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1.
Dev Biol ; 447(1): 3-13, 2019 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-29391166

RESUMEN

The journey of embryonic development starts at oocyte fertilization, which triggers a complex cascade of events and cellular pathways that guide early embryogenesis. Recent technological advances have greatly expanded our knowledge of cleavage-stage embryo development, which is characterized by an increased rate of whole-chromosome losses and gains, mixoploidy, and atypical cleavage morphokinetics. Embryonic aneuploidy significantly contributes to implantation failure, spontaneous miscarriage, stillbirth or congenital birth defects in both natural and assisted human reproduction. Essentially, early embryo development is strongly determined by maternal factors. Owing to considerable limitations associated with human oocyte and embryo research, the use of animal models is inevitable. However, cellular and molecular mechanisms driving the error-prone early stages of development are still poorly described. In this review, we describe known events that lead to aneuploidy in mammalian oocytes and preimplantation embryos. As the processes of oocyte and embryo development are rigorously regulated by multiple signal-transduction pathways, we explore the putative role of signaling pathways in genomic integrity maintenance. Based on the existing evidence from human and animal data, we investigate whether critical early developmental pathways, like Wnt, Hippo and MAPK, together with distinct DNA damage response and DNA repair pathways can be associated with embryo genomic instability, a question that has, so far, remained largely unexplored.


Asunto(s)
Aneuploidia , Blastocisto , Desarrollo Embrionario , Inestabilidad Genómica , Complicaciones del Embarazo , Animales , Blastocisto/metabolismo , Blastocisto/patología , Femenino , Humanos , Oocitos/metabolismo , Oocitos/patología , Embarazo , Complicaciones del Embarazo/genética , Complicaciones del Embarazo/metabolismo , Complicaciones del Embarazo/patología
2.
Genome Res ; 26(5): 567-78, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-27197242

RESUMEN

Dramatic genome dynamics, such as chromosome instability, contribute to the remarkable genomic heterogeneity among the blastomeres comprising a single embryo during human preimplantation development. This heterogeneity, when compatible with life, manifests as constitutional mosaicism, chimerism, and mixoploidy in live-born individuals. Chimerism and mixoploidy are defined by the presence of cell lineages with different parental genomes or different ploidy states in a single individual, respectively. Our knowledge of their mechanistic origin results from indirect observations, often when the cell lineages have been subject to rigorous selective pressure during development. Here, we applied haplarithmisis to infer the haplotypes and the copy number of parental genomes in 116 single blastomeres comprising entire preimplantation bovine embryos (n = 23) following in vitro fertilization. We not only demonstrate that chromosome instability is conserved between bovine and human cleavage embryos, but we also discovered that zygotes can spontaneously segregate entire parental genomes into different cell lineages during the first post-zygotic cleavage division. Parental genome segregation was not exclusively triggered by abnormal fertilizations leading to triploid zygotes, but also normally fertilized zygotes can spontaneously segregate entire parental genomes into different cell lineages during cleavage of the zygote. We coin the term "heterogoneic division" to indicate the events leading to noncanonical zygotic cytokinesis, segregating the parental genomes into distinct cell lineages. Persistence of those cell lines during development is a likely cause of chimerism and mixoploidy in mammals.


Asunto(s)
Blastocisto/metabolismo , Blastómeros/metabolismo , Linaje de la Célula/fisiología , Quimerismo/embriología , Genoma , Ploidias , Cigoto/metabolismo , Animales , Bovinos , Humanos
3.
BMC Vet Res ; 15(1): 104, 2019 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-30943959

RESUMEN

BACKGROUND: Usage of sexed semen that allows to choose the gender of the calves, is commonly practiced in livestock industry as a profitable breeding alternative, especially in dairy farming. The flow cytometric cell sorting is the only commercially available method for bovine sperm sexing. For validation of the sexing procedure several methods have been developed including sperm fluorescence in situ hybridisation techniques. Latter usually include the use of pre-labelled nucleotides for probe synthesis which is relatively expensive approach compared to combined application of aminoallyl-dUTP and chemical binding of fluorescent dyes. Here a sex determining dual colour bovine sperm fluorescence in situ hybridisation method is presented which is considered more cost-effective technique than the previously reported approaches. RESULTS: The reliability of sex chromosome identifying probes, designed in silico, was proven on bovine metaphase plate chromosomes and through comparison with a commercially available standard method. In the dual colour FISH experiments of unsexed and sexed bovine sperm samples the hybridisation efficiency was at least 98%, whereas the determined sex ratios were not statistically different from the expected. Very few cells carried both of the sex chromosome-specific signals (less than 0.2%). CONCLUSIONS: A protocol for a dual colour bovine sperm FISH method is provided which is cost-effective, simple and fast for sex determination of spermatozoa in bull semen samples.


Asunto(s)
Bovinos , Hibridación Fluorescente in Situ/veterinaria , Semen , Preselección del Sexo/veterinaria , Animales , Citometría de Flujo/métodos , Citometría de Flujo/veterinaria , Hibridación Fluorescente in Situ/métodos , Masculino , Preselección del Sexo/métodos , Espermatozoides
4.
Prenat Diagn ; 39(13): 1262-1268, 2019 12.
Artículo en Alemán | MEDLINE | ID: mdl-31691324

RESUMEN

OBJECTIVE: The study aimed to validate a whole-genome sequencing-based NIPT laboratory method and our recently developed NIPTmer aneuploidy detection software with the potential to integrate the pipeline into prenatal clinical care in Estonia. METHOD: In total, 424 maternal blood samples were included. Analysis pipeline involved cell-free DNA extraction, library preparation and massively parallel sequencing on Illumina platform. Aneuploidies were determined with NIPTmer software, which is based on counting pre-defined per-chromosome sets of unique k-mers from sequencing raw data. SeqFF was implemented to estimate cell-free fetal DNA (cffDNA) fraction. RESULTS: NIPTmer identified correctly all samples of non-mosaic trisomy 21 (T21, 15/15), T18 (9/9), T13 (4/4) and monosomy X (4/4) cases, with the 100% sensitivity. However, one mosaic T18 remained undetected. Six false-positive (FP) results were observed (FP rate of 1.5%, 6/398), including three for T18 (specificity 99.3%) and three for T13 (specificity 99.3%). The level of cffDNA of <4% was estimated in eight samples, including one sample with T13 and T18. Despite low cffDNA level, these two samples were determined as aneuploid. CONCLUSION: We believe that the developed NIPT method can successfully be used as a universal primary screening test in combination with ultrasound scan for the first trimester fetal examination.


Asunto(s)
Aneuploidia , Pruebas Prenatales no Invasivas/estadística & datos numéricos , Aberraciones Cromosómicas Sexuales , Programas Informáticos , Estonia , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Pruebas Prenatales no Invasivas/métodos , Embarazo , Salud Pública
5.
Hum Reprod ; 32(11): 2348-2357, 2017 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-29040498

RESUMEN

STUDY QUESTION: Is the rate and nature of chromosome instability (CIN) similar between bovine in vivo-derived and in vitro-cultured cleavage-stage embryos? SUMMARY ANSWER: There is a major difference regarding chromosome stability of in vivo-derived and in vitro-cultured embryos, as CIN is significantly lower in in vivo-derived cleavage-stage embryos compared to in vitro-cultured embryos. WHAT IS KNOWN ALREADY: CIN is common during in vitro embryogenesis and is associated with early embryonic loss in humans, but the stability of in vivo-conceived cleavage-stage embryos remains largely unknown. STUDY DESIGN, SIZE, DURATION: Because human in vivo preimplantation embryos are not accessible, bovine (Bos taurus) embryos were used to study CIN in vivo. Five young, healthy, cycling Holstein Friesian heifers were used to analyze single blastomeres of in vivo embryos, in vitro embryos produced by ovum pick up with ovarian stimulation (OPU-IVF), and in vitro embryos produced from in vitro matured oocytes retrieved without ovarian stimulation (IVM-IVF). PARTICIPANTS/MATERIALS, SETTING, METHODS: Single blastomeres were isolated from embryos, whole-genome amplified and hybridized on Illumina BovineHD BeadChip arrays together with the bulk DNA from the donor cows (mothers) and the bull (father). DNA was also obtained from the parents of the bull and from the parents of the cows (paternal and maternal grandparents, respectively). Subsequently, genome-wide haplotyping and copy-number profiling was applied to investigate the genomic architecture of 171 single bovine blastomeres of 16 in vivo, 13 OPU-IVF and 13 IVM-IVF embryos. MAIN RESULTS AND THE ROLE OF CHANCE: The genomic stability of single blastomeres in both of the in vitro-cultured embryo cohorts was severely compromised (P < 0.0001), and the frequency of whole chromosome or segmental aberrations was higher in embryos produced in vitro than in embryos derived in vivo. Only 18.8% of in vivo-derived embryos contained at least one blastomere with chromosomal anomalies, compared to 69.2% of OPU-IVF embryos (P < 0.01) and 84.6% of IVM-IVF embryos (P < 0.001). LARGE SCALE DATA: Genotyping data obtained in this study has been submitted to NCBI Gene Expression Omnibus (GEO; accession number GSE95358). LIMITATIONS REASONS FOR CAUTION: There were two main limitations of the study. First, animal models may not always reflect the nature of human embryogenesis, although the use of an animal model to investigate CIN was unavoidable in our study. Second, a limited number of embryos were obtained, therefore more studies are warranted to corroborate the findings. WIDER IMPLICATIONS OF THE FINDINGS: Although CIN is also present in in vivo-developed embryos, in vitro procedures exacerbate chromosomal abnormalities during early embryo development. Hence, the present study highlights that IVF treatment compromises embryo viability and should be applied with care. Additionally, our results encourage to refine and improve in vitro culture conditions and assisted reproduction technologies. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Agency for Innovation by Science and Technology (IWT) (TBM-090878 to J.R.V. and T.V.), the Research Foundation Flanders (FWO; G.A093.11 N to T.V. and J.R.V. and G.0392.14 N to A.V.S. and J.R.V.), the European Union's FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, SARM, EU324509 to J.R.V., T.V., O.T, A.D., A.S. and A.K.) and Horizon 2020 innovation programme (WIDENLIFE, 692065 to J.R.V., O.T., T.V., A.K. and A.S.). M.Z.E., J.R.V. and T.V. are co-inventors on a patent application ZL913096-PCT/EP2014/068315-WO/2015/028576 ('Haplotyping and copy-number typing using polymorphic variant allelic frequencies'), licensed to Cartagenia (Agilent Technologies).


Asunto(s)
Blastocisto/metabolismo , Técnicas de Cultivo de Embriones/veterinaria , Inestabilidad Genómica/fisiología , Animales , Blastómeros/fisiología , Bovinos , Femenino , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Inducción de la Ovulación/veterinaria
6.
Nature ; 478(7367): 97-102, 2011 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-21881559

RESUMEN

Both obesity and being underweight have been associated with increased mortality. Underweight, defined as a body mass index (BMI) ≤ 18.5 kg per m(2) in adults and ≤ -2 standard deviations from the mean in children, is the main sign of a series of heterogeneous clinical conditions including failure to thrive, feeding and eating disorder and/or anorexia nervosa. In contrast to obesity, few genetic variants underlying these clinical conditions have been reported. We previously showed that hemizygosity of a ∼600-kilobase (kb) region on the short arm of chromosome 16 causes a highly penetrant form of obesity that is often associated with hyperphagia and intellectual disabilities. Here we show that the corresponding reciprocal duplication is associated with being underweight. We identified 138 duplication carriers (including 132 novel cases and 108 unrelated carriers) from individuals clinically referred for developmental or intellectual disabilities (DD/ID) or psychiatric disorders, or recruited from population-based cohorts. These carriers show significantly reduced postnatal weight and BMI. Half of the boys younger than five years are underweight with a probable diagnosis of failure to thrive, whereas adult duplication carriers have an 8.3-fold increased risk of being clinically underweight. We observe a trend towards increased severity in males, as well as a depletion of male carriers among non-medically ascertained cases. These features are associated with an unusually high frequency of selective and restrictive eating behaviours and a significant reduction in head circumference. Each of the observed phenotypes is the converse of one reported in carriers of deletions at this locus. The phenotypes correlate with changes in transcript levels for genes mapping within the duplication but not in flanking regions. The reciprocal impact of these 16p11.2 copy-number variants indicates that severe obesity and being underweight could have mirror aetiologies, possibly through contrasting effects on energy balance.


Asunto(s)
Índice de Masa Corporal , Cromosomas Humanos Par 16/genética , Dosificación de Gen/genética , Obesidad/genética , Fenotipo , Delgadez/genética , Adolescente , Adulto , Anciano , Envejecimiento , Estatura/genética , Estudios de Casos y Controles , Niño , Preescolar , Estudios de Cohortes , Hibridación Genómica Comparativa , Discapacidades del Desarrollo/genética , Metabolismo Energético/genética , Europa (Continente) , Femenino , Duplicación de Gen/genética , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo , Cabeza/anatomía & histología , Heterocigoto , Humanos , Lactante , Recién Nacido , Masculino , Trastornos Mentales/genética , Persona de Mediana Edad , Mutación/genética , América del Norte , ARN Mensajero/análisis , ARN Mensajero/genética , Eliminación de Secuencia/genética , Transcripción Genética , Adulto Joven
7.
BMC Genomics ; 16: 703, 2015 Sep 16.
Artículo en Inglés | MEDLINE | ID: mdl-26376747

RESUMEN

BACKGROUND: Somatic mosaicism denotes the presence of genetically distinct populations of somatic cells in one individual who has developed from a single fertilised oocyte. Mosaicism may result from a mutation that occurs during postzygotic development and is propagated to only a subset of the adult cells. Our aim was to investigate both somatic mosaicism for copy-neutral loss of heterozygosity (cn-LOH) events and DNA copy number variations (CNVs) in fully differentiated tissues. RESULTS: We studied panels of tissue samples (11-12 tissues per individual) from four autopsy subjects using high-resolution Illumina HumanOmniExpress-12 BeadChips to reveal the presence of possible intra-individual tissue-specific cn-LOH and CNV patterns. We detected five mosaic cn-LOH regions >5 Mb in some tissue samples in three out of four individuals. We also detected three CNVs that affected only a portion of the tissues studied in one out of four individuals. These three somatic CNVs range from 123 to 796 kb and are also found in the general population. An attempt was made to explain the succession of genomic events that led to the observed somatic genetic mosaicism under the assumption that the specific mosaic patterns of CNV and cn-LOH changes reflect their formation during the postzygotic embryonic development of germinal layers and organ systems. CONCLUSIONS: Our results give further support to the idea that somatic mosaicism for CNVs, and also cn-LOHs, is a common phenomenon in phenotypically normal humans. Thus, the examination of only a single tissue might not provide enough information to diagnose potentially deleterious CNVs within an individual. During routine CNV and cn-LOH analysis, DNA derived from a buccal swab can be used in addition to blood DNA to get information about the CNV/cn-LOH content in tissues of both mesodermal and ectodermal origin. Currently, the real frequency and possible phenotypic consequences of both CNVs and cn-LOHs that display somatic mosaicism remain largely unknown. To answer these questions, future studies should involve larger cohorts of individuals and a range of tissues.


Asunto(s)
Variaciones en el Número de Copia de ADN , Genoma Humano , Pérdida de Heterocigocidad , Mosaicismo , Adulto , Autopsia , Femenino , Humanos , Masculino , Persona de Mediana Edad , Especificidad de Órganos/genética , Polimorfismo de Nucleótido Simple
8.
Twin Res Hum Genet ; 17(5): 405-10, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24909117

RESUMEN

Chromosome 17q21.31 microdeletion syndrome is a genomic disorder caused by a recurrent 600 kb long deletion. The deletion affects the region of a common inversion present in about 20% of Europeans. The inversion is associated with the H2 haplotype carrying additional low-copy repeats susceptible to non-allelic homologous recombination, and this haplotype is prone to deletion. No instances of 17q21.31 deletions inherited from an affected parent have been reported, and the deletions always affected a parental chromosome with the H2 haplotype. The syndrome is characterized clinically by intellectual disability, hypotonia, friendly behavior and specific facial dysmorphism with long face, large tubular or pear-shaped nose and bulbous nasal tip. We present monozygotic twin sisters showing the typical clinical picture of the syndrome. The phenotype of the sisters was very similar, with a slightly more severe presentation in Twin B. The 17q21.31 microdeletion was confirmed in both patients but in neither of their parents. Potential copy number differences between the genomes of the twins were subsequently searched using high-resolution single nucleotide polymorphism (SNP) and comparative genome hybridisation (CGH) arrays. However, these analyses identified no additional aberrations or genomic differences that could potentially be responsible for the subtle phenotypic differences. These could possibly be related to the more severe perinatal history of Twin B, or to the variable expressivity of the disorder. In accord with the expectations, one of the parents (the mother) was shown to carry the H2 haplotype, and the maternal allele of chromosome 17q21.31 was missing in the twins.


Asunto(s)
Haplotipos , Discapacidad Intelectual/genética , Polimorfismo de Nucleótido Simple , Gemelos Monocigóticos/genética , Adulto , Deleción Cromosómica , Cromosomas Humanos Par 17/genética , Femenino , Humanos , Discapacidad Intelectual/patología , Masculino , Síndrome de Smith-Magenis
9.
Hum Mol Genet ; 20(10): 1925-36, 2011 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-21349920

RESUMEN

The recently described DNA replication-based mechanisms of fork stalling and template switching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR) were previously shown to catalyze complex exonic, genic and genomic rearrangements. By analyzing a large number of isochromosomes of the long arm of chromosome X (i(Xq)), using whole-genome tiling path array comparative genomic hybridization (aCGH), ultra-high resolution targeted aCGH and sequencing, we provide evidence that the FoSTeS and MMBIR mechanisms can generate large-scale gross chromosomal rearrangements leading to the deletion and duplication of entire chromosome arms, thus suggesting an important role for DNA replication-based mechanisms in both the development of genomic disorders and cancer. Furthermore, we elucidate the mechanisms of dicentric i(Xq) (idic(Xq)) formation and show that most idic(Xq) chromosomes result from non-allelic homologous recombination between palindromic low copy repeats and highly homologous palindromic LINE elements. We also show that non-recurrent-breakpoint idic(Xq) chromosomes have microhomology-associated breakpoint junctions and are likely catalyzed by microhomology-mediated replication-dependent recombination mechanisms such as FoSTeS and MMBIR. Finally, we stress the role of the proximal Xp region as a chromosomal rearrangement hotspot.


Asunto(s)
Cromosomas Humanos X/genética , Replicación del ADN/genética , Isocromosomas/genética , Secuencia de Bases , Rotura Cromosómica , Hibridación Genómica Comparativa , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Polimorfismo Genético , Recombinación Genética , Alineación de Secuencia , Secuencias Repetidas en Tándem/genética
10.
Am J Med Genet A ; 161A(4): 865-70, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23495096

RESUMEN

The 2p15-p16.1 microdeletion syndrome is a novel, rare disorder characterized by developmental delay, intellectual disability, microcephaly, growth retardation, facial abnormalities, and other medical problems. We report here on an 11-year-old female showing clinical features consistent with the syndrome and carrying a de novo 0.45 Mb long deletion of the paternally derived 2p16.1 allele. The deleted region contains only three protein-coding RefSeq genes, BCL11A, PAPOLG, and REL, and one long non-coding RNA gene FLJ16341. Based on close phenotypic similarities with six reported patients showing typical clinical features of the syndrome, we propose that the critical region can be narrowed down further, and that these brain expressed genes can be considered candidates for the features seen in this microdeletion syndrome.


Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 2 , Anomalías Múltiples/diagnóstico , Anomalías Múltiples/genética , Proteínas Portadoras/genética , Niño , Facies , Femenino , Estudios de Asociación Genética , Humanos , Proteínas Nucleares/genética , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas c-rel/genética , Proteínas Represoras , Síndrome
11.
Nat Med ; 29(12): 3233-3242, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37996709

RESUMEN

Pregnancy loss is often caused by chromosomal abnormalities of the conceptus. The prevalence of these abnormalities and the allocation of (ab)normal cells in embryonic and placental lineages during intrauterine development remain elusive. In this study, we analyzed 1,745 spontaneous pregnancy losses and found that roughly half (50.4%) of the products of conception (POCs) were karyotypically abnormal, with maternal and paternal age independently contributing to the increased genomic aberration rate. We applied genome haplarithmisis to a subset of 94 pregnancy losses with normal parental and POC karyotypes. Genotyping of parental DNA as well as POC extra-embryonic mesoderm and chorionic villi DNA, representing embryonic and trophoblastic tissues, enabled characterization of the genomic landscape of both lineages. Of these pregnancy losses, 35.1% had chromosomal aberrations not previously detected by karyotyping, increasing the rate of aberrations of pregnancy losses to 67.8% by extrapolation. In contrast to viable pregnancies where mosaic chromosomal abnormalities are often restricted to chorionic villi, such as confined placental mosaicism, we found a higher degree of mosaic chromosomal imbalances in extra-embryonic mesoderm rather than chorionic villi. Our results stress the importance of scrutinizing the full allelic architecture of genomic abnormalities in pregnancy loss to improve clinical management and basic research of this devastating condition.


Asunto(s)
Aborto Espontáneo , Placenta , Embarazo , Femenino , Humanos , Primer Trimestre del Embarazo/genética , Aborto Espontáneo/genética , Prevalencia , Aberraciones Cromosómicas , Mosaicismo , ADN
12.
BMC Biotechnol ; 11: 17, 2011 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-21356118

RESUMEN

BACKGROUND: We present a comprehensive technological solution for bacterial diagnostics using tmRNA as a marker molecule. A robust probe design algorithm for microbial detection microarray is implemented. The probes were evaluated for specificity and, combined with NASBA (Nucleic Acid Sequence Based Amplification) amplification, for sensitivity. RESULTS: We developed a new web-based program SLICSel for the design of hybridization probes, based on nearest-neighbor thermodynamic modeling. A SLICSel minimum binding energy difference criterion of 4 kcal/mol was sufficient to design of Streptococcus pneumoniae tmRNA specific microarray probes. With lower binding energy difference criteria, additional hybridization specificity tests on the microarray were needed to eliminate non-specific probes. Using SLICSel designed microarray probes and NASBA we were able to detect S. pneumoniae tmRNA from a series of total RNA dilutions equivalent to the RNA content of 0.1-10 CFU. CONCLUSIONS: The described technological solution and both its separate components SLICSel and NASBA-microarray technology independently are applicative for many different areas of microbial diagnostics.


Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Bacteriano/genética , Replicación de Secuencia Autosostenida/métodos , Streptococcus pneumoniae/genética , Sondas ARN/genética , Programas Informáticos , Especificidad de la Especie
13.
Chemistry ; 17(10): 2903-15, 2011 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-21294195

RESUMEN

Fluorescent 2'-deoxynucleotides containing a protecting group at the 3'-O-position are reversible terminators that enable array-based DNA sequencing-by-synthesis (SBS) approaches. Herein, we describe the synthesis and full characterisation of four reversible terminators bearing a 3'-blocking moiety and a linker-dye system that is removable under the same fluoride-based treatment. Each nucleotide analogue has a different fluorophore attached to the base through a fluoride-cleavable linker and a 2-cyanoethyl moiety as the 3'-blocking group, which can be removed by using a fluoride treatment as well. Furthermore, we identified a DNA polymerase, namely, RevertAid M-MuLV reverse transcriptase, which can incorporate the four modified reversible terminators. The synthesised nucleotides and the optimised DNA polymerase were used on CodeLink slides spotted with hairpin oligonucleotides to demonstrate their potential in a cyclic reversible terminating approach.


Asunto(s)
Colorantes Fluorescentes/síntesis química , Fluoruros/química , Virus de la Leucemia Murina/enzimología , Sondas de Oligonucleótidos/síntesis química , ADN Polimerasa Dirigida por ARN/metabolismo , Cartilla de ADN/metabolismo , Colorantes Fluorescentes/química , Estructura Molecular , Sondas de Oligonucleótidos/química
14.
BMC Biotechnol ; 10: 34, 2010 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-20426847

RESUMEN

BACKGROUND: The hybridization of synthetic Streptococcus pneumoniae tmRNA on a detection microarray is slow at 34 degrees C resulting in low signal intensities. RESULTS: We demonstrate that adding specific DNA helper oligonucleotides (chaperones) to the hybridization buffer increases the signal strength at a given temperature and thus makes the specific detection of Streptococcus pneumoniae tmRNA more sensitive. No loss of specificity was observed at low temperatures compared to hybridization at 46 degrees C. The effect of the chaperones can be explained by disruption of the strong secondary and tertiary structure of the target RNA by the selective hybridization of helper molecules. The amplification of the hybridization signal strength by chaperones is not necessarily local; we observed increased signal intensities in both local and distant regions of the target molecule. CONCLUSIONS: The sensitivity of the detection of tmRNA at low temperature can be increased by chaperone oligonucleotides. Due to the complexity of RNA secondary and tertiary structures the effect of any individual chaperone is currently not predictable.


Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Oligonucleótidos/química , ARN Bacteriano/aislamiento & purificación , Secuencia de Bases , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Sensibilidad y Especificidad , Streptococcus pneumoniae/genética , Temperatura
15.
Mol Cell Endocrinol ; 510: 110816, 2020 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-32294491

RESUMEN

Human granulosa cells acquired as leftover from IVF treatment can be used to study infertility problems and are a valuable tool in the research of follicle maturation and ovulation. There is a need for more defined and long-term culture protocols for studying the response of granulosa cells upon treatment with selected hormones/chemicals. In the current study, we tested the effect of adding FGF2, IGF2 and FSH into defined basal medium in order to find culture conditions that would support proliferation of cumulus and mural granulosa cells along with the expression of common granulosa cell type markers such as FSHR, AMHR2, LHR and CYP19A1. We found that FGF2, IGF2 together with FSH helped to retain granulosa cell marker expression while supporting cell survival at least for two weeks of culture. The defined serum-free culture conditions for long-term culturing will be valuable in providing new standards in the research of human granulosa cells.


Asunto(s)
Técnicas de Cultivo de Célula , Factor 2 de Crecimiento de Fibroblastos/farmacología , Hormona Folículo Estimulante/farmacología , Células de la Granulosa/citología , Factor II del Crecimiento Similar a la Insulina/farmacología , Biomarcadores/metabolismo , Recuento de Células , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo/farmacología , Medio de Cultivo Libre de Suero , Células del Cúmulo/efectos de los fármacos , Células del Cúmulo/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Humanos
16.
Sci Rep ; 10(1): 2300, 2020 02 10.
Artículo en Inglés | MEDLINE | ID: mdl-32042028

RESUMEN

MicroRNAs (miRNAs) are known post-transcriptional regulators of various biological processes including ovarian follicle development. We have previously identified miRNAs from human pre-ovulatory ovarian granulosa cells that are expressed from the intronic regions of two key genes in normal follicular development: FSH receptor (FSHR) and CYP19A1, the latter encoding the aromatase enzyme. The present study aims to identify the target genes regulated by these miRNAs: hsa-miR-548ba and hsa-miR-7973, respectively. The miRNAs of interest were transfected into KGN cell line and the gene expression changes were analyzed by Affymetrix microarray. Potential miRNA-regulated genes were further filtered by bioinformatic target prediction algorithms and validated for direct miRNA:mRNA binding by luciferase reporter assay. LIFR, PTEN, NEO1 and SP110 were confirmed as targets for hsa-miR-548ba. Hsa-miR-7973 target genes ADAM19, PXDN and FMNL3 also passed all verification steps. Additionally, the expression pattern of the miRNAs was studied in human primary cumulus granulosa cell culture in relation to the expression of their host genes and FSH stimulation. Based on our findings we propose the involvement of hsa-miR-548ba in the regulation of follicle growth and activation via LIFR and PTEN. Hsa-miR-7973 may be implicated in the modulation of extracellular matrix and cell-cell interactions by regulating the expression of its identified targets.


Asunto(s)
Células del Cúmulo/fisiología , Regulación del Desarrollo de la Expresión Génica , MicroARNs/metabolismo , Folículo Ovárico/crecimiento & desarrollo , Adulto , Aromatasa/genética , Línea Celular Tumoral , Femenino , Hormona Folículo Estimulante/metabolismo , Perfilación de la Expresión Génica , Humanos , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia/genética , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Folículo Ovárico/citología , Fosfohidrolasa PTEN/genética , Cultivo Primario de Células , Receptores de HFE/genética , Adulto Joven
17.
BMC Biotechnol ; 9: 45, 2009 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-19445684

RESUMEN

BACKGROUND: Here we present a novel promising microbial diagnostic method that combines the sensitivity of Nucleic Acid Sequence Based Amplification (NASBA) with the high information content of microarray technology for the detection of bacterial tmRNA molecules. The NASBA protocol was modified to include aminoallyl-UTP (aaUTP) molecules that were incorporated into nascent RNA during the NASBA reaction. Post-amplification labeling with fluorescent dye was carried out subsequently and tmRNA hybridization signal intensities were measured using microarray technology. Significant optimization of the labeled NASBA protocol was required to maintain the required sensitivity of the reactions. RESULTS: Two different aaUTP salts were evaluated and optimum final concentrations were identified for both. The final 2 mM concentration of aaUTP Li-salt in NASBA reaction resulted in highest microarray signals overall, being twice as high as the strongest signals with 1 mM aaUTP Na-salt. CONCLUSION: We have successfully demonstrated efficient combination of NASBA amplification technology with microarray based hybridization detection. The method is applicative for many different areas of microbial diagnostics including environmental monitoring, bio threat detection, industrial process monitoring and clinical microbiology.


Asunto(s)
Colorantes Fluorescentes/química , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Bacteriano/química , Replicación de Secuencia Autosostenida/métodos , Sensibilidad y Especificidad , Streptococcus pneumoniae/metabolismo , Uridina Trifosfato/química
18.
PLoS One ; 14(11): e0225801, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31765427

RESUMEN

Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) has revolutionized our understanding of chromatin-related biological processes. The method, however, requires thousands of cells and has therefore limited applications in situations where cell numbers are limited. Here we describe a novel method called Restriction Assisted Tagmentation Chromatin Immunoprecipitation (RAT-ChIP) that enables global histone modification profiling from as few as 100 cells. The method is simple, cost-effective and takes a single day to complete. We demonstrate the sensitivity of the method by deriving the first genome-wide maps of histone H3K4me3 and H3K27me3 modifications of inner cell mass and trophectoderm of bovine blastocyst stage embryos.


Asunto(s)
Masa Celular Interna del Blastocisto/metabolismo , Inmunoprecipitación de Cromatina , Histonas/metabolismo , Trofoblastos/metabolismo , Animales , Blastocisto/citología , Blastocisto/metabolismo , Bovinos , Línea Celular , Cromatina/química , Cromatina/metabolismo , Fertilización In Vitro , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento , Histonas/genética , Humanos , Oocitos/citología , Procesamiento Proteico-Postraduccional , Análisis de Secuencia de ADN
19.
Nat Med ; 25(11): 1699-1705, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31686035

RESUMEN

Although chromosomal instability (CIN) is a common phenomenon in cleavage-stage embryogenesis following in vitro fertilization (IVF)1-3, its rate in naturally conceived human embryos is unknown. CIN leads to mosaic embryos that contain a combination of genetically normal and abnormal cells, and is significantly higher in in vitro-produced preimplantation embryos as compared to in vivo-conceived preimplantation embryos4. Even though embryos with CIN-derived complex aneuploidies may arrest between the cleavage and blastocyst stages of embryogenesis5,6, a high number of embryos containing abnormal cells can pass this strong selection barrier7,8. However, neither the prevalence nor extent of CIN during prenatal development and at birth, following IVF treatment, is well understood. Here we profiled the genomic landscape of fetal and placental tissues postpartum from both IVF and naturally conceived children, to investigate the prevalence and persistence of large genetic aberrations that probably arose from IVF-related CIN. We demonstrate that CIN is not preserved at later stages of prenatal development, and that de novo numerical aberrations or large structural DNA imbalances occur at similar rates in IVF and naturally conceived live-born neonates. Our findings affirm that human IVF treatment has no detrimental effect on the chromosomal constitution of fetal and placental lineages.


Asunto(s)
Inestabilidad Cromosómica/genética , Variaciones en el Número de Copia de ADN/genética , Desarrollo Embrionario/genética , Fertilización In Vitro/efectos adversos , Blastocisto/metabolismo , Linaje de la Célula/genética , Embrión de Mamíferos , Femenino , Feto , Genotipo , Humanos , Recién Nacido , Masculino , Placenta/metabolismo , Placenta/patología , Polimorfismo de Nucleótido Simple/genética , Embarazo
20.
Fertil Steril ; 109(6): 1127-1134.e1, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29935648

RESUMEN

OBJECTIVE: To compare the genomic profiles of blastocoel fluid (BF), inner cell mass (ICM), and trophectoderm (TE) cells derived from the same blastocyst. DESIGN: Prospective study. SETTING: Academic and in vitro fertilization units. PATIENT(S): Sixteen donated cryopreserved embryos at blastocyst stage. INTERVENTION(S): BF, TE, and ICM cells were retrieved from each blastocyst for chromosome analysis by means of next-generation sequencing (NGS). MAIN OUTCOME MEASURE(S): Aneuploidy screening and assessment of mosaicism in BF, TE and ICM samples with subsequent comparison of genomic profiles between the three blastocyst compartments. RESULT(S): Out of 16 blastocysts, 10 BF samples and 14 TE and ICM samples provided reliable NGS data for comprehensive chromosome analysis. Only 40.0% of BF-DNA karyotypes were fully concordant with TE or ICM, compared with 85.7% concordance between TE and ICM. In addition, BF-DNA was burdened with mosaic aneuploidies and the total number of affected chromosomes in BF was significantly higher compared with the TE and ICM. CONCLUSION(S): BF-DNA can be successfully amplified and subjected to NGS, but owing to increased discordance with ICM and TE, BF does not adequately represent the status of the rest of the embryo. To overcome biologic and technical challenges associated with BF sampling and processing, blastocentesis would require improvement in both laboratory protocols and aneuploidy calling algorithms. Therefore, TE biopsy remains the most effective way to predict embryonic karyotype, and the use of BF as a single source of DNA for preimplantation genetic screening is not yet advised.


Asunto(s)
Masa Celular Interna del Blastocisto/patología , Blastocisto/patología , Ectodermo/patología , Líquido Intracelular/química , Cariotipificación , Diagnóstico Preimplantación , Aneuploidia , Masa Celular Interna del Blastocisto/metabolismo , Células Cultivadas , Ectodermo/metabolismo , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Líquido Intracelular/metabolismo , Cariotipo , Cariotipificación/métodos , Cariotipificación/normas , Mosaicismo , Diagnóstico Preimplantación/métodos , Diagnóstico Preimplantación/normas , Reproducibilidad de los Resultados
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