RESUMO
Mutational screens are an effective means used in the functional annotation of a genome. We present a method for a mutational screen of the mouse X chromosome using gene trap technologies. This method has the potential to screen all of the genes on the X chromosome without establishing mutant animals, as all gene-trapped embryonic stem (ES) cell lines are hemizygous null for mutations on the X chromosome. Based on this method, embryonic morphological phenotypes and expression patterns for 58 genes were assessed, approximately 10% of all human and mouse syntenic genes on the X chromosome. Of these, 17 are novel embryonic lethal mutations and nine are mutant mouse models of genes associated with genetic disease in humans, including BCOR and PORCN. The rate of lethal mutations is similar to previous mutagenic screens of the autosomes. Interestingly, some genes associated with X-linked mental retardation (XLMR) in humans show lethal phenotypes in mice, suggesting that null mutations cannot be responsible for all cases of XLMR. The entire data set is available via the publicly accessible website (http://xlinkedgenes.ibme.utoronto.ca/).
Assuntos
Análise Mutacional de DNA/métodos , Predisposição Genética para Doença , Testes Genéticos/métodos , Fenótipo , Cromossomo X/genética , Aciltransferases , Animais , Sequência de Bases , Cromossomos Humanos X/genética , Genes Letais , Genes Ligados ao Cromossomo X , Doenças Genéticas Ligadas ao Cromossomo X/genética , Humanos , Proteínas de Membrana/genética , Deficiência Intelectual Ligada ao Cromossomo X/genética , Camundongos , Anotação de Sequência Molecular , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genéticaRESUMO
We present a passive microfluidic sperm selection strategy that collects motile sperm based on their preference to follow boundaries and turn corners. Clinical assessment of selected human sperm from the device revealed a strong correlation between high DNA integrity and the tendency for sperm to follow boundaries. Human sperm with preference to follow boundaries on the left- or right-hand sides have higher (>51%) DNA integrity than straight swimmers and significantly higher (>67%) DNA integrity than sperm in raw semen. Boundary following behaviour offers a strategy to selecting sperm with the highest DNA integrity to improve the success rate of assisted reproduction.
Assuntos
Separação Celular/instrumentação , Separação Celular/métodos , Motilidade dos Espermatozoides , Espermatozoides/fisiologia , DNA/análise , Desenho de Equipamento , Humanos , Dispositivos Lab-On-A-Chip , MasculinoRESUMO
Selection medium is important in sperm isolation for assisted reproductive technologies. Contrary to the naturally occurring human cervical mucus which has a high viscosity, most current practices for motility based sperm selection use a low viscosity medium. In this study, we used a microfluidic device to assess the effects of high viscosity media made with hyaluronic acid (HA) and methyl cellulose (MC) on bovine and human sperm motility and viability (sperm transferred directly from cryoprotectant). The microfluidic penetration test, viability, and motility were compared for sperm swimming in both HA and MC media with about 20cp viscosity (measured at 20 °C). Our resulted indicate that MC medium resulted in a significantly higher number of viable bovine sperm penetrating the medium as compared to HA. Furthermore, MC resulted in the selection of a sperm subpopulation with a 274% increase in sperm viability in comparison to the raw semen, while HA increased viability by only 133%. In addition to viability, bovine sperm motility parameters were significantly higher in the MC medium as compared with HA. Experiments with human sperm swimming in MC indicate that sperm swim slower and straighter at higher viscosities. In conclusion, the results indicate that in a micro-confined environment representative of the in vivo environment, MC is a preferred high viscosity medium to ensure the highest concentration of motile and viable sperm.
RESUMO
Sperm selection is essential to assisted reproductive technology (ART), influencing treatment outcomes and the health of offspring. The fundamental challenge of sperm selection is dictated by biology: a heterogeneous population of ~10(8) sperm per milliliter with a short lifetime in vitro. However, conventional sperm selection approaches result in less than 50% improvement in DNA integrity. Here, a clinically applicable microfluidic device is presented that selects sperm based on the progressive motility in 500 parallel microchannels. The result is a one-step procedure for semen purification and high DNA integrity sperm selection from 1 mL of raw semen in under 20 minutes. Experiments with bull sperm indicate more than 89% improvement in selected sperm vitality. Clinical tests with human sperm show more than 80% improvement in human DNA integrity, significantly outperforming the best current practices. These results demonstrate the presence of a sub-population of sperm with nearly intact chromatin and DNA integrity, and a simple clinically-applicable lab-on-a-chip method to select this population.
Assuntos
DNA/metabolismo , Técnicas Analíticas Microfluídicas , Análise do Sêmen , Espermatozoides , Adulto , Animais , Bovinos , Humanos , Masculino , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Análise do Sêmen/instrumentação , Análise do Sêmen/métodos , Espermatozoides/citologia , Espermatozoides/metabolismoRESUMO
BACKGROUND: The zinc finger protein Sp2 (specificity protein 2) is a member of the glutamine-rich Sp family of transcription factors. Despite its close similarity to Sp1, Sp3 and Sp4, Sp2 does not bind to DNA or activate transcription when expressed in mammalian cell lines. The expression pattern and the biological relevance of Sp2 in the mouse are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Whole-mount in situ hybridization of mouse embryos between E7.5 and E9.5 revealed abundant expression in most embryonic and extra-embryonic tissues. In order to unravel the biological relevance of Sp2, we have targeted the Sp2 gene by a tri-loxP strategy. Constitutive Sp2null and conditional Sp2cko knockout alleles were obtained by crossings with appropriate Cre recombinase expressing mice. Constitutive disruption of the mouse Sp2 gene (Sp2null) resulted in severe growth retardation and lethality before E9.5. Mouse embryonic fibroblasts (MEFs) derived from Sp2null embryos at E9.5 failed to grow. Cre-mediated ablation of Sp2 in Sp2cko/cko MEFs obtained from E13.5 strongly impaired cell proliferation. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that Sp2 is essential for early mouse development and autonomous proliferation of MEFs in culture. Comparison of the Sp2 knockout phenotype with the phenotypes of Sp1, Sp3 and Sp4 knockout strains shows that, despite their structural similarity and evolutionary relationship, all four glutamine-rich members of the Sp family of transcription factors have distinct non-redundant functions in vivo.
Assuntos
Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fator de Transcrição Sp2/fisiologia , Animais , Proliferação de Células , DNA/metabolismo , Evolução Molecular , Feminino , Hibridização In Situ , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Fatores de Tempo , Transcrição GênicaRESUMO
The ubiquitously expressed zinc finger transcription factors Sp1 and Sp3 play critical roles in embryonic development. Sp1 knockout mice die around embryonic day 10.5. Mice lacking Sp3 are postnatal lethal. Mice heterozygous for either Sp1 or Sp3 are apparently normal, although slightly smaller. Here, we show that compound heterozygosity of Sp1 and Sp3 results in embryonic lethality accompanied by a spectrum of developmental abnormalities, including growth retardation, morphological alterations of the lung, impaired ossification, anemia, and placental defects. Anemia in Sp1/Sp3 compound heterozygous mutant embryos is associated with impaired maturation of erythrocytes. Analyses of the placenta revealed a markedly reduced spongiotrophoblast layer and a severe disorganization of the labyrinth layer in Sp1/Sp3 compound heterozygous as well as in Sp3-deficient mutant embryos. Our findings demonstrate that a threshold of Sp1 and Sp3 activity is required for normal embryonic development, suggesting that Sp1 and Sp3 act cooperatively to regulate downstream targets.