Preimplantation genetic diagnosis for single gene disorders: experience with five single gene disorders

We report our experience of 14 preimplantation genetic diagnosis (PGD) cycles in eight couples carrying five different single gene disorders, during the last 18 months. Diagnoses were performed for myotonic dystrophy (DM), cystic fibrosis (CF) [ΔF508 and exon 4 (621+1 G>T)], fragile X and CF simultaneously, and two disorders for which PGD had not been previously attempted, namely neurofibromatosis type 2 (NF2) and Crouzon syndrome. Diagnoses for single gene disorders were carried out on ideally two blastomeres biopsied from Day 3 embryos. A highly polymorphic marker was included in each diagnosis to control against contamination. For the dominant disorders, where possible, linked polymorphisms provided an additional means of determining the genotype of the embryo hence reducing the risk of misdiagnosis due to allele dropout (ADO). Multiplex fluorescent polymerase chain reaction (F-PCR) was used in all cases, followed by fragment analysis and/or single-stranded conformation polymorphism (SSCP) for genotyping. Embryo transfer was performed in 13 cycles resulting in one biochemical pregnancy for CF, three normal deliveries (a twin and a singleton) and one early miscarriage for DM and a singleton for Crouzon syndrome. In each case the untransferred embryos were used to confirm the diagnoses performed on the biopsied cells. The results were concordant in all cases. The inclusion of a polymorphic marker allowed the detection of extraneous DNA contamination in two cells from one case. Knowing the genotype of the contaminating DNA allowed its origin to be traced. All five pregnancies were obtained from embryos in which two blastomeres were biopsied for the diagnosis. Our data demonstrate the successful strategy of using multiplex PCR to simultaneously amplify the mutation site and a polymorphic locus, fluorescent PCR technology to achieve greater sensitivity, and two-cell biopsy to increase the efficiency and success of diagnoses. Copyright © 2002 John Wiley & Sons, Ltd.     

Drosophila glue protein gene expression

The primary targets of steroid hormones are genes. For the ecdysone-controlled genes of Drosophila larval glue proteins proximal and distal control elements were identified by mutagenesis and sequence comparison. Their presence is required for the correct stage- and tissue-specific expression of these genes. The supposed function of these elements is described in a working model.     

Transfer-RNA,an early gene?

The theory of self-reproductive molecular systems involves the consequence that translation must have started from a selected distribution of RNA molecules, that comprised GC-rich sequences of a length < 100 nucleotides. This implies a joint function of messenger and adaptor, which both had to be recruited from the same mutant distribution. The reconstruction of tRNA precursors yields such a molecule showing some reverberation of a codon pattern GNC. These findings suggest that tRNA has been the earliest component of the translation machinery.     

重组基因酵母检测环境类雌激素污染物

介绍了利用重组基因酵母细胞检测环境类雌激素污染物的生物测定方法,并利用这种方法测定污泥、土壤、底泥、飘尘和家庭炉灶燃烧过程中排放的污染物中的雌激素活性,研究了样品不同的纯化方法对雌激素活性的影响.结果表明,重组受体基因/报道基因的酵母检测技术是一种筛选和定量分析环境样品中雌激素类污染物的快速、有效方法.酵母检测应结合分离纯化步骤,除去样品中的非活性和对酵母细胞有毒性的干扰物质.     

Novel mutations in the human CYP21 gene

The great majority of genetic defects underlying steroid 21-hydroxylase deficiency appear to result from intergenic recombinations between the homologous CYP21 and CYP21P genes. For a minority, novel sporadic point mutations have been detected. De novo mutations in CYP21 have also been reported, but only a few studies have systematically screened their occurrence. We here describe a population-based patient sample in order to estimate the rate of single-family (i.e. sporadic) and de novo germline mutations in the human CYP21 locus. Among 76 Finnish families were observed three single-family mutations and two de novo mutations in CYP21. The rates obtained, ∼5% and ∼2% for novel and de novo mutations, respectively, indicate that they are not rare and that their occurrence should not be ignored in genetic diagnostics of this disorder. Copyright © 2001 John Wiley & Sons, Ltd.     

PTA wastewater molecular toxicity detected with gene chip

The purified terephthalic acid （PTA） petrochemical wastewater molecular toxicity detected by use of Mouse Genome 430A 2.0 GeneChip was conducted in this research. The toxic dose to male mice was 0.03 g/（kg, d） of PTA in the wastewater. The mice liver total RNA was isolated as the temple for synthesis of eDNA and then the cDNA as the temple for synthesis of cRNA. Hybridizing the cRNA with the target genes on the gene chip, there were 232 genes expression levels up-regulated and 74 genes down-regulated discovered obviously. The foremost 40 genes for both the highest and the lowest expression levels involved endogenetic steroid and hormone metabolism, immune system, the leukocyte activity and inflammation, detoxification in liver, reproduction and growth hormone, regulation immune factors of anti-tumor and anti-infection and cancer to the mice sampled. The data suggest the PTA wastewater contained over 5 aromatics and their toxicities integrated were much higher than the pure chemical PTA. And the pure chemical PTA toxicities data cannot be used to evaluate the toxicity of the PTA wastewater instead.     

Prenatal detection of fetal hemoglobin E gene from maternal plasma

In order to provide a noninvasive prenatal diagnosis of the hemoglobin E (Hb E) related disorder, we have evaluated the possibility of identifying the fetal β^E-globin gene in maternal plasma. The analysis was performed during 8 to 18 weeks of gestation using DNA extracted from 200 µL of plasma from pregnant women whose husbands carried Hb E. The β^E-globin mutation in maternal plasma was detected by a nested PCR amplification followed by the Mnl I restriction analysis. The result was compared with that of routine analysis of the CVS specimens. Among the five pregnant women examined, the fetal β^E-globin gene was identified in maternal plasma in three of them and the result was completely concordant with the conventional CVS analysis. This simple noninvasive prenatal detection of the fetal β^E-globin gene should prove useful in a prevention and control program of Hb E/β-thalassemia in countries where the β^E-globin gene is prevalent. Copyright © 2003 John Wiley & Sons, Ltd.     

Fetal cerebral hemorrhage due to X-linked GATA1 gene mutation

We report a multiplex family with a GATA1 gene mutation responsible for a massive fetal cerebral hemorrhage occurring at 36 weeks. Two other stillbirth cousins presented with fetal hydrops and congenital hemochromatosis' phenotype at 37 and 12 weeks of gestation. Molecular screening revealed the presence of a c.613G>A pathogenic allelic variation in exon 4 of GATA1 gene in the 3 male siblings and their carrier mothers. The diagnosis of a GATA1 gene mutation may be suspected in cases of male fetuses with intracerebral bleeding, particularly if a history of prior fetal loss(es) and mild maternal thrombocytopenia are also present.     

转解毒酶基因工程菌的解毒作用研究

用已构建的可降解有机磷酸酯等杀虫药剂的转解毒酶基因工程菌,测定了对特异性底物--乙酸萘酯---NA-和--乙酸萘酯---NA-的分解作用,证明该工程菌高效地表达了解毒酶基因.将该工程菌细胞固定化后,通过对--NA和--NA的降解实验,测定了固定化细胞的酶活.用固定化细胞降解有机氯酸酯类的三氯杀虫酯-7504-、拟除虫菊酯类的溴氰菊酯和有机磷酸酯类的毒死蜱,结果表明,固定化细胞对该3类杀虫剂均具有一定的降解效果.用转解毒酶基因工程菌喂有机磷中毒的鸡,表明该转解毒酶基因工程菌对有机磷中毒的鸡,无论是急性中毒还是慢性中毒都有较明显的解毒作用.     

Reference gene validation for quantification of gene expression during final oocyte maturation induced by diethylstilbestrol and di-(2-ethylhexyl)-phthalate in common carp

Final oocyte maturation is the key step to successful spawning and fertilization.Quantitative real-time PCR(q PCR) is the technique of election to quantify the abundance of functional genes in such study. Reference gene is essential for correct interpretation of q PCR data. However, an ideal universal reference gene that is stable under all experimental circumstances has not been described. Researchers should validate their reference genes while performing q PCR analysis. The expression of 6 candidate reference genes: 18 s r RNA,28 s r RNA, Cathepsin Z, Elongation factor 1-α, Glyceraldehyde-3-phosphate dehydrogenase andβ-actin were investigated during final oocyte maturation induced by different compounds(DES and DEHP) in common carp(Cyprinus carpio). Four softwares(Bestkeeper, ge Norm,Norm Finder and Ref Finder) were used to screen the most stable gene in order to evaluate their expression stability. The results revealed that EF1α was highly stable expressed when final oocyte maturation was induced by DES, while gapdh was the most stable gene when final oocyte maturation was induced by DEHP. Stable expressed reference gene selection is critical for all q PCR analysis to get accurate target gene m RNA expression information.     

Overexpression of NADH oxidase gene from Deinococcus geothermalis in Escherichia coli

When using stable enzyme genes from a thermophile to create a biosensor in Escherichia coli, it is vital that these genes be overexpressed in order to provide a sufficient supply of enzymes. In this study, overexpression of the NADH oxidase (Nox) gene from the thermophile Deinococcus geothermalis was successfully achieved with the aim of creating a stable biosensor active at room temperatures. To do so, modification of 10 nucleotides, GAAATTAACT, upstream of the start codon of the Nox gene was necessary.     

微塑料颗粒对虹鳟免疫基因表达的影响

微塑料在海洋中分布广泛,鱼类是否会受到影响及受影响的程度暂并不明确.本文通过实验室产生2×105MP·L-1浓度的微塑料水体,研究当虹鳟暴露在含有不同粒径微塑料颗粒(0.2、1、20、40和90μm)的水体中2 h后,微塑料颗粒在鱼鳃的附着及其鳃组织免疫基因表达的变化特征.结果表明,微塑料颗粒会附着在虹鳟的鳃上,且在从内到外不同鳃片上均有分布,附着程度与微塑料颗粒的粒径大小有关.暴露2 h后虹鳟鳃组织上的部分免疫基因的表达发生变化,包括IFN-γ上调及IL-1β、S100A1和SAA的下调.本文通过对免疫基因表达变化的研究表明,水体中的微塑料颗粒会附着在虹鳟的鳃上,且会导致虹鳟出现免疫反应,论文成果可为相关领域的研究提供基础数据.     

Roadmap for an early gene therapy for cystic fibrosis airway disease

Gene therapy provides a mutation-independent approach to treat or even cure CF airway disease. To develop a clinical candidate for CF gene therapy, a thorough examination of preclinical efficacy in relevant cell and animal models is a prerequisite. For a long time, the CF field was struggling with a lack of appropriate animal models for CF airway pathology. Since 2008, many different and complementary animal models have been generated that develop hallmarks of CF airway disease, including the CF pig, ferret, and rat. With this, a new era has arisen that allows investigating the efficacy of gene therapy beyond molecular and electrophysiological end-points. Successful gene therapy most likely requires an appropriate time window. CF lung pathology progresses with age and therefore an early treatment would be beneficial to prevent irreversible damage. In that regard, newborn screening programs and prenatal diagnosis already provide a basis to facilitate future preventive gene-based treatment. If successful, gene therapy for CF airway disease would markedly reduce the treatment burden and improve life quality and life expectancy of CF patients.     

二氯乙腈对大肠杆菌基因表达的影响

为探究二氯乙腈(DCAN)对大肠杆菌(E.coli)基因表达的影响及相应的毒性作用,采用自组织映射(SOM)聚类及剂量效应关系分析方法考察了E.coli在不同剂量DCAN暴露120min过程中其基因表达状况.结果表明:随时间及浓度改变E.coli基因表达具有动态性;在DCAN浓度为1.429×10-3mg/L时,多个参...     

Organ targeted prenatal gene therapy—how far are we?

Prenatal gene therapy aims to deliver genes to cells and tissues early in prenatal life, allowing correction of a genetic defect, before long-term tissue damage has occurred. In contrast to postnatal gene therapy, prenatal application can target genes to a large population of dividing stem cells, and the smaller fetal size allows a higher vector-to-target cell ratio to be achieved. Early-gestation delivery may allow the development of immune tolerance to the transgenic protein which would facilitate postnatal repeat vector administration if needed. Targeting particular organs will depend on manipulating the vector to achieve selective tropism and on choosing the most appropriate gestational age and injection method for fetal delivery. Intra-amniotic injection reaches the skin, and other organs that are bathed in the fluid however since gene transfer to the lung and gut is usually poor more direct injection methods will be needed. Delivery to the liver and blood can be achieved by systemic delivery via the umbilical vein or peritoneal cavity. Gene transfer to the central nervous system in the fetus is difficult but newer vectors are available that transduce neuronal tissue even after systemic delivery. Copyright © 2011 John Wiley & Sons, Ltd.