakdown of the two pronuclei that encapsulate the parental genomes. While still within intact pronuclei, the parental genomes polarize towards each other, in a process driven by centrosomes, dynein, and microtubules. The maternal and paternal chromosomes eventually cluster at the pronuclear interface, in direct proximity to each other. Parental genome clustering often fails however, leading to massive chromosome segregation errors, incompatible with healthy embryo development. Nucleoli, which associate with chromatin, also cluster at the pronuclear interface in human zygotes. Defects in nucleolar clustering c...
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Papers by Andrea Lucas-Hahn