Name: rapid analysis of chromosome aberrations in mouse b lymphocytes by pna-fish
Uploaded: 2014-08-19T16:00:00.000+00:00
Duration: 7 min 54 s
Description: Watch this Scientific Journal Video about Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH at JoVE.com

This work is supported by NIH grant R00 CA160574 (SFB) and by a fellowship of the Rutgers Biotechnology Training Program (to SMM).

This procedure was approved by the Institutional Animal Care and Use Committee at Rutgers, the State University of New Jersey. Mice were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Scientists should consult their national and institutional animal organizations for established and approved guidelines.
Before beginning, prepare the solutions listed in Table 1.
1. B Cell Isolation and Activation
Euthanize a mouse using CO2 followed by cervical dislocation. Position the mouse so the left side of the body is up and spray the mouse with 70% ethanol until the fur is damp. Dissect the spleen and place in a 35 mm tissue culture dish with 2 ml wash buffer. In a laminar flow hood, gently break up the spleen in the tissue culture dish using the flat end of a 5 ml syringe.
<ol>
	<li>Insert a 70 &#956;m nylon mesh into a 50 ml tube and transfer the spleen sample in 2 ml wash buffer into the nylon mesh. Gently disrupt the spleen in the mesh using the flat end of the syringe.</li>
	<li>Rinse the back of the syringe and the 35 mm plate with 8 ml of wash buffer and filter through the 70 &#956;m nylon mesh. Centrifuge at 300 x g for 10 min at 4 &#176;C.</li>
	<li>Aspirate and discard the supernatant and resuspend the pellet in 3 ml of ACK lysis buffer. Pipette up and down briefly to disrupt clumps and incubate for 5 min. Add 10 ml of wash buffer and centrifuge at 300 x g for 10 min at 4 &#176;C.</li>
	<li>Aspirate and discard the supernatant and resuspend the pellet by tapping the bottom of the tube then by pipetting in 1 ml of wash buffer. Add 50 &#956;l of anti-CD43 MACS micro-beads and incubate on ice for 30 min. Add 10 ml of wash buffer, mix gently, and centrifuge at 300 x g for 10 min at 4 &#176;C. Add 10 ml of wash buffer, mix gently, and centrifuge at 300 x g for 10 min at 4 &#176;C.</li>
	<li>Set up a Mini-MACS column by placing a column on the MACS magnet. Position a labeled 15 ml conical tube below the column. Add 1 ml of wash buffer to column and allow to run through to the 15 ml tube.</li>
	<li>Aspirate the supernatant and resuspend in 1 ml of wash buffer. Load the sample in the column after the wash buffer has run through.</li>
	<li>After the sample has run through the column, add 1 ml wash buffer to wash the column. Add 7 ml of wash buffer directly to the collected sample in the 15 ml tube.</li>
	<li>Count the cells using a hemocytometer. Transfer the required volume of cells needed for 5,000,000 cells/well into a 50 ml tube. Centrifuge at 300 x g for 10 min at 4 &#176;C. Supplement B cell medium with LPS 1:500 and IL4 1:1,000.</li>
	<li>Aspirate the supernatant and add the appropriate volume of supplemented B cell medium needed for final concentration of 1,000,000 cells/ml.</li>
	<li>Plate cells in 6-well plates with 5 ml of cells at 1,000,000 cell/ml and place in a humidified cell culture incubator at 37 &#176;C, 5% CO2.</li>
</ol>
2. B Cell Fixation
<ol>
	<li>Add 50 &#956;l of colcemid to the well containing 5 ml of B cells (for a final concentration of 100 ng/ml) and incubate 1 hr at 37 &#176;C. Prewarm 75 mM KCl solution in a 37 &#176;C water bath. Transfer the B cells to a labeled 15 ml tube. Cover the label with tape and centrifuge at 300 x g for 10 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant leaving 1 ml in the tube and resuspend the pellet by pipetting. Add 5 ml of the prewarmed KCl drop by drop while tapping or pulsing on a vortex.</li>
	<li>Add 10 ml of KCl and mix by inverting. Incubate in a 37 &#176;C water bath for 15 min. Add 5 drops of fresh fixative and mix by inverting. Centrifuge at 300 x g for 10 min at 4 &#176;C. Aspirate the supernatant leaving 1 ml in the tube and resuspend pellet by pipetting up and down.</li>
	<li>Add 5 ml of fresh fixative drop by drop while tapping or pulsing on a vortex. Add an additional 10 ml of fixative and incubate for 30 min at room temperature.</li>
	<li>Centrifuge at 300 x g for 10 min at 4 &#176;C. Aspirate the supernatant leaving 1 ml in tube and resuspend by pipetting. Add 14 ml of fresh fixative and centrifuge at 300 x g for 10 min at 4 &#176;C.</li>
	<li>Repeat step 2.7 twice more.</li>
	<li>Add 14 ml of fresh fixative. Seal the caps with Parafilm and store overnight at -20 &#176;C.</li>
</ol>
3. Preparation of Metaphase Chromosome Spreads
<ol>
	<li>Set a regulated environmental chamber to 22.9 &#176;C and 52% humidity.</li>
	<li>Centrifuge the fixed B cells at 300 x g for 10 min at 4 &#176;C. Aspirate fixative leaving 70 &#956;l in the tube and resuspend by pipetting.</li>
	<li>Place labeled slides in the humidity chamber at a 35&#176; angle. Drop 35 &#956;l of the resuspended B cells onto a slide and drop two slides per sample. Allow the slides to dry in the humidity chamber for 30 min then store the slides in a slide box at 37 &#176;C.</li>
</ol>
4. Telomere PNA FISH
<ol>
	<li>Preparation of Probe
	<ol>
		<li>Pre-warm deionized formamide to 37 &#176;C. Mix 2 &#956;l of telomere PNA probe with 7 &#956;l deionized pre-warmed formamide and incubate at 37 &#176;C with shaking for 1 hr.</li>
		<li>Pre-warm FISH master mix to 37 &#176;C. Add 7 &#956;l pre-warmed FISH master mix to the mixture from step 4.1.1 and incubate at 37 &#176;C for 1-3 hr.</li>
		<li>Denature the probe mixture for 8 min at 80 &#176;C. Pre-anneal the probe for 1 hr at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Pretreatment of Chromosome Slides
	<ol>
		<li>Pre-warm a glass slide-staining jar containing 50 ml of 0.01 M HCl to 37 &#176;C in a water bath.</li>
		<li>Place slides in a different glass slide-staining jar containing 2x SSC for 5 min at room temperature.</li>
		<li>Add 2 &#956;l of pepsin stock to the pre-warmed glass slide-staining jar and mix by inverting. Transfer the slides to this glass slide-staining jar and incubate 90 sec at 37 &#176;C.</li>
		<li>Wash the slides 2x in a different glass slide-staining jar containing 1x PBS for 5 min each at room temperature, shaking. Then wash the slides for 5 min in 1x PBS/MgCl2 at room temperature, shaking.</li>
		<li>Transfer the slides to a glass slide-staining jar containing freshly-prepared 1% formaldehyde/1x PBS/MgCl2 for 10 min at room temperature.</li>
		<li>Wash the slides for 5 min in a different glass slide-staining jar containing 1x PBS at room temperature, shaking.</li>
		<li>Prepare an ethanol dehydration series by having separate glass slide-staining jars containing 70%, 90%, and 100% ethanol.</li>
		<li>Transfer the slides to each jar for 3 min, starting with 70% ethanol, then 90% ethanol, and then 100% ethanol. Place ethanol jars at -20 &#176;C when finished.</li>
		<li>Allow the slides to air dry by tapping excess ethanol onto a paper towel and propping slides at a 35&#176; angle. Once dry, mark an 18 x 18 mm area for hybridization on the back of the slides using a diamond pen.</li>
	</ol>
	</li>
	<li>Denaturation of Slides for FISH
	<ol>
		<li>Apply 120 &#956;l 70% deionized formamide/2X SSC to a 24 x 60 mm coverslip. Touch the slide to the coverslip. Denature the slide at 80 &#176;C on a hot plate for 90 sec.</li>
		<li>Quickly and carefully slide off the coverslip and place the slide in ice cold 70% ethanol for 3 min, followed by 90% ethanol, and 100% ethanol each for 3 min. Allow the slides to air dry.</li>
		<li>Prepare a humid chamber by lining a plastic box with a tight lid and wet tissue paper. The paper should be damp, but not saturated with water.</li>
		<li>In a humid chamber, apply the pre-annealed probe mix from step 4.1.3 to the marked area on the slide, cover with an 18 x 18 mm cover slip and incubate at 37 &#176;C for 1 hr.</li>
	</ol>
	</li>
	<li>Post-incubation Washes
	<ol>
		<li>Pre-warm 50% formamide/2x SSC, 1x SSC, and 4x SSC/0.1% Tween-20 in a 45 &#176;C water bath.</li>
		<li>Carefully remove the coverslips and wash the slides in pre-warmed 50% formamide/2x SSC 3xfor 5 min each in the dark and shaking. Wash the slides in pre-warmed 1x SSC 3xfor 5 min each, shaking. Finally, wash the slides in pre-warmed 4x SSC/0.1% Tween-20 3xfor 5 min each.</li>
		<li>Stain the slides for 3 min in DAPI in a light protected glass slide-staining jar.</li>
		<li>Wash the slides for 5 min in 2x SSC, shaking. Apply 35 &#956;l of Mowiol mounting medium, cover with 24 x 60 mm coverslips and store the slides at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
5. Microscopic Analysis of Chromosomes
<ol>
	<li>Analyze the metaphase slides using a standard epi-fluorescence microscope. Use an imaging platform with an automated stage such as the MetaSystems Metafer platform to obtain best results. For each metaphase spread, collect one image for DAPI fluorescence to visualize chromosomes, and one image for the fluorescent signal from the telomere probe. (e.g., Cy3- other fluors are available and work equally well.)</li>
	<li>Overlay the images with an image analysis program and analyze.</li>
</ol>

<ol>
	<li>Sancar, A., Lindsey-Boltz, L. A., Unsal-Kacmaz, K., Linn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+mammalian+DNA+repair+and+the+DNA+damage+checkpoints.">Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints.</a> Ann Rev Biochem. 73, 39-85 (2004).</li><li>Sperka, T., Wang, J., Rudolph, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+checkpoints+in+stem+cells,+ageing+and+cancer.">DNA damage checkpoints in stem cells, ageing and cancer.</a> Nature reviews Mol Cell Biol. 13 (9), 579-590 (2012).</li><li>Alexandrov, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signatures+of+mutational+processes+in+human+cancer.">Signatures of mutational processes in human cancer.</a> Nature. 500 (7463), 415-421 (2013).</li><li>Bunting, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=53BP1+inhibits+homologous+recombination+in+Brca1-deficient+cells+by+blocking+resection+of+DNA+breaks.">53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks.</a> Cell. 141 (2), 243-254 (2010).</li><li>Padilla-Nash, H. M., Barenboim-Stapleton, L., Difilippantonio, M. J., Ried, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+karyotyping+analysis+of+human+and+mouse+chromosomes.">Spectral karyotyping analysis of human and mouse chromosomes.</a> Nat Protoc. 1 (6), 3129-3142 (2006).</li><li>Callen, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+IgH-TCRalpha/delta+translocations+in+T+lymphocytes+mediated+by+RAG.">Chimeric IgH-TCRalpha/delta translocations in T lymphocytes mediated by RAG.</a> Cell Cycle. 8 (15), 2408-2412 (2009).</li><li>Boboila, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+end-joining+catalyzes+robust+IgH+locus+deletions+and+translocations+in+the+combined+absence+of+ligase+4+and+Ku70.">Alternative end-joining catalyzes robust IgH locus deletions and translocations in the combined absence of ligase 4 and Ku70.</a> Natl Acad Sci USA. 107 (7), 3034-3039 (2010).</li><li>Benn, P., Delach, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+lymphocyte+culture+and+chromosome+analysis.">Human lymphocyte culture and chromosome analysis.</a> Cold Spring Harb Protoc. 2008, (2008).</li><li>Nguyen, H. N., Reijo Pera, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metaphase+spreads+and+spectral+karyotyping+of+human+embryonic+stem+cells.">Metaphase spreads and spectral karyotyping of human embryonic stem cells.</a> Cold Spring Harb Protoc. 2008, (2008).</li><li>Schreck, R. R., Disteche, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+4.">Chapter 4.</a> Chromosome banding techniques. Unit 2, (2001).</li><li>Gilley, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-PKcs+is+critical+for+telomere+capping.">DNA-PKcs is critical for telomere capping.</a> Proc Natl Acad Sci USA. 98 (26), 15084-15088 (2001).</li><li>Bolzan, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+aberrations+involving+telomeres+and+interstitial+telomeric+sequences.">Chromosomal aberrations involving telomeres and interstitial telomeric sequences.</a> Mutagenesis. 27 (1), 1-15 (2012).</li><li>Bolzan, A. D., Telomeres Bianchi, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=interstitial+telomeric+repeat+sequences+and+chromosomal+aberrations.">interstitial telomeric repeat sequences and chromosomal aberrations.</a> Mutat Res. 612 (3), 189-214 (2006).</li><li>Poon, S. S., Lansdorp, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+18.">Chapter 18.</a> Quantitative fluorescence in situ hybridization (Q-FISH). Unit 18 14, (2001).</li><li>Morales, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=53BP1+and+p53+synergize+to+suppress+genomic+instability+and+lymphomagenesis.">53BP1 and p53 synergize to suppress genomic instability and lymphomagenesis.</a> Proc Natl Acad Sci USA. 103 (9), 3310-3315 (2006).</li><li>Halaby, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+interaction+of+Rnf8+and+p53+in+the+protection+against+genomic+instability+and+tumorigenesis.">Synergistic interaction of Rnf8 and p53 in the protection against genomic instability and tumorigenesis.</a> PLoS Genet. 9 (1), e1003259 (2013).</li></ol>

The authors have no conflicting interests.

Whereas activated B lymphocytes are particularly suited to preparation of mitotic chromosome spreads, other cell types can also be used. T lymphocytes share many of the advantages of B cells, as they can be purified from the spleen or lymph nodes, and have a high mitotic index when stimulated by growth in medium containing appropriate mitogens8. Embryonic Stem cells (ES cells) are also suited for metaphase chromosome analysis9. If these are not available, fibroblast cells can be used, although a lon...

A correction was made to: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH.&#160;Table 1 (the list of solutions to prepare) was omitted from the Protocol section. The contents of Table 1 are as follows:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Reagents</td>
			<td>Comments</td>
			<td>Storage</td>
		</tr>
		<tr>
			<td rowspan="3">B Cell Wash Buffer</td>
			<td>500 ml 1x Hanks Balanced Salt Solution</td>
			<td rowspan="3">Filter through 0.22 &#956;m stericup filtration unit</td>
			<td rowspan="3">4 &#176;C</td>
		</tr>
		<tr>
			<td>5 ml 50 &#176;C heat inactivated FCS</td>
		</tr>
		<tr>
			<td>5 ml 100x Pen/Strep</td>
		</tr>
		<tr>
			<td rowspan="8">B Cell Medium</td>
			<td>418.5 ml RPMI-1640</td>
			<td rowspan="8">Filter through 0.22 &#956;m stericup filtration unit</td>
			<td rowspan="8">4 &#176;C</td>
		</tr>
		<tr>
			<td>50 ml 50 &#176;C heat inactivated FCS</td>
		</tr>
		<tr>
			<td>5 ml 100x Pen/Strep</td>
		</tr>
		<tr>
			<td>5 ml glutamine</td>
		</tr>
		<tr>
			<td>5 ml nonessential amino acids</td>
		</tr>
		<tr>
			<td>5 ml sodium pyruvate</td>
		</tr>
		<tr>
			<td>1.8 ml 14.2 M 2-mercaptoethanol</td>
		</tr>
		<tr>
			<td>5 ml 1M HEPES</td>
		</tr>
		<tr>
			<td>Fixative</td>
			<td>3:1 v/v methanol/glacial acetic acid</td>
			<td></td>
			<td>Make fresh</td>
		</tr>
		<tr>
			<td rowspan="2">0.075 M KCl</td>
			<td>1.395 g KCl</td>
			<td rowspan="2"></td>
			<td rowspan="2">Room Temperature</td>
		</tr>
		<tr>
			<td>250 ml distilled H2O</td>
		</tr>
		<tr>
			<td rowspan="3">FISH Master Mix</td>
			<td>40 ml 50% Dextran sulfate</td>
			<td rowspan="3">Vortex the solution and leave in a shaking platform overnight</td>
			<td rowspan="3">Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>20 ml 20x SSC</td>
		</tr>
		<tr>
			<td>40 ml sterile distilled H2O</td>
		</tr>
		<tr>
			<td rowspan="3">70% Formamide/2x SSC</td>
			<td>10 ml 20x SSC</td>
			<td rowspan="3">Adjust to pH 7 with 1M HCl</td>
			<td rowspan="3">Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>20 ml distilled H2O</td>
		</tr>
		<tr>
			<td>70 ml deionized formamide</td>
		</tr>
		<tr>
			<td rowspan="2">0.01 M HCl</td>
			<td>1 ml 1M HCl</td>
			<td rowspan="2">Adjust to pH 2.0</td>
			<td rowspan="2">Room Temperature</td>
		</tr>
		<tr>
			<td>99 ml distilled H2O</td>
		</tr>
		<tr>
			<td rowspan="2">1x PBS/MgCl2</td>
			<td>50 ml 1M MgCl2</td>
			<td rowspan="2"></td>
			<td rowspan="2">Room Temperature</td>
		</tr>
		<tr>
			<td>950 ml 1x PBS</td>
		</tr>
		<tr>
			<td rowspan="3">50% Formamide/2x SSC</td>
			<td>20 ml 20x SSC</td>
			<td rowspan="3">Adjust to pH 7.25 
			Formamide used in this step does not have to be deionized.</td>
			<td rowspan="3">Make fresh</td>
		</tr>
		<tr>
			<td>80 ml distilled H2O</td>
		</tr>
		<tr>
			<td>100 ml formamide</td>
		</tr>
		<tr>
			<td rowspan="3">4x SSC/0.1% Tween20</td>
			<td>50 ml 20x SSC</td>
			<td rowspan="3"></td>
			<td rowspan="3">Room Temperature</td>
		</tr>
		<tr>
			<td>200 ml distilled H2O</td>
		</tr>
		<tr>
			<td>250 &#956;l Tween20</td>
		</tr>
		<tr>
			<td rowspan="2">DAPI staining solution (80 ng/ml)</td>
			<td>40 &#956;l 0.2mg/ml DAPI stock</td>
			<td rowspan="2"></td>
			<td rowspan="2">4 &#176;C in light protected staining jar</td>
		</tr>
		<tr>
			<td>100 ml 2x SSC</td>
		</tr>
		<tr>
			<td rowspan="5">Mowiol Mounting Medium</td>
			<td>6 g glycerol</td>
			<td rowspan="5">Incubate at 50 &#176;C with stirring for 2hrs</td>
			<td rowspan="5">Aliquot and store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>2.4 g Mowiol 4-88</td>
		</tr>
		<tr>
			<td>6 ml distilled H2O</td>
		</tr>
		<tr>
			<td>12 ml 0.2 M TrisCl pH 8.5</td>
		</tr>
		<tr>
			<td>230 &#956;l 1% Thimerosal (w/v in water)</td>
		</tr>
	</tbody>
</table>
Table 1. List of solutions.&#160;

A correction was made to: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH.

Erratum: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Cancer is caused by the accumulation of mutations affecting genes that regulate normal cell growth. Mutation is a consequence of changes to the structure and sequence of the genome caused by damage to DNA. DNA damage can occur through a variety of process, including exogenous agents such as ionizing radiation, and as a by-product of normal cellular metabolism, such as spontaneous deamination of nucleotide bases or damage occurring by contact with reactive oxygen species1.
Although mammalian cells possess a range of repair activities which can reverse DNA damage or restore the sequence at break sites, mutations nonetheless accumulate throughout the lifetime of a cell. DNA damage can furthermore contribute to senescence and loss of potency of stem cells, two processes which are associated with aging-associated disease2. Understanding the repair of DNA damage is therefore of central importance in addressing two significant issues in public health. Increasing evidence suggests that mammalian DNA repair pathways can contribute to the evolution of the cancer cell genome3,4, making it even more imperative to understand the processes involved in suppressing mutation at the molecular level.
Direct visualization of chromosome aberrations is a powerful and quantitative means of determining the extent of genomic instability in a particular cell type. Condensed chromosomes from cells at metaphase can be isolated and inspected using light or fluorescent microscopy. Such cytogenetic approaches have been in practice for several decades and can be used to demonstrate the appearance of translocations or specific types of chromosome aberrations associated with loss of DNA repair activities. The protocol lends itself to several potential extensions: chromosomes can be labeled with probes for spectral karyotyping (SKY) or multicolor fluorescent in situ hybridization (mFISH) to identify translocations5,6. These techniques also enable the frequency of chromosome translocations and the structure of complex chromosome translocations to be determined, which provides additional information beyond what is possible with this protocol. Alternatively, sequence-specific probes can be generated and used to test the frequency of DNA breakage at selected genomic sites7.
In this protocol, we describe preparation of metaphase chromosome spreads from B lymphocytes. A fluorescently-labeled peptide nucleic acid (PNA) probe for telomeric repeats is used, which efficiently marks telomeres in metaphase chromosome spreads This protocol has several advantages. B cells can be induced to grow at high mitotic index so that high-quality spreads can consistently be produced. B cells from genetically-modified mice are also much less likely to contain secondary genetic mutations that can confound the analysis of the contribution of specific genes to genomic integrity. The PNA-FISH approach can be completed in one day, and allows more accurate scoring of chromosome breaks. By using this approach, particularly in combination with specific equipment described in this protocol, it is possible to produce very consistent, high-quality spreads and rapidly analyze the rate and type of genomic instability.

<table><tbody><tr><td>50% Dextran sulfate</td><td>Intergen</td><td>S4030</td><td></td></tr><tr><td>Deionized formamide</td><td>Ambion</td><td>9342</td><td></td></tr><tr><td>Pepsin (5 g)</td><td>Sigma</td><td>P 6887&amp;nbsp;</td><td></td></tr><tr><td>FCS</td><td>Gemini</td><td>100-106</td><td></td></tr><tr><td>LPS (100 mg)</td><td>Sigma</td><td>L2630</td><td></td></tr><tr><td>IL-4 (5 &amp;mu;g)</td><td>Sigma</td><td>I1020</td><td></td></tr><tr><td>PNA Telomere Probe</td><td>PNA Bio Inc</td><td>F1002</td><td>(CCCTAACCCTAACCCTAA)</td></tr><tr><td>Colcemid</td><td>Roche</td><td>295892</td><td></td></tr><tr><td>Mowiol 4-88</td><td>Sigma</td><td>81381-50g</td><td></td></tr><tr><td>CD43 (ly-48_) micro-beads</td><td>Miltenyl Biotec.</td><td>130-049-801</td><td></td></tr><tr><td>DAPI</td><td>Sigma</td><td>32670-5MG</td><td></td></tr><tr><td>Eclipse E800</td><td>Nikon</td><td></td><td></td></tr><tr><td>AxioImager.Z2</td><td>Zeiss</td><td></td><td></td></tr><tr><td>CDS-5 Cytogenetic Drying Chamber</td><td>Thermotron</td><td></td><td></td></tr></tbody></table>

rapid analysis of chromosome aberrations in mouse b lymphocytes by pna-fish

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

Metaphase chromosomes derived from one cell should form a discrete cluster containing 40 chromosomes (if using mouse cells) (Figure 1A). Each chromosome has a centromere at one end, which is visible as a light blue sphere. In normal chromosomes, two telomere signals are seen at the end of the chromatids and two signals by each centromere. Interphase nuclei will be present on the slide as well. These will be visible as blue spheres, with red telomere signals, but no condensed chromosomes (see for example ...

Watch this Scientific Journal Video about Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH at JoVE.com

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

rapid-analysis-chromosome-aberrations-mouse-b-lymphocytes-pna

Research

JoVE Journal

Immunology and Infection

16.9K Views.  Rutgers, the State University of New Jersey.  DNA herstelmechanismen zijn essentieel om de genomische integriteit en preventie mutatie en kanker. Het doel van dit protocol is om instabiliteit van het genoom te kwantificeren door middel van directe observatie van chromosoomafwijkingen in metafase verspreidt van de muis B-cellen met behulp van fluorescente in situ hybridisatie (FISH) voor telomeer DNA herhalingen. 

Video: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

Genetics

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

FISH for Pre-implantation Genetic Diagnosis 

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

FISH for Pre-implantation Genetic Diagnosis

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

Medicine

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing ...

Cancer Research

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

Biology

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Analysis of Somatic Hypermutation in the JH4 intron of Germinal Center B cells from Mouse Peyer's Patches

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the variable coding exons of the Ig heavy and light chain gene loci. This process of somatic hypermutation (SHM) requires the enzyme activation-induced cytidine deaminase (AID), which converts deoxycytidines (C), into deoxyuridines (U). Processing the AID-generated U:G mismatches into mutations by the base excision and mismatch repair pathways introduces new Ig coding sequences that may produce a higher affinity Ig. Mutations in AID or DNA repair genes can block or significantly alter the types of mutations observed in the Ig loci. We describe a protocol to quantify JH4 intron mutations that uses fluorescence activated cell sorting (FACS), PCR, and Sanger sequencing. Although this assay does not directly measure Ig affinity maturation, it is indicative of mutations in Ig variable coding sequences. Additionally, these methods utilize common molecular biology techniques which&#160;analyze&#160;mutations in Ig sequences of multiple B cell clones. Thus, this assay is an invaluable tool in the study of SHM and Ig diversification.

Presented here is an assay to quantify somatic hypermutation within the immunoglobulin heavy chain gene locus using germinal center B cells from mouse Peyer&#8217;s patches.

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and T lymphocytes. Studies of zebrafish lymphopoiesis are difficult because antibodies recognizing D. rerio cell surface markers are generally not available, complicating isolation and characterization of different lymphocyte populations, including B-lineage cells. Transgenic lines with lineage-specific fluorophore expression are often used to circumvent this challenge. The transgenic lck:eGFP line has been used to study D. rerio T cell development, and has also been utilized to model T cell development and acute lymphoblastic leukemia (T-ALL). Although lck:eGFP fish have been widely used to analyze the T-lineage, they have not been used to study B cells. Recently, we discovered that many zebrafish B cells also express lck, albeit at lower levels. Consequently, lck:eGFP B cells likewise express low levels of GFP. Based on this finding, we developed a protocol to purify B-lineage cells from lck:eGFP zebrafish, which we report here. Our method describes how to utilize a fluorescent-activated cell sorter (FACS) to purify B cells from lck:eGFP fish or related lines, such as double-transgenic rag2:hMYC; lck:eGFP fish. In these lines, B cells, particularly immature B cells, express GFP at low but detectable levels, allowing them to be distinguished from T cells, which express GFP highly. B cells can be isolated from marrow, thymus, spleen, blood, or other tissues. This protocol provides a new method to purify D. rerio B cells, enabling studies focused on topics like B cell development and B lymphocyte malignancies.

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Transgenic lck:eGFP zebrafish express GFP highly in T lymphocytes, and have been used to study T cell development and acute lymphoblastic leukemia. This line can be used to study B cells, which express lck at lower levels. This protocol describes purification of malignant and non-malignant B cells from lck:eGFP zebrafish.

Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

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