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W Rens

Publications and source records attributed to W Rens.

35 records · Page 2Linked to original sources
PubMed1999

The integration of canine genetic maps with the canine karyotype using specific gene amplification of chromosome-specific DNA.

We have used a rapid approach to place markers that are already represented in current genetic maps onto individual chromosomes in species for which chromosome paints exist. PCR-based techniques are used to look for the presence of individual marker genes within each chromosome-specific DNA pool. The presence of a given marker within a DNA pool allows assignment of the complete radiation hybrid group, or linkage group from which the marker is drawn, to an individual chromosome. We have used this method with a new set of canine chromosome paints (Yang et al., 1999). In this way, we have assigned 39 of 44 published RH or syntenic RH groups to canine chromosomes, together with 33 of 40 canine linkage groups in a recently published map (Neff et al., 1999).

Animals↗
PubMed1999

Complete homology maps of the rabbit (Oryctolagus cuniculus) and human by reciprocal chromosome painting.

Fluorescence in situ hybridization (FISH) was used to construct a homology map to analyse the extent of evolutionary conservation of chromosome segments between human and rabbit (Oryctolagus cuniculus, 2n = 44). Chromosome-specific probes were established by bivariate fluorescence activated flow sorting followed by degenerate oligonucleotide-primed PCR (DOP-PCR). Painting of rabbit probes to human chromosomes and vice versa allowed a detailed analysis of the homology between these species. All rabbit chromosome paints, except for the Y paint, hybridized to human chromosomes. All human chromosome paints, except for the Y paint, hybridized to rabbit chromosomes. The results obtained revealed extensive genome conservation between the two species. Rabbit chromosomes 12, 19 and X were found to be completely homologous to human chromosomes 6, 17 and X, respectively. All other human chromosomes were homologous to two or sometimes three rabbit chromosomes. Many conserved chromosome segments found previously in other mammals (e.g. cat, pig, cattle, Indian muntjac) were also found to be conserved in rabbit chromosomes.

Animals↗
PubMed1999

The Beltsville sperm sexing technology: high-speed sperm sorting gives improved sperm output for in vitro fertilization and AI.

The Beltsville sperm sexing technology is currently the only effective means of altering the sex ratio of offspring in livestock. The method is based on the flow-cytometric separation of X- and Y-chromosome-bearing sperm based on X/Y DNA content difference. It is an effective means of producing progeny of predetermined sex in cattle, swine, sheep, and laboratory animals. The method involves treating sperm with a DNA-binding fluorochrome, Hoechst 33342, and flow-cytometrically sorting them into separate X and Y populations that can subsequently be used for surgical intratubal or intrauterine insemination, deep-uterine insemination, regular artificial insemination in some cases, in vitro fertilization to produce sexed embryos for transfer, and intracytoplasmic sperm injection of ova. Skewed sex ratios of 85 to 95% of one sex or the other have been repeatably achieved in most species. The method has been used worldwide to produce several hundred morphologically normal animal offspring of the predicted sex. It has also been validated in the laboratory using DNA reanalysis of the sorted sperm populations and by fluorescence in situ hybridization and PCR of individual sperm. We developed a new orienting nozzle that we have fitted to both conventional and high-speed cell sorters that have been modified for sperm sorting. Recently we completed the adaptation of the new orienting nozzle to a Cytomation MoFlo high-speed cell sorter modified for sperm. This adaptation of the nozzle has increased the overall production rate of sorted X and Y sperm from about .35 million/h to 5 or 6 million sperm/h (each population). Calves have been born from cows artificially inseminated using conventional technique and sexed sperm. In addition, numerous litters of pigs have been born after transfer of embryos produced from X or Y sorted sperm.

Animals↗
PubMed1998

A novel nozzle for more efficient sperm orientation to improve sorting efficiency of X and Y chromosome-bearing sperm.

Efficient high-resolution detection of DNA for flow cytometric sorting of X and Y chromosome-bearing sperm is dependent on effectively orientating the sperm head to the laser beam in orthogonally configured flow systems. Normally, a beveled needle is required to enlarge the fraction of properly orientated sperm (flat side facing the laser beam). In this report, a modification to a standard jet-in-air nozzle for improved sperm orientation is presented. Inside the modified nozzle (novel nozzle), orientation forces are applied lower in the nozzle than in the current beveled injection needle system. The nozzle was tested with sperm heads from several species. This study shows that use of the nozzle to orientate cattle, swine, rabbit, mouse, and human sperm effectively improves the percentage of sperm that are properly oriented. The percentage of sperm heads oriented by use of the former system (beveled needle) ranges around 30% for most species. With the newly designed nozzle, that percentage ranges around 60%. At least a twofold increase in analysis is achieved. It was found that, unlike results with the beveled needle, the percentage of properly oriented sperm was independent of the sample rate. The introduced nozzle is a significant improvement over the beveled needle system for the analysis and sorting of sperm on the basis of DNA content. In addition to the improvement in sorted sperm production brought about by the novel nozzle when fitted to standard-speed cell sorters, it clearly also has significant potential for improving the efficiency of the Beltsville Sperm Sexing Technology for separating X and Y chromosome-bearing sperm when adapted to high-speed cell-sorting systems.

Animals↗
PubMed1996

Slit-scan flow cytometry for consistent high resolution DNA analysis of X- and Y-chromosome bearing sperm.

This paper describes the application of slit-scan flow cytometry for accurate DNA analysis of X- and Y-chromosome bearing sperm. The introduction of the slit-scanning technique was initiated to improve the consistency in resolution of the X and Y population from donor to donor. An optimal resolution is essential for high purity sorting of X and Y sperm, as the difference in DNA content is small (3-4%) in most mammals. This difference is the discriminatory parameter for the flow cytometric sorting of the two populations. Our approach was to focus on the role of the sperm tail in the detection process. Slit-scan flow cytometric analysis allows the whole sperm to be spatially analyzed along the direction of flow. Sperm were stained with Dansyl Lysine, a UV excitable fluorescent membrane dye, which stained the head, midpiece, and principal piece. Analysis of these stained sperm showed that there was no difference between the relative number of sperm that travel headfirst or tailfirst through the detection zone of the flow cytometer. The influence of sperm with coiled tails on DNA analysis was also investigated. The proportion of sperm with coiled tails influences semen quality. The standard X-Y separation procedure uses Hoechst 33342, which stains all intact sperm, both living and dead. Propidium iodide was added to discriminate the dead sperm population. Slit-scan analysis showed that measurement of a sample containing a high proportion of living sperm with coiled tails results in an inferior DNA histogram and reduced X-Y resolution. Sperm with coiled tails can result in a lower detected fluorescence intensity, but the reason for this is unclear. Slit-scan flow cytometry allows exclusion of sperm with coiled tails from the analysis, resulting in a restoration of high resolution of X- and Y-chromosome bearing sperm populations.

Animals↗
PubMed1994

Slit-scanning technique using standard cell sorter instruments for analyzing and sorting nonacrocentric human chromosomes, including small ones.

We have investigated the performance of two types of standard flow cell sorter instruments, a System 50 Cytofluorograph and a FACSTar PLUS cell sorter, for the on-line centromeric index (CI) analysis of human chromosomes. To optimize the results, we improved the detection efficiency for centromeres in two ways. A higher efficiency was obtained first by elongation of the chromosomes and second by introducing a high resolution lens system for laser beam focusing. In the two-parameter flow karyotype of CI and DNA content of human chromosomes, distinct peaks are produced not only by the larger chromosomes 1-8 and X, but by the smaller nonacrocentric chromosomes 9-12 and 16-20 as well. As the chromosomes 9-12 cannot be distinguished by other flow karyotyping methods, we discriminated and sorted chromosomes 12 and 10 from 9 and 11 to investigate the capacity for the separation of chromosomes in this group. A purity of at least 90% was achieved; in the isolated population the fraction chromosomes 12 was 55%; the remaining 45% were chromosomes 10 (40%) and unidentifiable chromosomes (5%).

Cell Separation↗
PubMed1994

Construction of mouse chromosome-specific DNA libraries and their use for the detection of X-ray-induced aberrations.

We describe here the development of mouse chromosome-specific DNA libraries and their use in the detection of radiation-induced chromosome aberrations by fluorescence in situ hybridization. Large metacentric chromosomes, resulting from a translocation involving chromosomes 1, 11 and 13, were flow-sorted. Using a slit-scan technique for morphometric analysis, metacentric chromosomes were separated from normal acrocentric chromosomes and their aggregates. DNA from the metacentric chromosomes was amplified by PCR using the linker/adaptor method. In this pilot study, mouse was whole-body irradiated with 1, 2 and 3 Gy and aberrations were scored in metaphase spreads of splenocytes cultured in vitro. The results indicate that directly after radiation exposure, stable and unstable aberrations are induced at about equal frequencies in the splenocytes. The availability of chromosome-specific probes for mouse may prove very useful when analysing the behaviour of stable aberrations, as well as the testing of many suspected mutagenic carcinogens and aneugens in vivo for induction of chromosomal translocations and non-disjunction, respectively.

Animals↗
PubMed1994

Flow cytometric detection of chromosome abnormalities by measuring centromeric index, DNA content, and DNA base composition.

This paper highlights two improvements of the on-line centromeric index (CI) analysis for the detection of chromosome abnormalities. On-line CI versus DNA content analysis of an EBV-transformed cell line, with a deletion (11)(p13p15.1), of a patient with aniridia and Wilms' tumour demonstrates the first improvement of the method of on-line CI analysis for flow karyotyping and sorting; a reciprocal translocation, insertion, or deletion can, when the cell type contains not more than a few of these types of abnormalities, be traced to the p-arm or q-arm of the relevant chromosome. On-line CI analysis was also performed with chromosomes isolated from a transitional cell carcinoma of the bladder. Cytogenetic analysis of this cell line showed numerous chromosomal abnormalities. Chromosomes of this cell line were also karyotyped by bivariate flow cytometry using a different set of parameters: Hoechst 33,258 fluorescence intensity (HOfl) versus chromomycin A3 fluorescence intensity (CAfl). A comparison of these results reveals the second improvement of the CI method for the detection of chromosome abnormalities; bivariate analysis of CI versus propidium fluorescence (PIfl) are complementary to bivariate analysis of HOfl versus CAfl. Chromosomes with distributions that fuse together in the HO/CA flow karyotype may be distinguished as individual peaks on the basis of their CI values.

Aniridia↗
PubMed1993

Identification of a tumor marker chromosome by flow sorting, DNA amplification in vitro, and in situ hybridization of the amplified product.

A method combining flow sorting and molecular cytogenetic techniques for the identification of unknown marker chromosomes is described. In this study, the bladder tumor cell line J82 was used, which was known to carry a marker chromosome of the size of chromosome 7 in every cell. From the cytogenetic analysis of Q-banded metaphase cells, it was shown to be composed of approximately 40% presumably the greater part of chromosome 20 and for the rest microscopically unidentifiable material. This marker chromosome was found using flow cytometric analysis to form an independent peak and hence was suitable for isolation using dual-parameter sorting after staining with Hoechst 33258 and chromomycin A3. Subsequently, the marker was isolated by dual-parameter sorting. DNA amplification of 300 isolated chromosomes by polymerase chain reaction (PCR) using the Alu-primer Bk33 and the LINES-primer LH5 was carried out. After purification of the amplified product, a yield of 5 microns of DNA was obtained. The DNA was labelled using Bio-11-dUTP and applied to human lymphocyte metaphase cells in a suppressive in situ hybridization procedure. Fluorescence was visible over chromosome 20 and over the distal one-half of 6p. Together the fluorescent regions accounted for only approximately 60% of the marker length, indicating a possible duplication of chromosome 20 material. This was confirmed by applying bicolor in situ hybridization using chromosome 6- and 20-specific DNA libraries to metaphase cells of the J82 cells.

Aneuploidy↗
PubMed1993

Time-optimized analysis of slit-scan chromosome profiles on a general-purpose personal computer.

Slit-scan flow cytometry provides a method to analyze large numbers of metaphase chromosomes in a relatively short time due to morphological features. The high detection rate requires fast computing for on-line analysis. Up to now, this has been achieved using special-purpose computers, parallel systems or other complex hardware. Here, we describe an algorithm that can be implemented on a general-purpose personal computer. Digitized chromosome profiles can be classified by several criteria especially for the detection of chromosome abnormalities in biological dosimetry. A data set of approximately 4600 profiles was used. Programming in assembler results in an average computing time of about 600 microseconds per profile. Thus on-line evaluation of slit-scanning data appears to become feasible for many flow cytometers running nowadays.

Algorithms↗
PubMed1993

Effectiveness of pulse-shape criteria for the selection of dicentric chromosomes by slit-scan flow cytometry and sorting.

A method was developed to detect dicentric chromosomes by slit-scan flow cytometry. The two centromeres of dicentric chromosomes are represented by the two dips in the trimodal fluorescence profile. A trimodal profile can, however, also be generated by aggregates of chromosomes. We tested the effectiveness of slit-scan profile criteria that were applied to discriminate between trimodal profiles generated by dicentrics and trimodal profiles generated by artefacts. A Profile-Dip Counter (PDC) module was designed that can assess, in real time, the number of dips in slit-scan profiles. The PDC module was used in combination with a Cytofluorograph System 50 cell sorter for slit-scan sorting of chromosomes prepared from irradiated V79 cells. Chromosomes corresponding to trimodal profiles were sorted individually onto slides for subsequent visual inspection by fluorescence microscopy. The isolated chromosomes were stretched by treatment with trypsin to increase the efficiency for centromere detection. When fixed with glutaraldehyde, chromosomes could be sorted intact on slides. We found that trimodal profiles are generated by dicentric chromosomes as well as by monocentric and aggregated chromosomes. When stringent pulse-shape criteria were applied for the selection of profiles, the yield of dicentric chromosomes was 70% of the sorted chromosomes.

Animals↗
PubMed1992

Semi-automated detection of aberrant chromosomes in bivariate flow karyotypes.

A method is described that is designed to compare, in a standardized procedure, bivariate flow karyotypes of Hoechst 33258 (HO)/Chromomycin A3 (CA) stained human chromosomes from cells with aberrations with a reference flow karyotype of normal chromosomes. In addition to uniform normalization of normal and abnormal flow karyotypes, the main purpose is detection of structurally abnormal chromosomes in often complex karyotypes of tumor cells. The method, which has been implemented in a computer program, consists of a comparison of individual chromosome peaks with the positions of peaks in the flow karyotype constituted by normal chromosomes and takes into account the natural variability in base composition of normal chromosomes among healthy individuals. Flow-karyotypes are normalized using an iterative fitting procedure, using corrections for (1) amplification of HO and CA fluorescence, (2) cross-talk between the fluorescence signals of HO and CA, and (3) offset of the HO and CA origin. Flow karyotypes of two cell lines, one with a simple deletion and the other with more complex karyotypic changes, were analyzed. The results of flow analysis were found to be in general agreement with the cytogenetic analysis of quinacrine banded karyotypes.

Cells, Cultured↗
PubMed1992

Detection of recurrent chromosome abnormalities in Ewing's sarcoma and peripheral neuroectodermal tumor cells using bivariate flow karyotyping.

Bivariate flow karyotyping can be used for the detection of recurrent chromosome abnormalities in tumor cells. For this purpose 2 cell lines originally derived from Ewing's sarcomas and 4 cell lines from peripheral neuroectodermal tumors were used. The characteristic t(11;22) was known to be present in 5 cell lines. The remaining cell line was known to have a variant t(2;11;22;21) translocation. Metaphase chromosomes were stained with the fluorescent dyes Hoechst 33258 and Chromomycin A3 and analyzed subsequently using bivariate flow cytometry. The resulting bivariate flow karyotypes of the tumor cells were normalized by a standardized procedure using a computerized method and compared with a reference flow karyotype of normal chromosomes. In 5 cell lines two recurring abnormal chromosome peaks were identified at positions expected for the der(11) and der(22) chromosomes characteristic for the reciprocal t(11;22)(q24;q12). In the remaining cell line with the variant t(2;11;22;21), only the peak representing the der(22) was identifiable. It is concluded that bivariate flow karyotyping can be used for the semiautomated detection of recurrent translocations and the assessment of their variability among different tumors.

Bisbenzimidazole↗
PubMed1991

Bivariate flow karyotyping of human chromosomes: evaluation of variation in Hoechst 33258 fluorescence, chromomycin A3 fluorescence, and relative chromosomal DNA content.

The total variation of chromosome peak positions, in bivariate distributions of Hoechst 33258 and chromomycin A3 fluorescence of 19 healthy individuals, was compared with the experimental variation, determined from 23 bivariate distributions of chromosomes prepared separately from a single cell lineage. The experimental variation in Hoechst and chromomycin fluorescence and the relative chromosomal DNA content were determined from experiments performed over several days. The additional variance contributed by time was the same as the daily variance. The accuracy by which the relative chromosomal DNA content can be calculated from bivariate peak positions was investigated. A least squares method was used to fit the distributions of relative DNA content, obtained, respectively, from mono- and bivariate flow analyses of chromosomes from the same cell lineage. In general the DNA contents match quite well, but for a few chromosomes a difference was found, statistically discernible at the 5% level. The average relative chromosomal DNA content of the chromosomes from the 19 normal individuals, calculated from bivariate peak positions, showed a linear relation with the estimates published by other investigators.

Bisbenzimidazole↗
PubMed1990

On-line sorting of human chromosomes by centromeric index, and identification of sorted populations by GTG-banding and fluorescent in situ hybridization.

Using slit-scan flow cytometry, the shape of human metaphase chromosomes, as expressed in their centromeric index (CI), and the DNA content of the chromosomes have been used as parameters in bivariate flow karyotyping. The resolution of the DNA vs CI flow karyogram of the larger chromosomes up to chromosome 13 is much higher than the resolution obtained in the DNA-based monovariate flow karyogram. Chromosome length appears to be an important factor in the resolution of the DNA vs CI-based flow karyogram. A method has been developed to obtain chromosomes in suspension that are long enough for adequate analysis. Several chromosomes that cannot be distinguished or are difficult to discriminate in the DNA-based karyogram can now be distinguished as individual peaks, e.g., chromosomes 1 and 2. The peak of chromosomes 9-12 can be separated into two peaks formed by chromosomes 9 and 11, and 10 and 12, respectively. The advantage of the system applied in this study is that the DNA vs CI analysis is performed on-line, allowing chromosomes to be sorted on the bases of their CI. Pulse shapes of the selected chromosomes can be recorded simultaneously with the transmission of the sorting command. The purity of the sorted fraction can be estimated from the off-line inspection of these pulse shapes. Fractions of chromosome 1 have been sorted out on the basis of the CI information, centrifuged on slides, fixed and subsequently banded with trypsin and Giemsa or hybridized with the chromosome 1 specific probe, pUC 1.77. The observed purity under the selected conditions ranges from 80%-99% and is in accordance with the estimates of the purities made on the basis of the simultaneously recorded pulse shapes. Fixation of the chromosome suspension prior to flow cytometric analysis and sorting appears to be essential for the preservation of their morphology and has no adverse influence on the resolution of Giemsa banding or on the quality of in situ hybridization.

Cell Line↗
PubMedDate not supplied

Single UV excitation of Hoechst 33342 and propidium iodide for viability assessment of rhesus monkey spermatozoa using flow cytometry.

Many fluorescent probes excited by visible light have been used to assess sperm quality by flow cytometry. Developing a viability evaluation method using UV excited stains would be useful for multiparameter analysis of sperm function. This investigation was conducted to determine the efficacy of Hoechst 33342 (H342) and propidium iodide (PI) dual staining for evaluating rhesus monkey sperm viability through use of flow cytometry and excited by a single UV laser. The results showed that the live cells stained only with H342 strongly correlated with expected sperm viability, and flow cytometric analyses were highly correlated with fluorescence microscopic observation. Using H342/PI/SYBR-14 triple staining method, it was found that the live/dead sperm distributions were completely concordant in both H342/PI and SYBR-14/PI assays. In addition, this dual staining was extended with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) to simultaneously analyze viability and acrosome integrity of sperm cryopreserved using two different extenders, TTE and TEST, and indicated that TTE offered better preservation of plasma and acrosome integrity than TEST. Therefore, the H342/PI dual staining provides an accurate technique for evaluating viability of rhesus monkey sperm and should be valuable for multiparameter flow cytometric analysis of sperm function.

Animals↗