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K J Schimenti

Publications and source records attributed to K J Schimenti.

10 recordsLinked to original sources
PubMed2000

Mouse mutants from chemically mutagenized embryonic stem cells.

The drive to characterize functions of human genes on a global scale has stimulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain and interlocus variation in mutation induction, and extensive husbandry requirements. To overcome these obstacles and develop new methods for generating mouse mutants, we devised protocols to generate germline chimaeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribosyl transferase). The spectrum of mutations induced by EMS and the frameshift mutagen ICR191 was consistent with that observed in other mammalian cells. Chimaeras derived from ES cells treated with EMS transmitted mutations affecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditional mutagenesis, including the ability to conduct shortened breeding schemes and to screen for mutant phenotypes directly in ES cells or their differentiated derivatives.

Abnormalities, Drug-Induced↗
PubMed1998

Factors affecting ectopic gene conversion in mice.

Duplicated genes and repetitive sequences are distributed throughout the genomes of complex organisms. The homology between related sequences can promote nonallelic (ectopic) recombination, including gene conversion and reciprocal exchange. Resolution of these events can result in translocations, deletions, or other harmful rearrangements. In yeast, ectopic recombination between sequences on nonhomologous chromosomes occurs at high frequency. Because the mammalian genome is replete with duplicated sequences and repetitive elements, high levels of ectopic exchange would cause aneuploidy and genome instability. To understand the factors regulating ectopic recombination in mice, we evaluated the effects of homology length on gene conversion between unlinked sequences in the male germline. Previously, we found high levels of gene conversion between lacZ transgenes containing 2557 bp of homology. We report here that genetic background can play a major role in ectopic recombination; frequency of gene conversion was reduced by more than an order of magnitude by transferring the transgenes from a CF1 strain background to C57BL/6J. Additionally, conversion rates decreased as the homology length decreased. Sequences sharing 1214 bp of sequence identity underwent ectopic conversion less frequently than a pair sharing 2557 bp of identity, while 624 bp was insufficient to catalyze gene conversion at significant levels. These results suggest that the germline recombination machinery in mammals has evolved in a way that prevents high levels of ectopic recombination between smaller classes of repetitive sequences, such as the Alu family. Additionally, genomic location appeared to influence the availability of sequences for ectopic recombination.

Animals↗
PubMed1998

Meiotic prophase arrest with failure of chromosome synapsis in mice deficient for Dmc1, a germline-specific RecA homolog.

DMC1 is a meiosis-specific gene first discovered in yeast that encodes a protein with homology to RecA and may be component of recombination nodules. Yeast dmc1 mutants are defective in crossing over and synaptonemal complex (SC) formation, and arrest in late prophase of meiosis I. We have generated a null mutation in the Dmc1 gene in mice and show that homozygous mutant males and females are sterile with arrest of gametogenesis in the first meiotic prophase. Chromosomes in mutant spermatocytes fail to synapse, despite the formation of axial elements that are the precursor to the SC. The strong similarity of phenotypes in Dmc1-deficient mice and yeast suggests that meiotic mechanisms have been highly conserved through evolution.

Adenosine Triphosphatases↗
PubMed1997

Molecular analysis of gene conversion in spermatids from transgenic mice.

Investigations into the mechanisms and properties of gene conversion in mammals are greatly restricted by the inability to recover all the products of a meiosis. Additionally, the study of this process has been hampered by the lack of visible markers to detect gene conversion, especially when the events are rare. In previous work, we developed a transgenic system for detection and quantitation of gene conversion events in the germline of mice (Murti, J.R., Bumbulis, M., Schimenti, J.C., 1992. High frequency germline gene conversion in transgenic mice. Mol. Cell. Biol. 12, 2545-2552) that could be exploited as an assay for recombinogenic chemicals (Murti, J.R, Schimenti, K.J., Schimenti, J.C., 1994. A recombination-based transgenic mouse system for genotoxicity testing. Mutat. Res. 307, 583-595). A specific intrachromosomal gene conversion event between two complementarily defective lacZ genes resulted in the production of beta-galactosidase in spermatids, enabling a measurement of conversion frequency. Here, we report that the anticancer drug, cisplatin, increased gene conversion in meiotic stage cells in these transgenic mice. Furthermore, a method was developed for direct molecular analysis of transgene conversion events in single or pooled lacZ-positive spermatids. The ability to identify gametes that have undergone a rare gene conversion event, followed by molecular amplification of the recombinant gene, should make it possible to investigate the mechanisms of genetic recombination in mammals in greater detail than previously possible.

Animals↗
PubMed1997

Evidence for cyclophosphamide-induced gene conversion and mutation in mouse germ cells.

Cyclophosphamide (CP) is a widely used antineoplastic drug. It tests positive in several genotoxicity assays, including those with endpoints such as chromosomal aberrations in mammalian cells, mitotic recombination in Drosophila melanogaster, and dominant lethal mutations in rodents. We have explored the effects of CP on genome stability of mouse (Mus domesticus) spermatogenic cells, using a recombination-based transgenic assay called MUSCATEER. In this system, intrachromosomal gene conversion events between two mutually defective lacZ genes generates beta-galactosidase activity in spermatids. The frequency of gene conversion events is determined by scoring spermatids stained with the lacZ substrate, X-gal. A dose-dependent induction of lacZ-positive spermatids was observed following single intraperitoneal CP exposures of 10, 100, and 200 mg/kg. At 200 mg/kg, there was a 25-fold increase over baseline. Treatment of a control transgenic line containing only a frame-shifted lacZ transgene provided an indication that CP also induced reversion mutations. The timing of the response indicated that the induction of recombination and/or mutation occurred primarily in meiotic stage cells. These results demonstrate potent germline mutagenicity of CP, and validate the utility and sensitivity of genetic recombination as a rapid indicator of genotoxicity in whole animals.

Animals↗
PubMed1994

A recombination-based transgenic mouse system for genotoxicity testing.

It is well established that mutagens induce recombination in cultured cells and experimental organisms. Presumably, this is a consequence of the DNA-damage-triggering cellular-repair mechanisms. The relationship between recombination and mutagenicity has been exploited in submammalian organisms, such as yeast, to assay the ability of chemical agents and radiation to induce a form of recombination called gene conversion--the non-reciprocal transfer of genetic information. This work has demonstrated the efficacy of predicting mutagenicity on the basis of recombination induction. Here, we describe the utilization of a transgenic mouse system for efficient detection of germ-line gene-conversion events as a mutagen-screening tool. These mice contain two mutually defective reporter (lacZ) genes under the regulatory control of a spermatogenesis-specific promoter. A particular intrachromosomal gene conversion event must occur for the generation of functional lacZ activity. Conversion events are visualized by histochemical staining or flow cytometric analysis of transgenic spermatids. The highly mutagenic compound chlorambucil induced a several fold percentage-wise increase of lacZ-positive spermatids, whereas acrylamide, a weak genotoxin, produced no marked increase in converted spermatids. The results indicate that recombination-based transgenic mouse models for genotoxin screening present a viable option for inexpensive and rapid whole-animal mutagen testing. The particular mice we describe may ultimately prove to be a useful tool for identifying agents which can cause heritable genetic mutations in humans.

Acrylamide↗
PubMed1992

Fixation of mammalian cells for flow cytometric evaluation of DNA content and nuclear immunofluorescence.

Mammalian tissue culture cells were fixed with 3 different alcoholic fixatives--acetone:methanol, EtOH, and MeOH. The quality of the resulting DNA histograms was evaluated by comparison of CV, G1/G2 ratio, G1 mode, cell aggregation, and debris formation; 81-90% MeOH (final concentration) was determined to be the optimal fixative by these criteria. A procedure was then examined using a prefix with paraformaldehyde followed by MeOH (PF/MeOH). This procedure produced cell preparations with reduced debris and aggregation, equivalent mode and ratio, but increased CV when compared with MeOH fixation. Both MeOH and PF/MeOH fixation procedures were then compared for their utility in dual staining for DNA and intracellular immunofluorescence for a nuclear protein, SV40 T antigen (Tag). Since alcohols are known to affect immunofluorescence staining of some antigens, fixation with paraformaldehyde followed by Triton X-100 permeabilization (PF/TX) was also included in this comparison to generalize the study by providing an alternative to MeOH permeabilization. The three procedures were evaluated for the quality of the sample by measuring the same descriptors of the DNA parameter as in the alcohol study. PF/TX consistently produced samples with decreased DNA CV and less debris and aggregation compared to MeOH methods. Two criteria were used to evaluate immunofluorescence--the amount of Tag measured and reproducibility. All MeOH methods were equivalently reproducible with CV's less than 3%. PF/TX was slightly less so with a CV of less than 6%. In contrast, different levels of Tag were measured for each procedure. For mouse 3T3 cells infected with a recombinant retroviral vector encoding T antigen, the level of T antigen measured after PF/MeOH was 21% greater than in MeOH fixed cells, and the level in PF/TX fixed cells was 37% less. The fraction of fluorescence specific to T antigen for these cells was 79-83% for all procedures. The lower levels measured after fixation by PF/TX were shown to be due to epitope masking. Why higher levels are measured with PF/MeOH procedures is unknown at present but may be due to antigen retention. Therefore, each of these fixation methods may be used with confidence in reliability but they are not equivalent with respect to the molecular architecture of the nucleus. It is postulated that PF/TX permeabilizes cells but cells retain native supramolecular structure, whereas MeOH based fixatives disrupt this structure and randomize availability of epitope to antibody. If so, the two procedures could be used as complementary procedures to study gene expression and function.

Acetone↗
PubMed1992

Reticulocyte quantification by flow cytometry, image analysis, and manual counting.

Reticulocyte counting by flow cytometry with thiazole orange was compared to manual or automated counting of new methylene blue stained blood smears. Forty-nine samples were compared for manual counting from randomly chosen clinical samples. Two hundred and eighty-nine samples from bone marrow transplant patients were compared during the period before and through chemo-irradiation and engraftment. The slopes of correlation plots were less than 1 when flow cytometric data were the dependent variable, suggesting that thiazole orange is less sensitive than new methylene blue. In a third study, 407 samples from bone marrow transplant patients were compared after increasing the thiazole orange concentration. The reticulocyte fluorescence distribution was divided into four groups of the brightest (youngest) 40, 60, 80, and 100% of reticulocytes. The slopes from regression analysis were 0.25, 0.49, 0.78, and 1.14, respectively. This demonstrates that thiazole orange is more sensitive than new methylene blue because the window of analysis includes an increased fraction of mature reticulocytes. In addition, the precision of each assay as measured. The rank order of precision from high to low was flow cytometry > image analysis >> manual counting.

Benzothiazoles↗
PubMed1992

Kinetics of erythrogenesis after bone marrow transplantation.

To determine the kinetics of bone marrow erythrogenesis after bone marrow transplantation, the authors counted reticulocytes (by blood smear and flow cytometry) and compared those data with neutrophil and platelet recovery in 23 consecutive bone marrow transplant patients. The earliest indication of marrow recovery after allogeneic and autologous bone marrow transplantation was defined as the second increasing cell count after the lowest recorded count, provided that the trend continued upward. Recovery of marrow function was detected earlier in 10 of 23 patients using reticulocyte counts than by either neutrophil or platelet count alone. Specifically, in 8 of these 10 patients, recovery of erythropoiesis was determined earlier by flow cytometric examination than by the blood smear method. On the other hand, combining the data using the earliest value of platelet, neutrophil, and reticulocyte counts indicated that the mean day of recovery in our patient population was determined to be 12.1 +/- 4 days after marrow infusion. In patients undergoing autologous and allogeneic bone marrow transplantation, serial neutrophil and reticulocyte count determinations are complementary in early clinical detection of successful engraftment.

Adolescent↗