Metaphase spread definition betting

Construction of the vector and characteristics of the mutation were previously described by Skarnes et al. The serial section was then counterstained with hematoxylin along with TUNEL staining, performed using an in situ cell death detection kit Roche Diagnostics as previously described [ 55 ]. Only clearly stained cells were considered as apoptotic and only tubules cut perpendicular to the length of the tubule round tubules in sections were evaluated.

Significant differences were assessed by statistical analysis using non-parametric Mann Whitney test with the threshold of significance set at 0. Preparation and immunofluorescence of spermatocyte spreads Spermatocyte spreads were prepared following the procedures described by Manterola et al. Briefly, seminiferous tubules were isolated, placed in a petri dish, and mechanically disaggregated using forceps.

The slides were then slowly dried in a humid chamber for 3h, washed with Photo-Flo 0. Chromosome spreads were examined using a Nikon eclipse E microscope equipped with epifluorescence optics, and the images were photographed with a high-definition cooled color camera head DS-Fi1c.

All images were processed with Adobe Photoshop CS5 software. Briefly, we identified changes in chromosome morphology of autosomes and sex chromosomes, such as the appearance of excrescences on the AEs of sex chromosomes and the widening of SC attachment plates on the autosomes, as well as the synapsis behavior of autosomes and sex chromosomes, with special regard to the length of the pairing region between X and Y chromosomes. Both immunofluorescent signals and background were quantified and the final intensities were calculated by subtracting the background immunofluorescent signal from the immunofluorescent signal from the protein.

It is important to note that in spread preparations, apoptotic cells exhibit a particular morphology that does not resemble a normal spermatocyte [ 56 ] and thus, we discarded those cells from our analysis. Statistical significance between mice was calculated by the one-way analysis of variance ANOVA , followed by Tuckey post test. A Z-test for two proportions was calculated to determine statistical differences between the signal intensity of BRDT in the chromatin of autosomes and in the chromatin of the sex chromosomes.

The threshold of significance was set at 0. No statistically significant differences in the distribution of the results between animals of the same genotype were observed. Using the same measurement tool, the position of each MLH1 foci was calculated by its distance to the centromere and to the distal telomere, respectively. The relative interference distance between two MLH1 foci on an SC was determined by following the method described by Anderson et al.

Primer sets are provided in S1 Table. The acidic ribosomal phosphoprotein P0 Arbp gene was used as an internal control for data normalization and the fold change between wild type and mutant samples calculated. All samples were analyzed in triplicate.

All the dataset generated, including DEGs and genes with no differential expression no-change were then grouped per chromosome by using the genome database biomaRt, obtaining the chromosome localization and expression pattern DEG or no-change for each gene. From the obtained list, we used R to filter the genes that were only expressed in testes by using the transcript profile of developing mouse testes described by Scultz et al.

Ggplot2 was used to generate the graphs of the gene-expression profile of the X chromosome at 17dpp and 20dpp testes. The MNase sensitivity assay and salt extraction analysis of chromatin were performed by following the procedure described by Kishi et al. Briefly, seminiferous tubules from 6 testes per each genotype were suspended in 5 ml of buffer A 0. Then, each sucrose layer was carefully removed and the nuclear pellet was suspended in 5 ml of buffer C 20 mM Tris-HCl pH 7.

All MNase assay experiments were repeated three times with four animals per genotype per experiment. Analysis of the relative intensity of the bands from three different chromatin digestions per each genotype was performed using the gel analyzer tool on Image J. Background was subtracted prior to the analysis of the bands. Statistical analysis was performed using 2-way Anova with Bonferroni posttests.

Chromatin isolation from pachytene spermatocytes and MNase digestion of native chromatin to obtain a mononucleosome fraction Chromatin from pachytene spermatocytes was obtained following the procedure described by Getun et al. Briefly, pachytene spermatocytes were re-suspended in 1ml lysis buffer without calcium 10 mM Tris pH 7.

To standardize the amount of MNase necessary to obtain only a mono-nucleosome fraction, chromatin was divided in 6 equal amounts and digested with increasing concentrations of MNase NU, Sigma-Aldrich 0. Tiled qPCR analysis of the sub-region of the pseudoautosomal region PAR hotspot Tiled primer pairs covering the region comprising from , to , kb in the X chromosome, were designed using the PCRTiler software [ 59 ].

Primer sets are provided in S2 Table. Primer pairs were tested and those that produced multiples amplicons in the melting curves were discarded. Only primer pairs with similar amplification efficiency were used. Supporting information S1 Fig. Depletion of BRDT does not affect the global DSBs repair during zygonema and chromosome synapsis and desynapsis in zygonema and diplonema. XY indicates the sex chromosomes. Error bars indicate standard deviation. TIF S4 Fig. H3K4me3 signal is not observed in pachynema and early diplonema, but is readily detected in late diplotene spermatocytes throughout all the chromatin of autosome chromosomes but not in the XY.

Lack of BRDT produces a different pattern of mis-regulation of gene expression in autosomes versus the X chromosome. The elevated expression of all genes except Ddx3x was confirmed. There is a significant increase in the expression of Nxf2, Fmr1, Gm and Huwe1.

Triplicates per each reaction were performed. TIF S7 Fig. Flow cytometry analysis performed on peripheral blood spleen and marrow specimens revealed similar but more pronounced phenotypes in these animals compared with the younger animal cohort. Encouraged by these results, we decided to generate mice with a more potent disruption of the cohesin complex. Our rationale for this is the fact that SMC1A and STAG2 mutations found in patients potentially generate strong loss of function alleles because these genes are located on the X chromosome.

Short-term exposure to doxycycline 22 d caused splenomegaly not depicted and lineage skewing Fig. Diseased animals demonstrated signs of anemia, as exemplified by peripheral blood counts Fig. In addition, our study describes the first in vivo models that demonstrate a tumor-suppressive role for the cohesin complex.

Fusion genes and mutations in many lineage-specific transcription factors and epigenetic regulators have been uncovered. Recently, mutations in the cohesin Rad21, Smc1a, Smc3, and Stag2 complex have been identified in de novo and secondary AML samples, as well as many solid tumors Leiserson et al.

Many of these mutations are predicted and shown here to result in loss of function of the affected protein. This is very surprising, as all cohesin genes are essential in every organism tested from yeast to mice. These findings support the notion that cohesin proteins are present at high levels both in actively cycling and quiescent tissues Wendt et al.

In tumors, mutations in Smc3 and Rad21 are usually heterozygous, leaving one copy of the gene unaffected. Stag2 and Smc1a reside on chromosome X and are therefore not compensated by a wild-type protein when mutated in male patients. Similar in females, all mutations found in Stag2 were reported to be on the active transcribed X chromosome Solomon et al. How cells deficient for a cohesin subunit manage to correctly separate their chromosomes remains to be understood. To model the loss of cohesin as seen in human malignancies, we generated three independent loss of function cohesin animals.

As standard mouse knockout techniques create complete null alleles, we opted to generate three mouse models with inducible shRNA targeting cohesin complex members in vivo Premsrirut et al. In these animals, we managed to efficiently suppress cohesin transcripts and reduce cohesin protein expression and function similar to that seen in human malignancies.

Surprisingly, even in mice where we induced systemic cohesin knockdown, we did not observe detrimental effects, although we never reached a complete loss of expression in this model. We were surprised by the initial lack of survival phenotypes in cells with a very efficient reduction of the cohesin complex members.

One explanation is the fact that during metaphase only a very small fraction of cohesin is required for SCC Waizenegger et al. Apparently, the small amount of cohesin protein that remains in our shRNA mouse is most likely sufficient for proper SCC and successful mitosis. This is consistent with the fact that we did not observe overwhelming defects in SCC in cohesin knockdown cells.

At this moment, no consensus exists on whether mutations in cohesin in human malignancies lead to genomic instability Solomon et al. As no differences are observed in steady state SCC and DNA damage response, we hypothesize that loss of cohesin complex member expression in human disease mainly affects higher-order chromatin structure and gene expression. The genome of eukaryotes is organized into structural domains that help establish regulatory interactions Lieberman-Aiden et al.

The cohesin complex is one of the main factors involved in the stability of the three-dimensional organization of the chromatin. When cohesin is removed from cells, a general unfolding and disorganization of the chromatin is observed with associated changes in gene expression Seitan et al.

Changes in higher-order chromatin structure have been observed in disease, including cancer Rickman et al. It is therefore not unconceivable that loss of cohesin in cancer leads to disruption of chromatin organization. Cohesin serves as a docking station for transcription factors, and its absence could therefore cause transcription factors to aberrantly activate genes Yan et al.

We postulate that cohesin mutations in AML mainly impact genome organization, adding a new layer of complexity to an already complicated disease. The chromatin accessibility data and correlation with gene expression changes presented here support this hypothesis. During the progression of the disease, clonal evolution is a common occurrence Ding et al. Recently, several studies have identified mutations in the originating stem cell in AML Genovese et al.

Cohesin genes are among the genes that are mutated in the stem cells that are found in the bone marrow of AML patients Ding et al. Here, we provide evidence that loss of cohesin indeed leads to increased self-renewal of stem cells in vitro. Cohesin knockdown mice display a skewing in their stem cell compartment in the bone marrow and show signs of extramedullary hematopoiesis in the spleen.

All these phenotypes have been reported before in mouse models for AML Lee et al. This is the first example of cohesin loss of function in a mammalian system. Strikingly, efficient but not complete silencing of cohesin is not detrimental to cellular homeostasis but leads to myeloproliferative disease, suggesting that there are defined thresholds of cohesin expression and activity.

This notion is consistent with the findings illustrated in the accompanying paper in this issue by Viny et al. Our study is the first evidence that mutation of cohesin is a driving event in the initiation and progression of cancer and not merely a bystander.

Furthermore, this suggests that cohesin loss could be therapeutically exploited. Cells carrying mutations in the cohesin complex are potentially more vulnerable to certain stressors or insults. The identification of cohesin synthetic lethal interactions could aid the development of new drugs, specifically targeting cohesin mutant cells. For this to be successfully introduced into the clinic, it is a prerequisite that AML patients are preselected based on their cohesin mutation status.

Such a new class of drugs is very welcome in a disease such as AML, where very few targeted therapies are available at this moment. Protein levels were measured using the Bradford assay. Virus was produced in T cells. Supernatant containing the virus was harvested 36, 48, and 60 h after transfection. Mouse bone marrow cells were isolated from femur, tibia, and pelvic bones. Viral transduction was monitored 48 h after virus addition by FACS analysis.

CFU assay. RNA sequencing and analysis. Libraries were sequenced at a HiSeq Ilumina. Next, sequencing data analysis was performed as follows. Reads were aligned to mm9 reference genome using TopHat, and Cuffdiff was used to find differentially expressed genes Trapnell et al. Metaphase spreads. Cells were grown in the presence of 0. Cells were harvested and resuspended in ice-cold 0. Slides were air-dried and stained with DAPI. Metaphases were acquired using Metafer Metasystems microscope and software.

These shRNAs were tested in 3T3 cells for their knockdown efficiency. Targeted ES cells were injected using tetraploid complementation technique. FACS analysis. Peripheral blood was extracted by retroorbital bleeding and blood was collected in EDTA-coated tubes. Bone marrow was isolated the tibia, femur, and pelvic bones by spinning cleaned bones for 2 min at 8, rpm. Staining for cell surface antigens was performed by adding fluorochrome- or biotin-conjugated antibodies on ice between 20 min and overnight on ice in the dark.

Statistical tests and error bars. Statistically significant differences are indicated with asterisks in figures with the accompanying p-value in the legend. Error bars in figures indicate SD for the number of replicates as indicated in the figure legend. ATAC sequencing was performed as described earlier Buenrostro et al.

The resulting library was sequenced in HiSeq Reads were aligned with bowtie on mm9. HTSeq was used to measure count density at peak sites Anders et al. Accession numbers for high-throughput sequencing experiments. Online supplemental material. Table S1, included as a separate Excel file, lists targeting sequences of shRNAs used in retroviral plasmids. Acknowledgments We would like to thank Peter van Galen and Bradley Bernstein Broad Institute for help with the epigenetic mapping in small populations.

We thank members of the Aifantis laboratory for helpful discussion. Prem K. Premsrirut is a founder, officer, and shareholder of Mirimus Inc. The authors declare no additional competing financial interests. Author contributions: J. Mullenders and I. Aifantis conceived the experiments and analyzed the data and wrote the manuscript. Mullenders, K. Wang, and C. Kayembe managed the shRNA mouse colony. Mullenders performed in vitro CFU experiments.

Mullenders and P. Rocha performed karyotype analysis. Lhoumaud, M. Keller, J. Pae, B. Aranda-Orgilles, P. Premsrirut, and L. Cimmino provided technical assistance. Aranda-Orgilles, R. Raviram, Y. Gong, and A. Tsirigos provided bioinformatics support. Hoehn analyzed the histology of diseased animals. Skok and R. Bonneau are supervising R.

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Indications for this laboratory test include, but are not limited to Genetic evaluation of an unborn child prenatal to rule out chromosomal defects in early pregnancy Postnatal diagnosis of developmental delay and congenital anomalies in infants and children Chromosomal abnormalities in cancers A normal human cell contains 23 pairs of chromosomes, including 22 pairs of autosomes and one pair of sex chromosomes XX or XY.

Any deviation from 23 pairs and the well-established structure of human chromosomes is associated with various diseases. For example, a discrepancy in chromosome numbers is called aneuploidy which involves either Missing chromosomes or One or more extra chromosomes The most common aneuploidies are Down syndrome with trisomy 21 extra copy of chromosome Figure 1 and Turner syndrome with monosomy X missing or partially missing one of the X chromosomes. Figure 1: Chromosomal number abnormalities Aneuploidy Trisomy 21 in Down syndrome.

Modified from Image source. Chromosomal structural abnormalities include Figure 2 Deletion: Part of a chromosome is missing Duplication: Part of a chromosome is repeated Inversion: Part of the chromosome is inverted within the chromosome Translocation: Material between two different chromosomes is exchanged Figure 3 Insertion: Addition of material from another chromosome.

Metaphase spread technique helps investigate such deviations from normal chromosomal numbers and structure. Figure 2: Chromosomal structural abnormalities. Created by author with Biorender. Translocation of gene ABL resulting in Philadelphia chromosome in chronic myelogenous Leukemia , a type of blood cancer.

Image source. How does the metaphase spread technique work? DNA in human cells exists in complex with various proteins, forming long strands of chromatin within the nucleus. Therefore, in the metaphase spread technique, cells isolated from clinical samples are cultured and treated with an agent eg: colcemid, Nocodozole that arrests the cells in metaphase Figure 4.

Figure 4: Steps in Metaphase spread technique. The swollen cells are fragile therefore should be handled with care and hence can easily rupture when dropping them on the slide at a later stage step 8. The sample is then fixed—a preservation technique used to prevent degradation by internal and external environmental factors—by adding a freshly prepared mixture of Methanol to acetic acid fixative.

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