三唑磷作用下翡翠贻贝抑制性消减杂交文库构建与分析

采用抑制性差减杂交技术,构建了三唑磷农药急性胁迫下翡翠贻贝雌贝性腺正反向消减杂交cDNA文库.正反向文库共有117个表达序列标签（expressed sequence tags,ESTs）与NCBI数据库中已知功能蛋白具较高同源性,假定蛋白12种,另有195个ESTs未检索到同源序列.正向文库中含有细胞周期蛋白、性激素合成蛋白、DNA修复蛋白、能量代谢相关蛋白等一系列与生殖、应激适应和代谢相关的转录本信息,而反向文库中的差异ESTs主要涉及DNA复制、转录和翻译调控基因以及骨架蛋白.结果表明三唑磷对翡翠贻贝的毒性涉及到生殖调控、能量代谢、应激适应、转录翻译调控等生理代谢的多个方面,正向文库中发现的几种生殖调控基因（细胞周期蛋白、周期蛋白依赖性蛋白激酶、合子DNA复制许可因子、精子特异性蛋白、雌激素硫酸转移酶和卵黄膜卵透明带域蛋白）可能是三唑磷生殖干扰毒性作用过程中的关键基因,这为有机磷农药的生殖毒性探索提供了基础数据.     

Preimplantation genetic diagnosis using FISH for carriers of Robertsonian translocations: the Portuguese experience

Preimplantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis for couples at risk of transmitting genetic disorders to their offspring. We present a fluorescence in situ hybridization (FISH) analysis of embryos obtained after seven PGD cycles in six couples with Robertsonian translocations and male factor infertility: 4 der(13;14), 1 der(14;21) and 1 der(15;21). Of 74 metaphase II (MII) injected oocytes, 61 (82.4%) fertilized normally and cleaved. Of these, 37/61 (60.7%) embryos were of high morphological quality with ≥6 blastomeres. After biopsy of 44 embryos at day 3 of development, seven degenerated, seven arrested in development and 30/44 (68.2%) evolved, of which 25/30 (83.3%) reached the morula/blastocyst stage. Analysis of biopsied blastomeres showed 23/44 (52.3%) of normal/balanced embryos, of which 15 (11 at the morula/blastocyst stage) were transferred in six cycles. One term pregnancy was achieved, which ended by cesarean section at 37 weeks of gestation, giving birth to two healthy newborn. Analysis of 49 embryos (excluding 12 inconclusive cases) showed a predominance of alternate segregation (38/49, 77.6%) over adjacent segregation (7/49, 14.3%), with one (2%) being a polyploid mosaic and three (6.1%) chaotic. Copyright © 2002 John Wiley & Sons, Ltd.     

Aspects of biopsy procedures prior to preimplantation genetic diagnosis

Today, preimplantation genetic diagnosis (PGD) is offered in over 40 centres worldwide for an expanded range of genetic defects causing disease. This very early form of prenatal diagnosis involves the detection of affected embryos by fluorescent in situ hybridization (FISH) (sex determination or chromosomal defects) or by polymerase chain reaction (PCR) (monogenic diseases) prior to implantation. Genetic analysis of the embryos involves the removal of some cellular mass from the embryos (one or two blastomeres at cleavage-stage or some extra-embryonic trophectoderm cells at the blastocyst stage) by means of an embryo biopsy procedure. Genetic analysis can also be performed preconceptionally by removal of the first polar body. However, additional information is then often gained by removal of the second polar body and/or a blastomere from the embryo. Removal of polar bodies or cellular material from embryos requires an opening in the zona pellucida, which can be created in a mechanical way (partial zona dissection) or chemical way (acidic Tyrode's solution). However, the more recent introduction of laser technology has facilitated this step enormously. Different biopsy procedures at different preimplantation stages are reviewed here, including their pros and cons and their clinical applications. The following aspects will also be discussed: safety of zona drilling by laser, use of Ca²⁺/Mg²⁺-free medium for decompaction, and removal of one or two cells from cleavage-stage embryos. Copyright © 2001 John Wiley & Sons, Ltd.     

Pregnancy after preimplantation genetic diagnosis for Sanjad–Sakati syndrome

Sanjad–Sakati syndrome (SSS) is an autosomal recessive disorder characterized by congenital hypoparathyroidism, growth and mental retardation. In Saudi Arabia, the disease is caused by a deletion of 12 bp (155-166nt) in the tubulin-specific chaperone E gene. In a family with two affected siblings with SSS, preimplantation genetic diagnosis (PGD) was performed. Fluorescent PCR (F-PCR) was utilized to check the heterozygosity and the homozygosity status of the parents and the affected children, respectively. F-PCR was then optimized for single-cell analysis by using peripheral blood lymphocytes. The patient underwent a cycle with intra-cytoplasmic sperm injection. A total of 11 embryos were obtained and biopsied. There were five heterozygous, three homozygous affected and three normal embryos. One heterozygous and one normal embryo were transferred because of their very good quality (morula). A singleton pregnancy was obtained, and amniosynthesis confirmed the presence of the heterozygous fetus. These results show for the first time, the feasibility of PGD for SSS. Copyright © 2004 John Wiley & Sons, Ltd.     

β-Glucuronidase activity in mouse oocytes,mouse preimplantation embryos,and human gametes

A microfluorimetric assay was designed to monitor β-glucuronidase (β-gluc) activity during the early development of mouse embryos and was also applied to measure β-gluc activity in human sperm cells and single human oocytes. Mouse oocytes and preimplantation embryos at different stages of development, failed fertilized human oocytes, and human sperm cells were collected, and β-gluc activity was analysed. In the mouse, β-gluc activity could be measured starting from the morula stage on in a pooled sample of 15 embryos and at blasto-cyst formation in one single embryo. β-Gluc activity was low in human sperm cells. The enzyme could be readily demonstrated in 87 per cent of non-fertilized human oocytes. Considerable variation in β-gluc activity was noticed between individual oocytes.     

Normal pregnancy after preimplantation DNA diagnosis of a dystrophin gene deletion

To perform preimplantation DNA diagnosis for Duchenne muscular dystrophy (DMD) in a female carrier of a dystrophin gene deletion of exons 3–18, we developed a polymerase chain reaction (PCR)-based assay of exon 17 sequences. Exon 17 was efficiently amplified in all 50 single blastomeres of normal control embryos and in five blastomeres of one male embryo of the DMD carrier obtained after a first preimplantation diagnosis (PID) for gender determination. In ten blastomeres of another two male embryos of the DMD carrier, no PCR signals were observed, probably as a result of the deletion. After intracytoplasmic sperm injection, embryos were analysed for exon 17 and three of the four embryos showing normal PCR signals were replaced, resulting in a singleton pregnancy. Prenatal diagnosis showed a female karyotype and DNA analysis indicated that the fetus was not a DMD carrier.     

Rapid sexing of human embryos by non-radioactive in situ hybridization: Potential for preimplantation diagnosis of X-linked disorders

Sixty spare human embryos at various stages of preimplantation development were prepared for cytogenetic analysis. Fluorescent staining of those with metaphases allowed scoring for the presence of a Y chromosome. In situ hybridization was then performed using a biotinylated Y-specific sequence, and the probe was detected by a standard streptavidinlinked alkaline phosphatase system. This enabled comparison of the chromosomal sex with that obtained after in situ hybridization in 28 embryos, and the sexing result obtained by the two methods was concordant in all cases. A further 21 embryos in which no metaphase chromosomes were obtained were sexed by biotinylated in situ hybridization only. Overall, 66 per cent of male interphase nuclei demonstrated a Y-specific hybridization signal. Results were obtained in under 24 h, which may permit the sexing of an embryo biopsied during cleavage and the transfer of sexed embryos at the blastocyst stage to the mother's uterus in the same cycle as oocytes are collected for in vitro fertilization.     

蚯蚓金属硫蛋白定量PCR检测方法及其分子诊断

根据GenBank中蚯蚓(Eisenia fetida)金属硫蛋白(MT)基因序列设计合成引物,采用RT-PCR扩增出目的基因片段,将目的基因片段插入pEASY-Blunt中制备质粒标准品.通过不同梯度稀释倍数的质粒标准品,建立了目的基因MT与内参基因β-actin的SYBR Green I荧光定量RT-PCR检测方法.通过土壤染毒培养实验,将蚯蚓分别暴露于50,500,1000mg/kg 重金属Cd染毒的自然土壤中培养14,28d,研究蚯蚓体内MT mRNA转录水平的表达变化.结果表明,重组质粒测序后显示目的片段序列同源性好,证明所设计的MT、β-actin引物能成功用于蚯蚓MT mRNA的检测.两基因构建的荧光定量标准曲线线性关系分别为0.994,0.999,且PCR扩增效率也皆接近于100%.染毒实验发现,Cd可诱导蚯蚓体内MT mRNA的表达,其表达水平与Cd污染暴露呈现出剂量-效应和时间-效应关系,表明蚯蚓MT基因可作为潜在分子标志物,诊断环境中Cd污染及其暴露水平.     

Detection of both the normal and mutant alleles in single cells of individuals heterozygous for the sickle cell mutation—prelude to preimplantation diagnosis

As a preliminary step to preimplantation diagnosis of sickle cell disease in unfertilized eggs or 8-cell embryos of heterozygous parents, we established quality control for detection of the mutant and normal alleles of the beta-haemoglobin gene using single buccal cells. Efficient polymerase chain reaction (PCR) amplification of a 680 base pair sequence of the beta-globin gene spanning the site of the sickle cell mutation was obtained for 79 per cent of single heterozygous cells. In 71 per cent of cases, both alleles were detected. With this current efficiency, we predict that a clinical preimplantation diagnosis at the 8-cell embryo stage could be carried out safely and reliably for a couple at risk of transmitting sickle cell disease to their children.     

Superovulation fails to increase human blastocyst yield after uterine lavage

Uterine lavage affords the potential for non-invasive human blastocyst recovery, with obvious potential for preimplantation genetic diagnosis. In an effort to duplicate in women the multiple blastocyst recovery per cycle that can be achieved in several other species, we initiated a programme in which fertile women underwent superovulation, followed by lavage and embryo collection. We superovulated 15 fertile women, aged 21–40, in 29 cycles using one of four regimens. Insemination was by either intercourse or artificial intracervical donor insemination with cryopreserved sperm from men of proven fertility. In 28 of 29 cycles, the uterus was lavaged daily for 1, 2, or 3 days between 5 and 10 days after human chorionic gonadotropin (hCG) administration or luteinizing hormone (LH) surge. Almost total fluid volume was recovered in every lavage. There were no retained pregnancies and no complications. Surprisingly, only two morulae, one blastocyst, and four unfertilized ova were recovered. Thus, alterations in ovulation induction, insemination timing, or lavage techniques must be contemplated in order to increase the blastocyst yield and thus fulfil the potential of uterine lavage for preimplantation diagnosis.     

Preimplantation diagnosis

Research towards preimplantation diagnosis of genetic disease was initiated in the UK. in the mid 1980s with the aim of helping those couples who would prefer selection to occur at this stage rather than during pregnancy. Following in vitro fertilisation, (IVF), biopsy and removal of 1 or 2 of the totipotent cells from the cleavage stage 3 day old embryo provides the material for molecular genetic diagnosis without interfering with development. Earliest applications were in the avoidance of X-linked disease by sexing embryos and selecting females for transfer to the mother. Initially, polymerase chain reaction (PCR) amplification of DNA from the biopsied blastomeres was performed using primers specific for sequences derived from the Y chromosome and this led to the birth of several normal girls. To reduce the risk of misdiagnosis due to amplification failure, PCR based methods for sexing the embryo now employ both X and Y specific sequences, but the preferred method is currently considered to be fluorescent in situ hybridisation (FISH) with fluorochrome labelled DNA probes to the embryonic nuclei that have been fixed and spread on slides. Dual FISH with probes from X and Y chromosomes allows unequivocal diagnosis of sex and determination of chromosome copy number, avoiding transfer of embryos with abnormal numbers of sex chromosomes, including those with only the maternal X that would be at 50% risk for the X-linked disease. The application of FISH for preimplantation diagnosis has also led to the realisation that chromosomal mosaicism is common at the cleavage stage of development, a finding that has important implications for diagnosis of both dominant single gene disorders and trisomies, as well as for our understanding of early human development. Cloning and sequencing of the relevant genes has enabled the development of methods for the diagnosis of certain recessive single gene disorders in cleavage stage embryos. PCR based methods have to be developed for each condition, sometimes for each family if there is heterogeneity. Preimplantation diagnosis has been successful so far for cystic fibrosis, Tay Sachs disease, and Lesch-Nyhan syndrome. Worldwide, 32 pregnancies have been established following all types of preimplantation diagnosis and with 29 babies born, there is no evidence for any adverse effect on development.     

An approach to preimplantation diagnosis of β-thalassaemia

Using the polymerase chain reaction (PCR), it was possible to amplify a single copy fragment of the β-globin gene from 2–32 human embryonic cells obtained from arrested preimplantation embryos. For the detection of β-thalassaemia mutations, allele specific priming of the PCR using nested primers was employed using approximately 10 pg of DN A from individuals known to carry these mutations. This approach was successful in detecting the presence or absence of five Asian Indian β-thalassaemia mutations that were selected for this study. In spite of meticulous precautions against contamination, false-positive amplification was observed, a problem that will have to be overcome before this approach can be used in clinical practice.     

Preimplantation genetic diagnosis of the fragile X syndrome by use of linked polymorphic markers

Fragile X syndrome is the most common cause of familial mental retardation. The most common mutation is expansion of a triplet (CGG)_n repeat in the 5′ untranslated region of the FMR1 gene on Xq27.3. The expansion is refractory to PCR due to preferential amplification of the smaller allele in heterozygous cells and the high GC content of the repeat and surrounding sequences. Direct detection of the normal parental alleles in preimplantation embryos has been used for preimplantation genetic diagnosis (PGD) of this disorder. However, this approach is only suitable for approximately 63% of couples due to the heterozygosity of the repeat in the normal population. As an alternative we investigated the use of polymorphic markers flanking the mutation to track the normal and premutation carrying maternal chromosomes in preimplantation embryos. Using a panel of 11 polymorphisms, six (CA)_n repeats and five single nucleotide polymorphisms, diagnosis was developed for 90% of referred couples. Multiplex amplification of informative markers was tested in 300 single buccal cells from interested couples with efficiency and allele drop out (ADO) rates ranging from 69% to 96% and 6% to 18%, respectively. Use of this approach is accurate and applicable to a larger number of patients at risk of transmitting fragile X to their offspring. Copyright © 2001 John Wiley & Sons, Ltd.     

Preimplantation diagnosis: A patient perspective

A new dimension in the prevention of birth defects will be achieved when genetic diseases can be routinely diagnosed in embryos prior to implantation. The impressions and attitudes towards preimplantation diagnosis were studied in prospective patients, women at high reproductive risk for a genetic disease. Their perspective highlighted not only the advantages and disadvantages of this new approach, but also those changes necessary in order for preimplantation diagnosis to become a useful and practical technique. The data presented are based on information obtained by a mailed questionnaire answered by 58 women. The main benefit of preimplantation diagnosis for these high-risk women would be the ability to undertake a pregnancy without having to be subjected to the physical and/or emotional trauma of elective termination. Their major concerns related to possible damage to the embryo following biopsy, the cost of the procedure, and the low success rate of completed pregnancies. Other issues to be addressed before preimplantation diagnosis could begin to compare favourably with existing forms of prenatal testing were that the methods of obtaining oocytes or embryos should be simple, well tolerated, highly efficient, and low in maternal risk, and that the genetic analysis of embryonic or extraembryonic cells should be unequivocally accurate.     

水中轮状病毒实时定量PCR外标准品的构建

采用细胞培养和T-A克隆技术,在轮状病毒主要结构蛋白VP7基因序列上设计合成引物,经PCR扩增后将特异性产物连接入pGEM-T-easy载体中,经过酶切鉴定和测序分析获得轮状病毒cDNA标准品.利用常规PCR和实时定量PCR方法对所获得的cDNA标准品进行特异性、稳定性和重复性指标的检验.结果表明,利用此标准品制备的标准曲线具有较高的扩增效率和良好的线性关系(斜率为-3.353,R2=0.995);实时定量PCR熔解曲线分析表明,温度在81℃±0.5℃的PCR产物是轮状病毒VP7序列上的特异性产物,表明此标准品是轮状病毒特异性的;同时,所制备的标准品在实时定量PCR检测中具有较大的线性范围(9×100～9×1011 copies/μL,每个反应最低可以检测到9个拷贝数的轮状病毒cDNA,表明利用该标准品进行实时定量PCR分析时具有较高的检测灵敏性;此外,该标准品具有较高的稳定性和重复性(3次独立实验CV值在0.2%～0.9%之间),可长期稳定保存.构建的标准品可用作检测水环境中轮状病毒的实时荧光定量PCR的外标准品.     

Preimplantation genetic diagnosis for aneuploidy screening in early human embryos: a review

Embryonic aneuploidies may be responsible for pregnancy failure in many IVF patients. In recent years, fluorescent in situ hybridisation (FISH) for multiple chromosomes has been used to document a high frequency of chromosomal errors and aneuploidy in human preimplantation embryos and, after embryo biopsy, to select embryos that are more likely to implant. Such studies suggest that women with recurrent miscarriage and advanced maternal age may benefit most from preimplantation genetic diagnosis with aneuploidy screening (PGD-AS). The success of PGD-AS is likely to be enhanced by new technologies, such as comparative genomic hybridisation, which enable full karyotyping of single cells. Copyright © 2002 John Wiley & Sons, Ltd.     

镉对长江华溪蟹金属硫蛋白mRNA表达的诱导及低分子量壳聚糖的联合作用

采用实时荧光定量PCR法,研究了不同浓度镉（Cd2＋）对长江华溪蟹（Sinopotamon yangtsekiense）金属硫蛋白（metallothionein,简称MT）在肝胰腺、鳃、肌肉和心脏中mRNA表达的诱导作用及低分子量壳聚糖（lowmolecular weight chitosan,简称LMWC）的联合作...     

Comparative Analysis of Viral Concentration Methods for Detecting the HAV Genome Using Real-Time RT-PCR Amplification

Hepatitis A is a major infectious disease epidemiologically associated with foodborne and waterborne outbreaks. Molecular detection using real-time RT-PCR to detect the hepatitis A virus (HAV) in contaminated vegetables can be hindered by low-virus recoveries during the concentration process and by natural PCR inhibitors in vegetables. This study evaluated three virus concentration methods from vegetables: polyethylene glycol (PEG) precipitation, ultrafiltration (UF), and immunomagnetic separation (IMS). UF was the most efficient concentration method, while PEG and IMS were very low for the recovery rate of HAV. These results demonstrate that UF is the most appropriate method for recovering HAV from contaminated vegetables and that this method combined with the real-time RT-PCR assay may be suitable for routine laboratory use.