Trophoblast-like cells sorted from peripheral maternal blood using flow cytometry: A multiparametric study involving transmission electron microscopy and fetal dna amplification

Three monoclonal antibodies (MAbs) against trophoblast (GB17, GB21, and GB25) and flow cytometry were used to sort trophoblast-like cells (TLCs) from peripheral blood of pregnant women. Sorted TLCs were processed for electron microscopy and fetal DNA amplification of the Y-specific sequences from mothers carrying male fetuses. At the ultra-structural level, most of the nucleated cells had the morphology of leucocytes, suggesting maternal contaminants, and we did not find the characteristic features of the free inter-villous trophoblast cells. Nevertheless, polymerase chain reaction (PCR) analysis showed an amplification of Y-specific sequences in two out of three samples of sorted TLCs. These results suggest that besides the maternal leucocytes, sufficient trophoblast nucleated fetal cells can be obtained using cell enrichment by sorting. This sensitive method holds promise for non-invasive prenatal diagnosis of fetal sex and if sufficient Y(positive) nuclei are found, for the diagnosis of selected numerical chromosome abnormalities.     

Characterization of microbial communities from coastal waters using microarrays

Molecular methods, including DNA probes, were used to identify and enumerate pathogenic Vibrio species in the Chesapeake Bay; our data indicated that Vibrio vulnificus exhibits seasonal fluctuations in number. Our work included a characterization of total microbial communities from the Bay; development of microarrays that identify and quantify the diversity of those communities; and observation of temporal changes in those communities. To identify members of the microbial community, we amplified the 16S rDNA gene from community DNA isolated from a biofilm sample collected from the Chesapeake Bay in February, 2000. The resultant 75 sequences were 95% or more similar to 7 species including two recently described Shewanella species, baltica and frigidimarina, that have not been previously isolated from the Chesapeake. When the genera of bacteria from biofilm after culturing are compared to those detected by subcloning amplified 16S fragments from community DNA, the cultured sample exhibited a strong bias. In oysters collected in February, the most common bacteria were previously unknown. Based on our 16S findings, we are developing microarrays to detect these and other microbial species in these estuarine communities. The microarrays will detect each species using four distinct loci, with the multiple loci serving as an internal control. The accuracy of the microarray will be measured using sentinel species such as Aeromonas species, Escherichia coli, and Vibrio vulnificus. Using microarrays, it should be possible to determine the annual fluctuations of bacterial species (culturable and non-culturable, pathogenic and non-pathogenic). The data may be applied to understanding patterns of environmental change; assessing the health of the Bay; and evaluating the risk of human illness associated with exposure to and ingestion of water and shellfish.     

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.     

A routine method for storing lymphocytes for repeated isolation of DNA

A protocol for easy storage and later expansion of lymphocyte populations is given. Compared with methods using transformed cell lines, the method has a number of advantages for repeated production of cells for the isolation of DNA in amounts sufficient for use in diagnostic DNA technology.     

In vitro characteristics of human fetal cells obtained from chorionic villus sampling and amniocentesis

In vitro characteristics of human fetal cells have been investigated after chorionic villus sampling at the first trimester and amniocentesis at the second trimester of pregnancy. Light microscopy revealed heterogeneous morphology of cell types in both the chorionic villus culture and the amniotic fluid cultures. Based on the experiments performed, chorionic villus cells are more sensitive to pronase, trypsin, and versene during subculture and have a higher DNA content per single cell and release more [¹²⁵I]-Beta-human chorionic gonadotropin into culture medium than those found in amniotic fluid cells. The practical applications of this study are discussed.     

DNA polymorphism and globin chain analysis in the prenatal diagnosis of β-thalassaemia major in Taiwan

Thirty-six pregnancies in 25 families at risk of β-thalassaemia major received prenatal diagnosis. Chorionic villus sampling or amniocentesis was done in 35 pregnancies to obtain fetal cells for DNA linkage study, for which Southern blotting and DNA hybridization were used to detect seven restriction fragment length polymorphisms (RFLPs) within the β-globin gene cluster: ϵ-HincII, ^Gγ-HindIII, ^Aγ-HindIII, Φβ-HincII, 3′Φβ-HincII, β-AvaII, and 3′β BarnHI. β-Thalassaemia major was diagnosed in seven and excluded in 22 pregnancies. In the remaining six cases, β-thalassaemia major could not be excluded. In these six pregnancies and another one with late booking, ultrasound-guided cordocentesis was performed at the 22nd to 27th week of gestation. Globin chain composition was determined with urea-acetic acid-Triton X-100-12 per cent polyacrylamide gel electrophoresis. β-Thalassaemia major was diagnosed in two fetuses and excluded in the other five. Eleven fetuses (in which β-thalassaemia major was excluded) have been delivered and are healthy at more than 5 months old DNA linkage analysis coupled with globin chain electrophoresis provides an effective way for prenatal diagnosis of β-thalassaemia major, although these methods are being replaced by more direct detection techniques using oligonucleotide probes.     

Circulating ‘trophoblast’ cells in pregnancy have maternal genetic markers

A syncytiotrophoblast-associated antigen identified by the monoclonal antibody (McAb) H315 is detectable on the surface of a low proportion of peripheral blood cells in pregnant women, raising the possibility of a new approach to prenatal diagnosis of genetic disorders. We aimed at verifying the trophoblastic origin of H315⁺ cells and their use for prenatal diagnosis of β-thalassaemia. H315 ⁺ cells were separated from the peripheral blood of pregnant women: the DNA obtained from these cells in two selected cases was shown to have genetic markers indistinguishable from those of the mother and definitely different from the fetus. Our results suggest that H315 antigen is expressed by maternal cells and that prenatal diagnosis on peripheral blood of the mother using H315 McAb is not feasible.     

Prenatal diagnosis of 21-hydroxylase deficiency by rflp analysis of the 21-hydroxylase,complement C4, and HLA class II genes

In 19 pregnancies at risk for 21-hydroxylase deficiency (21OHD) in 18 families with at lea one affected child, prenatal diagnosis was performed by RFLP analysis using the enzymi Taq I and EcoRI and the DNA probes specific for the 21 OH genes, the closely linke complement C4 genes and the highly polymorphic HLA class II genes DRB, DQB, and DPI For fetal DNA analysis either chorionic villi or cultivated amniotic cells were used. In all 1 cases, a clear prenatal diagnosis was possible either with the 21OH probe alone or in mo cases, by combining the results of the different closely linked loci.     

Prenatal diagnosis of cystic fibrosis using linked DNA probes

This paper presents data collected in Europe on 107 prenatal diagnoses of cystic fibrosis (CF) using linked DNA markers. To date, 38 children have been born without CF, as predicted, demonstrating the present rapid move from research to clinical genetic service.     

丙烯酰胺对斑马鱼各器官的毒性作用及生殖细胞的DNA损伤

在我国,聚丙烯酰胺(PAM)主要应用于石油开采,其长期暴露于环境中可以降解成有毒的丙烯酰胺(AM)。为探究在降解过程中AM的毒性作用,选择斑马鱼作为受试动物,进行AM长期毒性暴露40 d,考察了对斑马鱼的肝脏、脑组织、心脏、腮等器官的影响情况。结果表明:在2.04 mg·L~(-1)、6.12 mg·L~(-1)和18.36 mg·L~(-1)暴露浓度下,形态学观察斑马鱼的鳃丝,鳃小片和鳃细胞有严重的受损现象;随着浓度的升高斑马鱼肝脏、脑组织和心脏中MDA含量、LDH活力的升高,SDH和Na+-K+-ATPase活力的降低,均对其肝脏、脑组织和心脏造成氧化损伤作用,从而影响了斑马鱼体内细胞能量代谢过程。采用彗星试验检测斑马鱼的生殖腺细胞DNA损伤,结果显示暴露于浓度为2.04 mg·L~(-1)~18.36 mg·L~(-1)的AM后,斑马鱼的DNA损伤均表现为显著差异(P0.01)。上述研究结果均确定了AM对斑马鱼的毒性效应并可造成其生殖腺细胞的DNA损伤。