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ABSTRACT: STUDY QUESTION: How does tetraploidy develop in hydatidiform moles (HMs), and what is the frequency of the different origins? SUMMARY ANSWER: Most molar pregnancies with tetraploid cells appear to be produced by somatic endoreduplications, while a minority originate from a tetraploid zygote. The frequency of zygotic tetraploidy was estimated to be 0.7%. WHAT IS KNOWN ALREADY: The parental origin of the genome in tetraploid HMs has only been evaluated in a few cases, most showing three genome sets from the father (PPPM). Estimates of the proportion of HMs that are tetraploid vary between 2 and 28%. STUDY DESIGN, SIZE, DURATION: From 1986 to 2010, unfixed samples of clinically suspected molar pregnancies were forwarded to the Danish Mole Project. For this cohort study 442 samples fulfilled the following criteria for inclusion: macroscopic appearance of HM and ≥10 vesicular chorionic villi with a diameter of ≥1 mm. PARTICIPANTS/MATERIALS, SETTING, METHODS: Of 403 karyotyped samples, 21 cases disclosed ≥2 tetraploid metaphases. The 21 cases were scrutinized by karyotyping, flow cytometry (FC) and DNA-marker analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Among 20 HMs, 3 showed the genotype PPPM: one with the sex chromosomes XXYY and two with XXXY, indicating that they originated in tetraploid zygotes. In 14 androgenetic, one likely androgenetic and two mosaics, the tetraploid cells likely developed by endoreduplications of diploid cells. One case did not fulfil the histopathological criteria for HM. LIMITATIONS, REASONS FOR CAUTION: As an inclusion criterion was the macroscopic observation of vesicular chorionic villi, some non-molar hydropic placentas may have been included and some early moles may have been excluded. WIDER IMPLICATIONS OF THE FINDINGS: In future, studies to determine that an HM is tetraploid and discriminate cases of mosaicism and to deduce the origin of the tetraploidy must use the techniques of karyotyping, DNA-marker analysis and FC in combination. STUDY FUNDING/COMPETING INTEREST(S): No external funding was sought to support this study. None of the authors has any conflict of interest to declare.
Human Reproduction 04/2013; · 4.47 Impact Factor
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Reproduction Fertility and Development 03/2013; · 2.11 Impact Factor
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ABSTRACT: Vitamin D3 analogues are widely used topical and oral remedies for various ailments such as psoriasis, osteoporosis and secondary hyperparathyroidism. In topical treatment, high skin permeability and cellular uptake are key criteria for beneficial effects due to the natural barrier properties of skin. In this study, we wish to establish an in vivo model that allows the comparison of permeability and activity of vitamin D3 analogues in human skin. We generate a bipartite, genetic sensor technology that combines efficient lentivirus-directed gene delivery to xenotransplanted human skin with vitamin D3-induced expression of a luciferase reporter gene and live imaging of animals by bioluminescence imaging. Based on the induction of a transcriptional activator consisting of the vitamin D receptor fused to the Gal4 DNA-binding domain, the vitamin D3-responsive sensor facilitates non-invasive and rapid assessment of permeability and functional properties of vitamin D3 analogues. By topical application of a panel of vitamin D3 analogues onto 'sensorized' human skin, the sensor produces a drug-induced readout with a magnitude and persistence that allow a direct comparative analysis of different analogues. This novel genetic tool has great potential as a non-invasive in vivo screening system for further development and refinement of vitamin D3 analogues.
Experimental Dermatology 03/2013; 22(3):178-183. · 3.54 Impact Factor
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Rozh H Al-Mashhadi,
Charlotte B Sørensen,
Peter M Kragh,
Christina Christoffersen,
Martin B Mortensen,
Lars P Tolbod,
Troels Thim,
Yutao Du,
Juan Li,
Ying Liu, [......],
Mette Schmidt,
Gabor Vajta,
Torben Larsen,
Stig Purup, Lars Bolund,
Lars B Nielsen,
Henrik Callesen,
Erling Falk,
Jacob Giehm Mikkelsen,
Jacob F Bentzon
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ABSTRACT: Lack of animal models with human-like size and pathology hampers translational research in atherosclerosis. Mouse models are missing central features of human atherosclerosis and are too small for intravascular procedures and imaging. Modeling the disease in minipigs may overcome these limitations, but it has proven difficult to induce rapid atherosclerosis in normal pigs by high-fat feeding alone, and genetically modified models similar to those created in mice are not available. D374Y gain-of-function mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene cause severe autosomal dominant hypercholesterolemia and accelerates atherosclerosis in humans. Using Sleeping Beauty DNA transposition and cloning by somatic cell nuclear transfer, we created Yucatan minipigs with liver-specific expression of human D374Y-PCSK9. D374Y-PCSK9 transgenic pigs displayed reduced hepatic low-density lipoprotein (LDL) receptor levels, impaired LDL clearance, severe hypercholesterolemia, and spontaneous development of progressive atherosclerotic lesions that could be visualized by noninvasive imaging. This model should prove useful for several types of translational research in atherosclerosis.
Science translational medicine 01/2013; 5(166):166ra1. · 7.80 Impact Factor
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Martien A M Groenen,
Alan L Archibald,
Hirohide Uenishi,
Christopher K Tuggle,
Yasuhiro Takeuchi,
Max F Rothschild,
Claire Rogel-Gaillard,
Chankyu Park,
Denis Milan,
Hendrik-Jan Megens, [......],
Simon White,
Xun Xu,
Martine Yerle,
Guojie Zhang,
Jianguo Zhang,
Jie Zhang,
Shuhong Zhao,
Jane Rogers,
Carol Churcher,
Lawrence B Schook
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ABSTRACT: For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model.
Nature 11/2012; 491(7424):393-398. · 36.28 Impact Factor
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ABSTRACT: We present a new approach to indel calling which explicitly exploits that indel differences between a reference and a sequenced sample make the mapping of reads less efficient. We assign all unmapped reads with a mapped partner to their expected genomic positions and then perform extensive de novo assembly on the regions with many unmapped reads to resolve homozygous, heterozygous and complex indels by exhaustive traversal of the de Bruijn graph. The method is implemented in the software SOAPindel and provides a list of candidate indels with quality scores. We compare SOAPindel to Dindel, Pindel and GATK on simulated data and find similar or better performance for short indels (<10 bp) and higher sensitivity and specificity for long indels. A validation experiment suggests that SOAPindel has a false positive rate around 10% for long indels (>5 bp) while still providing many more candidate indels than other approaches.
Genome Research 09/2012; · 13.61 Impact Factor
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ABSTRACT: Recombinant adeno-associated virus (rAAV) vectors have been extensively used for experimental gene therapy of inherited human diseases. Several advantages, such as simple vector construction, high targeting frequency by homologous recombination, and applicability to many cell types, make rAAV an attractive approach for targeted genome editing. Combined with cloning by somatic cell nuclear transfer (SCNT), this technology has recently been successfully adapted to generate gene-targeted pigs as models for cystic fibrosis, hereditary tyrosinemia type 1, and breast cancer. This review summarizes the development of rAAV for targeted genome editing in mammalian cells and provides strategies for enhancing the rAAV-mediated targeting frequency by homologous recombination. We discuss current development and application of the rAAV vectors for targeted genome editing in porcine primary fibroblasts, which are subsequently used as donor cells for SCNT to generate cloned genetically designed pigs and provide positive perspectives for the generation of gene-targeted pigs with rAAV in the future.
Journal of Genetics and Genomics 06/2012; 39(6):269-74. · 1.88 Impact Factor
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ABSTRACT: Production of transgenic animals via somatic cell nuclear transfer (SCNT) has been adapted worldwide, but this application is somewhat limited by its relatively low efficiency. In this study, we used handmade cloning (HMC) established previously to produce transgenic pigs that express the functional nematode fat-1 gene. Codon-optimized mfat-1 was inserted into eukaryotic expression vectors, which were transferred into primary swine donor cells. Reverse transcriptase PCR (RT-PCR), gas chromatography, and chromosome analyses were performed to select donor clones capable of converting n-6 into n-3 fatty acids. Blastocysts derived from the clones that lowered the n-6/n-3 ratio to approximately 1:1 were transferred surgically into the uteri of recipients for transgenic piglets. By HMC, 37% (n=558) of reconstructed embryos developed to the blastocyst stage after 7 days of culture in vitro, with an average cell number of 81±36 (n=14). Three recipients became pregnant after 408 day-6 blastocysts were transferred into four naturally cycling females, and a total of 14 live offspring were produced. The nematode mfat-1 effectively lowered the n-6/n-3 ratio in muscle and major organs of the transgenic pig. Our results will help to establish a reliable procedure and an efficient option in the production of transgenic animals.
Cellular reprogramming. 06/2012; 14(3):258-66.
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Jian Li,
Kai Wang,
Fei Gao,
Thomas D Jensen,
Shengting T Li,
Paula M DeAngelis,
Steen Kølvraa,
Charlotte Proby,
Ola Forslund, Lars Bolund,
Ole Petter F Clausen
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ABSTRACT: Keratoacanthoma (KA) is a benign keratinocytic neoplasm that spontaneously regresses after 3-6 months and shares features with squamous cell carcinomas (SCCs). Furthermore, there are reports of KAs that have metastasized, invoking the question of whether KA is a variant of SCC (Hodak et al., 1993). To date, no reported criteria are sensitive enough to discriminate reliably between KA and SCC, and consequently there is a clinical need for discriminating markers. Our previous study analyzed 132 KAs and 29 SCCs and revealed significantly different regions of genomic aberrations using chromosomal comparative genomic hybridization (CGH). In the present study, we applied array CGH to investigate 98 KAs and 22 SCCs from the above samples. The result shows that all KAs and SCCs have some degree of genetic aberrations. The distribution of numbers of aberrant clones per sample differed significantly between KAs and SCCs (P<0.02), which also demonstrated recurrent aberrations that differed significantly (P<0.001), as illustrated by unsupervised cluster analysis. Classifiers for clinicopathological parameters of KAs were established based on t-test statistics and permutation tests. Tumor size, fibrosis, and inflammation, which are related to the developmental stages of KAs, showed significant (t-test, permutation test) associations with aberrations of selected genomic regions. This suggests chromosomal instability during the whole life cycle of KAs.
Journal of Investigative Dermatology 04/2012; 132(8):2060-6. · 6.31 Impact Factor
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Peter M. Kragh,
Anders Lade Nielsen,
Juan Li,
Yutao Du,
Lin Lin,
Mette Schmidt,
Ingrid Brück Bøgh,
Ida E. Holm,
Jannik E. Jakobsen,
Marianne G. Johansen,
Stig Purup, Lars Bolund,
Gábor Vajta,
Arne Lund Jørgensen
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ABSTRACT: In an effort to develop a porcine model of Alzheimer’s disease we used handmade cloning to produce seven transgenic Göttingen
minipigs. The donor fibroblasts had been stably transfected with a plasmid cassette containing, as transgene, the cDNA of
the neuronal variant of the human amyloid precursor protein gene with the Swedish mutation preceded by beta-globin sequences
to induce splicing and a human PDGFbeta promoter fragment to drive transcription. Transgene insertion had occurred only at
the GLIS3 locus where a single complete copy of the transgene was identified in intronic sequences in opposite direction. Similar and
robust levels of the transgene transcript were detected in skin biopsies from all piglets and the sequence of full-length
transcript was verified. Consistent with PDGFbeta promoter function, high levels of transgene expression, including high level
of the corresponding protein, was observed in brain tissue and not in heart or liver tissues. A rough estimate predicts that
accumulation of the Aβ peptide in the brain may develop at the age of 1–2years.
Transgenic Research 04/2012; 18(4):545-558. · 2.75 Impact Factor
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ABSTRACT: During the last two decades, pigs have been used to develop some of the most important large animal models for biomedical research. Advances in pig genome research, genetic modification (GM) of primary pig cells and pig cloning by nuclear transfer, have facilitated the generation of GM pigs for xenotransplantation and various human diseases. This review summarizes the key technologies used for generating GM pigs, including pronuclear microinjection, sperm-mediated gene transfer, somatic cell nuclear transfer by traditional cloning, and somatic cell nuclear transfer by handmade cloning. Broadly used genetic engineering tools for porcine cells are also discussed. We also summarize the GM pig models that have been generated for xenotransplantation and human disease processes, including neurodegenerative diseases, cardiovascular diseases, eye diseases, bone diseases, cancers and epidermal skin diseases, diabetes mellitus, cystic fibrosis, and inherited metabolic diseases. Thus, this review provides an overview of the progress in GM pig research over the last two decades and perspectives for future development.
Journal of Inherited Metabolic Disease 03/2012; 35(4):695-713. · 3.58 Impact Factor
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Yong Hou,
Luting Song,
Ping Zhu,
Bo Zhang,
Ye Tao,
Xun Xu,
Fuqiang Li,
Kui Wu,
Jie Liang,
Di Shao, [......],
Zuhong Lu,
Ning Gu,
Goodman Laurie, Lars Bolund,
Karsten Kristiansen,
Jian Wang,
Huanming Yang,
Yingrui Li,
Xiuqing Zhang,
Jun Wang
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ABSTRACT: Tumor heterogeneity presents a challenge for inferring clonal evolution and driver gene identification. Here, we describe a method for analyzing the cancer genome at a single-cell nucleotide level. To perform our analyses, we first devised and validated a high-throughput whole-genome single-cell sequencing method using two lymphoblastoid cell line single cells. We then carried out whole-exome single-cell sequencing of 90 cells from a JAK2-negative myeloproliferative neoplasm patient. The sequencing data from 58 cells passed our quality control criteria, and these data indicated that this neoplasm represented a monoclonal evolution. We further identified essential thrombocythemia (ET)-related candidate mutations such as SESN2 and NTRK1, which may be involved in neoplasm progression. This pilot study allowed the initial characterization of the disease-related genetic architecture at the single-cell nucleotide level. Further, we established a single-cell sequencing method that opens the way for detailed analyses of a variety of tumor types, including those with high genetic complex between patients.
Cell 03/2012; 148(5):873-85. · 32.40 Impact Factor
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Xun Xu,
Yong Hou,
Xuyang Yin,
Li Bao,
Aifa Tang,
Luting Song,
Fuqiang Li,
Shirley Tsang,
Kui Wu,
Hanjie Wu, [......],
Ning Gu,
Xiuqing Zhang,
Laurie Goodman, Lars Bolund,
Jian Wang,
Huanming Yang,
Karsten Kristiansen,
Michael Dean,
Yingrui Li,
Jun Wang
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ABSTRACT: Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer and has very few mutations that are shared between different patients. To better understand the intratumoral genetics underlying mutations of ccRCC, we carried out single-cell exome sequencing on a ccRCC tumor and its adjacent kidney tissue. Our data indicate that this tumor was unlikely to have resulted from mutations in VHL and PBRM1. Quantitative population genetic analysis indicates that the tumor did not contain any significant clonal subpopulations and also showed that mutations that had different allele frequencies within the population also had different mutation spectrums. Analyses of these data allowed us to delineate a detailed intratumoral genetic landscape at a single-cell level. Our pilot study demonstrates that ccRCC may be more genetically complex than previously thought and provides information that can lead to new ways to investigate individual tumors, with the aim of developing more effective cellular targeted therapies.
Cell 03/2012; 148(5):886-95. · 32.40 Impact Factor
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Yingrui Li,
Xun Xu,
Luting Song,
Yong Hou,
Zesong Li,
Shirley Tsang,
Fuqiang Li,
Kate McGee Im,
Kui Wu,
Hanjie Wu, [......],
Jingxiang Li,
Wen Wang,
Zuhong Lu,
Xiuqing Zhang, Lars Bolund,
Karsten Kristiansen,
Jian Wang,
Huanming Yang,
Michael Dean,
Jun Wang
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ABSTRACT: Cancers arise through an evolutionary process in which cell populations are subjected to selection; however, to date, the process of bladder cancer, which is one of the most common cancers in the world, remains unknown at a single-cell level.
We carried out single-cell exome sequencing of 66 individual tumor cells from a muscle-invasive bladder transitional cell carcinoma (TCC). Analyses of the somatic mutant allele frequency spectrum and clonal structure revealed that the tumor cells were derived from a single ancestral cell, but that subsequent evolution occurred, leading to two distinct tumor cell subpopulations. By analyzing recurrently mutant genes in an additional cohort of 99 TCC tumors, we identified genes that might play roles in the maintenance of the ancestral clone and in the muscle-invasive capability of subclones of this bladder cancer, respectively.
This work provides a new approach of investigating the genetic details of bladder tumoral changes at the single-cell level and a new method for assessing bladder cancer evolution at a cell-population level.
GigaScience. 01/2012; 1(1):12.
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Xiaodong Fang,
Yulian Mou,
Zhiyong Huang,
Yong Li,
Lijuan Han,
Yanfeng Zhang,
Yue Feng,
Yuanxin Chen,
Xuanting Jiang,
Wei Zhao, [......],
Liangxue Lai,
Guojie Zhang,
Yingrui Li,
Jun Wang, Lars Bolund,
Huanming Yang,
Jian Wang,
Shutang Feng,
Songgang Li,
Yutao Du
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ABSTRACT: The pig is an economically important food source, amounting to approximately 40% of all meat consumed worldwide. Pigs also serve as an important model organism because of their similarity to humans at the anatomical, physiological and genetic level, making them very useful for studying a variety of human diseases. A pig strain of particular interest is the miniature pig, specifically the Wuzhishan pig (WZSP), as it has been extensively inbred. Its high level of homozygosity offers increased ease for selective breeding for specific traits and a more straightforward understanding of the genetic changes that underlie its biological characteristics. WZSP also serves as a promising means for applications in surgery, tissue engineering, and xenotransplantation. Here, we report the sequencing and analysis of an inbreeding WZSP genome.
Our results reveal some unique genomic features, including a relatively high level of homozygosity in the diploid genome, an unusual distribution of heterozygosity, an over-representation of tRNA-derived transposable elements, a small amount of porcine endogenous retrovirus, and a lack of type C retroviruses. In addition, we carried out systematic research on gene evolution, together with a detailed investigation of the counterparts of human drug target genes.
Our results provide the opportunity to more clearly define the genomic character of pig, which could enhance our ability to create more useful pig models.
GigaScience. 01/2012; 1(1):16.
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Xiaodong Fang,
Yulian Mou,
Zhiyong Huang,
Yong Li,
Lijuan Han,
Yanfeng Zhang,
Yue Feng,
Yuanxin Chen,
Xuanting Jiang,
Wei Zhao, [......],
Liangxue Lai,
Guojie Zhang,
Yingrui Li,
Jun Wang, Lars Bolund,
Huanming Yang,
Jian Wang,
Shutang Feng,
Songgang Li,
Yutao Du
GigaScience. 01/2012;
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Nicklas Heine Staunstrup,
Johannes Madsen,
Maria Nascimento Primo,
Juan Li,
Ying Liu,
Peter M Kragh,
Rong Li,
Mette Schmidt,
Stig Purup,
Frederik Dagnæs-Hansen,
Lars Svensson,
Thomas K Petersen,
Henrik Callesen, Lars Bolund,
Jacob Giehm Mikkelsen
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ABSTRACT: Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or β1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human β1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to severe skin damage-induced inflammation and should be of great potential in studies aiming at the development and refinement of topical therapies for cutaneous inflammation including psoriasis.
PLoS ONE 01/2012; 7(5):e36658. · 4.09 Impact Factor
