This work was supported in part by grants from the National Institute of Environmental Health Sciences (ES014443 and ES017274) and AstraZeneca to KSR.

NOTE: All steps should be carried out at room temperature unless otherwise specified. Please refer to Reagents section for details on how to prepare individual reagents.
1. Labeling L1 Probes
NOTE: Probes can be labeled by chemical or PCR labeling.
<ol>
	<li>Chemical labeling
	<ol>
		<li>Make 0.7-1% agarose gel in Tris-acetate-EDTA (TAE) buffer, heat in a microwave until melted, allow the gel to cool, and then add ethidium bromide (0.5 &#181;g/ml). Pour into gel tray, add combs and allow the gel to solidify at room temperature.</li>
		<li>Label the SNeo probe (1-2 &#181;g) with CY3 or FITC at 37 &#176;C for 1 hr according to the manufacturer&#39;s protocol. 
		NOTE: SNeo denotes the Spliced Neomycin gene expressed after a full cycle of retrotransposition. The probe is ~1,000 bp in size. SNeo can be PCR amplified from any vector or from genomic DNA. See Table of Materials and Reagents for information on Streptavidin-CY3/FITC labeling reagents.</li>
		<li>Mix the labeled SNeo probe solution with 1x DNA loading buffer, load into each well and run at 75-100 volts for 15-20 min.</li>
		<li>Visualize the probe band using a Ultra-Violet (UV) Illuminator. Note that the size of the SNeo probe can be adjusted and that Lock nuclei acid (LNA) can be also be added (Figure 2).</li>
		<li>Cut labeled SNeo probe from the gel with a clean blade, solubilize and purify the SNeo probe from the gel using a PCR Clean-Up kit according to manufacturer&#39;s protocol. 
		CAUTION: Cover every exposed part of the body with protective shields (i.e., laboratory coats and UV clear face mask) when viewing and cutting the gel. See Table of Material and Reagents for information to gel purify PCR products.</li>
		<li>Re-run 5 &#181;l of SNeo labeled probe with an un-labeled SNeo probe on a 0.7% agarose gel (see 1.1.1) to confirm size increase and loss of signal intensity (Figure 2).</li>
		<li>Quantify the amount of labeled SNeo probe by measuring the absorbance at 260 nm and dilute the SNeo probe to 10 ng/&#181;l aliquots in nuclease free H2O. Store aliquots at -20 &#176;C.</li>
	</ol>
	</li>
	<li>PCR Labeling (Alternative method for probe labeling)
	<ol>
		<li>Combine SNeo specific primers (5&#39;-ggatagcattgggagatatacct-3&#39; and 5&#39;-attgaacaagatg gattgcacgc-3&#39;) with PCR reagents into a single tube: 13.6 &#181;l nuclease free H2O; 0.1 &#181;l dATP, dCTP, dGTP (10 nM); 0.05 &#181;l dTTP (10 nM); 0.05 &#181;l dUTP-biotin/dUTP-FITC/CY3 (10 nM); 4 &#181;l Go Tag 5x buffer; 0.4 &#181;l Go Tag Polymerase; 1 &#181;l SNeo template (10 ng). See Table of Materials and Reagents for information on PCR reagents.</li>
		<li>Adjust the amount of each reagent by multiplying by the total number of samples.</li>
		<li>Use the following PCR cycling parameters to amplify and label SNeo probes: 95 &#176;C for 2 min; 35 cycles of 95 &#176;C for 30 sec, 62 &#176;C for 30 sec, 72 &#176;C for 60 sec; 72 &#176;C for 2 min; Hold at 4 &#176;C indefinitely. 
		NOTE: The annealing temperature can be adjusted or gradient PCR can be completed to determine optimal annealing temperatures for other probes.</li>
		<li>Make 0.7-1% gel as in section 1.1.1, load and visualize the probe band as in section 1.1.4.</li>
		<li>Cut and purify the labeled SNeo probe as in section 1.1.5.</li>
		<li>Re-run 5 &#181;l of SNeo labeled probe with un-labeled SNeo probe to confirm size increase and loss of signal intensity (see Figure 2).</li>
		<li>Quantify the amount of labeled SNeo probe by measuring absorbance at 260 nm and dilute SNeo probe to 10 ng/&#181;l aliquots in nuclease free H2O.</li>
	</ol>
	</li>
</ol>
2. Preparing Chromosome Spreads
<ol>
	<li>Generate stable clones expressing vector backbone (control) or retrotransposition competent L1 using standard selection methodologies.12,16 While non-episomal reporters were used to correlate findings with studies of transcription, episomal vectors could also be used. Grow cells stably expressing control or L1 vector (1 x 106 cells per 10 cm plate, with adjustments in plate size made as needed) in complete growth media. For HepG2 cells, use RPMI-1600, 10% FBS and 200 &#181;g/ml of hygromycin. Grow cultures to 70% confluence at 37 &#176;C and 5% CO2. 
	NOTE: The choice of growth medium is dependent on cell type. Given that the plasmids used here carry selection cassettes for both hygromycin and neomycin, stable selection of clones could also be done with neomycin after selection on hygromycin, but the amount of neomycin needs to be optimized to establish tolerance levels for each cell type.</li>
	<li>Add colcemid (0.4 &#181;g/ml) to culture medium and incubate for 90 min to arrest cells at metaphase. If using different cell types, determine the optimal concentration of colcemid and time of exposure (60, 90, and 120 min) for optimal metaphase arrest empirically.</li>
	<li>Wash cells 2x with 10 ml of 1x Dulbecco&#39;s PBS (DPBS), trypsinize by adding 3-5 ml of 0.25% trypsin solution to the cells and incubate for 5 min to detach the cells. Inactivate trypsin with an equal amount of media containing 10% FBS.</li>
	<li>Centrifuge the cells for 2 min at 1,000 x g at 4 &#176;C, aspirate the medium from the cell pellet and wash the cells with DPBS. Aspirate all DPBS, leaving 200 &#181;l of DPBS to re-suspend the cells. Ensure cells are mixed well and that all clumps are dispersed. Use flickering or gentle pipetting to mix and disperse clumps and avoid vortexing.</li>
	<li>Add 5 ml of hypotonic solution (i.e., 75 mM KCl) that is pre-warmed to 37 &#176;C drop-wise while rotating the 15 ml tube horizontally and incubate at 37 &#176;C for 20 min. See Table of Materials and Reagents for information on how to obtain and make hypotonic solution.</li>
	<li>Centrifuge cells at 120 x g for 5 min at 4 &#176;C and repeat steps 2.4 - 2.5 3x leaving approximately 200 &#181;l of hypotonic solution to re-suspend the cells after each wash. Ensure that at the end of the 3 washes, the pellet is visibly white and swollen.</li>
	<li>Re-suspend the pellet in 200 &#181;l of Carnoy Fixative solution and make 1: 2, 1: 4, 1: 8, 1:16 dilutions in Carnoy Fixative solution. Drop 10 &#181;l of each dilution onto a dry clean slide from approximately 1 cm above and immediately expose the spread-free side to hot steam from boiling water for 30 sec. See Table of Materials and Reagents for information on how to make Carnoy Fixative solution.</li>
	<li>Dry the spreads at room temperature, stain with 0.1 &#181;g/ml Hoechst-33342 by immersion for 15-20 min in Coplin Jar and wash 3x with DPBS. See Table of Materials and Reagents for information on how to make the Hoechst-33342 solution.</li>
	<li>View spreads on fluorescence/phase-contrast microscope at 40X magnification. After optimizing spread preparation, spreads need not be stained with Hoechst-33342. View spreads on phase-contrast microscope at 10X magnification to determine spread quality and separation as outlined in the Results section (see Figure 3).</li>
	<li>Select and circle good spreads with a Diamond Point Marker on the opposite side of the slide (i.e., spread-free side).</li>
	<li>Store spreads at -20 &#176;C for up to a month. Avoid exposure to moisture.</li>
</ol>
3. Fluorescence In Situ Hybridization (FISH)
<ol>
	<li>Stabilizing and dehydrating spreads 
	NOTE: Equilibrate metaphase chromosome spreads to room temperature if stored at -20 &#176;C. See Table of Materials and Reagents for information on how to obtain and make reagents.
	<ol>
		<li>Incubate metaphase chromosome spreads with 200 &#181;l RNase A for 1 hr at 37 &#176;C.</li>
		<li>Wash slides in 2x-SSC buffer twice for 5 min each followed by rinsing with 10 mM HCl solution.</li>
		<li>Incubate spreads with 1% pepsin for 10 min at 37 &#176;C, rinse with deionized H2O and wash twice with 2x-SSC buffer for 5 min each.</li>
		<li>Incubate spreads with 4% paraformaldehyde for 10 min and wash in 2x SSC buffer twice for 5 min each.</li>
		<li>Dehydrate spread by incubating for 2 min in ethanol series: 70%, 80%, and 95% ethanol.</li>
		<li>Air-dry slides.</li>
	</ol>
	</li>
	<li>Hybridization Spreads with L1 -labeled retrotransposition probes (i.e., SNeo) 
	CAUTION: For direct-labeled probes, keep away from light at all times. See Table of Materials and Reagents for information on how to obtain and make reagents.
	<ol>
		<li>Add 30 ng of SNeo to hybridization buffer, heat at 72 &#176;C for 10 min and cool for 2 min at room temperature.</li>
		<li>Add 30 &#181;l of SNeo probe solution to each spread, cover with coverslip and seal the edges with rubber cement. Ensure no bubble formation.</li>
		<li>Heat the slide at 72 &#176;C for 5 min on a heat block, gradually drop the temperature to 37 &#176;C, and incubate overnight in a dark humidified chamber at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Washing and Viewing (or Addition of Secondary Antibody) 
	CAUTION: For direct-labeled probes, keep away from light at all times. 
	NOTE: See Table of Materials and Reagents for information on how to prepare. Use of sufficient volume to cover the entire chromosome spread is recommended. Remember that excess volume will be lost when coverslip is added.
	<ol>
		<li>Immerse slides in 2x SSC buffer to remove coverslips.</li>
		<li>Wash slides by immersion in 2x-SSC buffer at 45 &#176;C for 5 min.</li>
		<li>Wash slides by immersion in wash buffer at 45 &#176;C for 5 min, 2x.</li>
		<li>Wash slides by immersion in 0.1x SSC buffer at 45 &#176;C for 10 min.</li>
		<li>Wash slides by immersion in 2x SSC buffer at 45 &#176;C for 10 min. 
		NOTE: Place a Coplin Jar containing recommended buffer in a water bath, adjust temperature to 45 &#176;C.</li>
		<li>Cool slides to room temperature and equilibrate slides in detection buffer.</li>
		<li>For direct labeled probes, skip to step 3.4.10.</li>
		<li>For indirect labeled probes, block in blocking buffer for 20-30 min and wash 3x in DPBS.</li>
		<li>Incubate with 50 &#181;l of secondary antibody (e.g., 5 &#181;g/ml streptavidin-FITC, or &#945;CY3 in blocking buffer) for 1 hr.</li>
		<li>Wash slides in 2x SSC for 5 min twice.</li>
		<li>Counterstain with Hoechst-33342 solution (0.1 &#181;g/ml) for 10 min. 
		NOTE: this staining is done to stain chromosomes for co-localization with probe.</li>
		<li>Wash in DPBS 3x, add a drop of mounting medium, place a coverslip and seal the edges with nail polish.</li>
		<li>Analyze L1 retrotransposition using fluorescence microscope at 40X magnification.</li>
	</ol>
	</li>
</ol>

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The authors declare no potential competing interests.

Methodologies such as whole genome sequencing, inverse PCR and southern blotting have been employed to study L1 retrotransposition. Although these methodologies are extremely valuable in locating where L1 insertions occur within genomes, a confounding challenge for all of them is the need for in-silico programing to reassemble sequences. The FISH methodology described here is designed to complement these methods, especially in the case of studies requiring analyses of ectopic L1 retrot...

Human Long Interspersed Nuclear Element-1 (Line-1 or L1) is an autonomous mobile element that propagates within the genome through a &#34;copy and paste&#34; retrotransposition mechanism. A typical human L1 is ~6 kb long and consists of a 5&#39;UTR (Untranslated Region) that serves as a promoter, two open reading frames (ORFs): L1-ORF1 and L1-ORF2, and a 3&#39;UTR with a polyA tail. L1-ORF1 protein has three distinct domains: a coil-coil domain, RNA recognition Motif and C-terminal domain, while L1-ORF2 protein has endonuclease, reverse transcriptase and cysteine rich domains1,2,3,4,5. L1-ORF1 exhibits nucleic acid chaperone activities, while L1-ORF2 provides enzymatic activities, with both proteins required for retrotransposition1,2,6.
A cycle of L1 retrotransposition starts with transcription from the 5&#39;UTR of L1 bicistronic mRNA by RNA polymerase II, translocation into the cytoplasm, and translation. In the cytoplasm, L1-ORF1 exhibits either cis-binding where it packages its own RNA or trans-binding where it packages other RNAs (i.e., SINE/SVAs/pseudogenes) to form a ribonucleoprotein particle (RNP) with L1-ORF2p7,8. RNPs translocate into the nucleus where the endonuclease activity of L1-ORF2p nicks genomic DNA (gDNA) to expose an OH- group9, that is in turn used by reverse transcriptase to prime and reverse synthesize RNA to DNA from the 3&#39; end. During reverse synthesis, the second strand of DNA is nicked 7-20 bp from the original nick site to create staggered breaks which are filled to form the signature L1 insertion sequences (e.g., TTTTAA) called target site duplications (TSD)9,10. This process is known as target prime reverse transcription (TPRT) and leads to insertion of full or truncated copies of L1/other DNAs with TSDs at both ends of the inserted sequence. L1 retrotransposition has also been shown to be mediated through TPRT-like non-homologous end-joining in some cells types11.
The L1 retrotransposition vector employed in our studies is non-episomal and consists of tagged L1-ORF1&#38;2p driven by combined CMV-L1-5&#39;UTR promoters (Figure 1)12. Earlier versions of this construct have been described in studies using yeast and human cell cultures13,14,15. Two distinct CMV promoters located at the 5&#39; and 3&#39; ends of the vector, with the 3&#39; end placed in reverse orientation to drive expression of neomycin after splicing and integration. The retrotransposition indicator cassette at the 3&#39; end consists of a neomycin gene inserted antisense to L1-ORFs and rendered inactive by separation into two halves by a globin intron with spliced donor (SD) and spliced acceptor (SA) sites (Figure 1). Upon integration into a chromosome, L1 is transcribed from the common promoter to produce an mRNA that consists of a bicistronic mRNA and inactive neomycin mRNA. During RNA processing, the globin intron is spliced out of the neomycin gene to restore a fully functional neomycin gene. The hybrid mRNA is packaged into a RNP in cis and translocated into the nucleus where it is integrated into the genome as either full length or truncated insertions using TPRT.
Here we describe a methodology that uses fluorescence in situ hybridization (FISH) with probes specifically directed at the spliced neomycin gene (SNeo) to track L1-retrotransposiition patterns and insertion rates at the single nucleus level. The efficiency and specificity of detection was confirmed using retrotransposition competent and incompetent constructs and probes to detect SNeo or the neomycin and globin intron junction16. This methodology accounts for some of the shortcomings of cell culture based retrotransposition assays, such as multiple insertions, colony resistance and favorable clone expansion.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Labeling Probes&lt;/strong&gt;</td></tr><tr><td>Go Tag DNA polymerase</td><td>Promega</td><td>M3178</td><td></td></tr><tr><td>GO Tag 10x colorless buffer</td><td>Promega</td><td>M3178</td><td></td></tr><tr><td>Individual dNTP</td><td>Sigma</td><td>DNTP10</td><td>Adjust the concentration of dATP, dGTP, dCTP to 2&amp;nbsp;mM and dTTP to 1.5&amp;nbsp;mM in nuclease free water.</td></tr><tr><td>dUTP-16-Biotin (0.5&amp;nbsp;mM)</td><td>Roche</td><td>11093070910</td><td></td></tr><tr><td>dUTP-FITC (0.5&amp;nbsp;mM)</td><td>Thermo Scientific</td><td>R0101</td><td></td></tr><tr><td>dUTP-CY3 (0.5&amp;nbsp;mM)</td><td>Sigma</td><td>GEPA53022</td><td></td></tr><tr><td>MIRUS FISH Labeling Kit</td><td>MIRUS</td><td>MIR3625</td><td></td></tr><tr><td>MIRUS FISH Labeling Kit</td><td>MIRUS</td><td>MIR3225</td><td></td></tr><tr><td>NucleoSpin PCR Clean-Up kit&amp;nbsp;</td><td>Qiagen</td><td>1410/0030</td><td>Any PCR clean reagent can be used in place of NucleoSpin PCR Clean Up Kit</td></tr><tr><td>PCR Machine</td><td></td><td></td><td>Any Thermocycler machine can be used to amplify the label product.</td></tr><tr><td>Agarose</td><td>Sigma</td><td>A9539</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Preparing chromosome Spreads&lt;/strong&gt;</td></tr><tr><td>Colcemid</td><td>Life Technologies&amp;nbsp;</td><td>15212-012</td><td>Add Colcemid directly to growth media to a final concentration of 0.4&amp;nbsp;&amp;micro;g/ml</td></tr><tr><td>1&amp;nbsp;M KCl</td><td>Sigma</td><td>P9541</td><td>Dilute 1&amp;nbsp;M KCl solution to 75&amp;nbsp;mM solution to make Hypotonic Solution</td></tr><tr><td>Carnoy Fixative Solution</td><td></td><td></td><td>Mix 3 volumes of methanol and 1 volume of acetic acid to make Carnoy Fixative Solution. Make Fresh everytime.</td></tr><tr><td>Methanol</td><td>Sigma</td><td>322415</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma</td><td>320099</td><td></td></tr><tr><td>Hoechst-33342</td><td>Thermo Scientific</td><td>62249</td><td>Dissolve Hoechst-33342 in PBS to final concentration of 1&amp;nbsp;mg/ml.</td></tr><tr><td>Diamond Point Marker</td><td>Thermo Scientific</td><td>750</td><td></td></tr><tr><td>Frosted Slides</td><td>Thermo Scientific</td><td>2951-001</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Life Technologies&amp;nbsp;</td><td>R001100</td><td>To detech cells, incubate cell in Trypsin for 5 min and inactive with equal volume of complete media.</td></tr><tr><td>Cover slides</td><td>VWR</td><td>48366067</td><td></td></tr><tr><td>Coplin Jars</td><td>Thermo Scientific</td><td>107</td><td></td></tr><tr><td>Heat Block</td><td></td><td></td><td>Any heat block can be used for this purpose, though blocks fitted for heating slides are recommended.</td></tr><tr><td>Beaker (600&amp;nbsp;ml)</td><td>Sigma</td><td>CLS1003600</td><td>Fill the beaker with water, heat to boil, use the hot steam to burst chromosomes.</td></tr><tr><td>Nikon Microscope</td><td>Nikon</td><td>125690</td><td>Any microscope can be used to look at spread quality.</td></tr><tr><td colspan="4">&lt;strong&gt;Reagents and Materials for FISH&lt;/strong&gt;</td></tr><tr><td>Trisodium citrate</td><td>Sigma</td><td>S1804</td><td></td></tr><tr><td>Sodium Chroride</td><td>Sigma</td><td>S3014</td><td></td></tr><tr><td>Formamide</td><td>Sigma</td><td>F9037</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>F1379</td><td></td></tr><tr><td>Detran Sulfate</td><td>Sigma</td><td>D8906</td><td></td></tr><tr><td>SDS</td><td>Sigma</td><td>L3771</td><td></td></tr><tr><td>Salmon Sperm DNA</td><td>Life Technologies&amp;nbsp;</td><td>15632-011</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A8531</td><td></td></tr><tr><td>HCl (N)</td><td>Sigma</td><td>38283</td><td></td></tr><tr><td>Ethyl Alcohol</td><td>Sigma</td><td>459844</td><td></td></tr><tr><td>Methanol</td><td>Sigma</td><td>322415</td><td></td></tr><tr><td>DPBS</td><td>Life Technologies&amp;nbsp;</td><td>14190-250</td><td></td></tr><tr><td>NaOH</td><td>Sigma</td><td>S8045</td><td></td></tr><tr><td>Rnase A</td><td>Sigma</td><td>R4642</td><td></td></tr><tr><td>Pepsin</td><td>Sigma</td><td>P6887</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Hoechst-33342</td><td>Thermo Scientific</td><td>62249</td><td>Dissolve Hoechst-33342 in PBS to final concentration of 1&amp;nbsp;mg/ml.</td></tr><tr><td>Rubber Cement/Cytobond Sealant&amp;nbsp;</td><td></td><td>2020-00-1</td><td></td></tr><tr><td>Seven Coplin Jars</td><td>Thermo Scientific</td><td>107</td><td></td></tr><tr><td>Dark Humidified chamber</td><td>Sigma</td><td>CLS2551</td><td></td></tr><tr><td>Cover slides</td><td>VWR</td><td>48366067</td><td></td></tr><tr><td>Dry Digital Heat Block</td><td>VWR</td><td>13259</td><td></td></tr><tr><td>Fluorescence Microscope</td><td></td><td></td><td></td></tr><tr><td>20x Saline-Sodium Citrate (20x SSC)</td><td></td><td></td><td>Combine 175 g of NaCl, and 88.2&amp;nbsp;g of trisodium citrate with 800 ml of molecular grade H&lt;sub&gt;2&lt;/sub&gt;O. Stir while adjusting to pH 7. Once the all salts dissolve, adjust the volume to 1 L and filter through 0.22 &amp;micro;m Filter paper. Store at 4 &amp;deg;C.</td></tr><tr><td>1&amp;nbsp;mg/ml of Rnase A</td><td></td><td></td><td>Dilute 20x SSC buffer to 2x SSC buffer. Dissolve RNase A in 2x SSC buffer to a final concentration of 1&amp;nbsp;mg/ml. Make fresh solution every time.</td></tr><tr><td>1% Pepsin</td><td></td><td></td><td>Dissolve pepsin (W/V) in 10 mM HCl to a final concentration of 1% solution. Make fresh every time.</td></tr><tr><td>4% Paraformaldehyde (4% PFA)</td><td></td><td></td><td>Weigh 4.0 g of PFA in fume hood, add 50 ml of 1xPBS, heat to 60&amp;nbsp;&amp;deg;C while stirring and adjust the pH with drops of NaOH until all PFA dissolves and the solution becomes clear. Adjust the volume to 100&amp;nbsp;ml, filter through 0.22 &amp;micro;m Filter paper and store 5 ml aliquots at -20&amp;nbsp;&amp;deg;C. Thaw aliquot of PFA at 37 &amp;deg;C for 10-15 min for subsequent uses. Adjust filter size depending on amount of paraformaldehyde.</td></tr><tr><td>Wash Buffer</td><td></td><td></td><td>Combine 100 ml of Formamide (20%) with 2.5 ml of 20x SSC, adjust to pH 7 with 1.0 M HCl and bring the volume to 500 ml with molecular grade H&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>Hybridization Buffer</td><td></td><td></td><td>Combine 50% formamide, 10% dextran sulfate, 0.1% SDS, and 300 ng/ml Salmon Sperm DNA in 2x SSC buffer. Amount of Formamide determine how focused chromatids are; titrate the amount.</td></tr><tr><td>Detection Buffer</td><td></td><td></td><td>Dilute 20x SSC buffer to 4x and add 0.2% Tween-20 to make detection buffer.</td></tr><tr><td>Blocking Buffer</td><td></td><td></td><td>Combine 5% bovine serum albumin (BSA) with 0.2% Tween-20 in 4x SSC buffer.</td></tr><tr><td>Dehydrating Solution</td><td></td><td></td><td>Make 70%, 80%, and 95% ethanol solutions.</td></tr></tbody></table>

analysis of line-1 retrotransposition at the single nucleus level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

A schematic diagram of the L1 retrotransposition vector is presented in Figure 1. The vector consists of a neomycin gene in antisense orientation to L1 ORFs that is interrupted by a globin intron in sense orientation and sandwiched by SD and SA sites. When stably integrated into a chromosome, L1 mRNA is transcribed from the combined CMV and L1-5&#39;UTR promoter (Figure 1). During RNA processing, the globin intron is spliced out...

Watch this Scientific Journal Video about Analysis of LINE-1 Retrotransposition at the Single Nucleus Level at JoVE.com

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Here we employ FISH methodology to track LINE-1 retrotransposition at the single nuclei level in chromosome spreads of HepG2 cell lines stably expressing synthetic LINE-1.

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

analysis-of-line-1-retrotransposition-at-the-single-nucleus-level

Research

JoVE Journal

Biology

8.4K Views.  University of Arizona College of Medicine.  Hier maken we gebruik van FISH methodologie op te sporen LINE-1 retrotransposition bij de enkele kernen niveau chromosoom spreads van HepG2-cellijnen stabiel tot expressie synthetische LINE-1. 

Video: Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

Genetics

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis. An extension of the MARCM method, called twin-spot MARCM (tsMARCM), labels both of the twin cells derived from a common progenitor with two distinct colors. This technique was developed to enable the retrieval of useful information from both hemi-lineages. By comprehensively analyzing different pairs of tsMARCM clones, the tsMARCM system permits high-resolution neural lineage mapping to reveal the exact birth-order of the labeled neurons produced from common progenitor cells. Furthermore, the tsMARCM system also extends gene function studies by permitting the phenotypic analysis of identical neurons of different animals. Here, we describe how to apply the tsMARCM system to facilitate studies of neural development in Drosophila.

Here, we present a protocol for a mosaic labeling technique that permits the visualization of neurons derived from a common progenitor cell in two distinct colors. This facilitates neural lineage analysis with the capability of birth-dating individual neurons and studying gene function in the same neurons of different individuals.

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

Developmental Biology

Detection of Retrotransposition Activity of Hot LINE-1s by Long-Distance Inverse PCR

Long interspersed nuclear elements 1 (LINE-1s) are the only family of mobile genetic elements in the human genome that can move autonomously. They do so by a process called retrotransposition wherein they transcribe to form an mRNA intermediate which is then consequently inserted into the genome by reverse transcription. Despite being silent in normal cells, LINE-1s are highly active in different epithelial tumors. De novo LINE-1 insertions can potentially drive tumorigenesis, and hence it is important to systematically study LINE-1 retrotransposition in cancer. Out of ~150 retrotransposition-competent LINE-1s present in the human genome, only a handful of LINE-1 loci, also referred to as &#34;hot&#34; LINE-1s, account for the majority of de novo LINE-1 insertion in different cancer types. We have developed a simple polymerase chain reaction (PCR)-based method to monitor retrotransposition activity of these hot LINE-1s. This method, based on long-distance inverse (LDI)-PCR, takes advantage of 3&#180; transduction, a mechanism by which a LINE-1 mobilizes its flanking non-repetitive region, which can subsequently be used to identify de novo LINE-1 3&#180; transduction events stemming from a particular hot LINE-1.

This article outlines a simple PCR-based assay to monitor the activity of an active LINE-1 retrotransposon and to map de novo retrotranspositions in a given genome. Using the MCF7 cell line, we demonstrate herein how this method can be applied to detect activity of a LINE-1 located at 22q12.1.

Long interspersed nuclear elements 1 (LINE-1s) are the only family of mobile genetic elements in the human genome that can move autonomously. They do so ...

Cancer Research

RNA Next-Generation Sequencing and a Bioinformatics Pipeline to Identify Expressed LINE-1s at the Locus-Specific Level

Long INterspersed Elements-1 (LINEs/L1s) are repetitive elements that can copy and randomly insert in the genome resulting in genomic instability and mutagenesis. Understanding the expression patterns of L1 loci at the individual level will lend to the understanding of the biology of this mutagenic element. This autonomous element makes up a significant portion of the human genome with over 500,000 copies, though 99% are truncated and defective. However, their abundance and dominant number of defective copies make it challenging to identify authentically expressed L1s from L1-related sequences expressed as part of other genes. It is also challenging to identify which specific L1 locus is expressed due to the repetitive nature of the elements. Overcoming these challenges, we present an RNA-Seq bioinformatic approach to identify L1 expression at the locus specific level. In summary, we collect cytoplasmic RNA, select for polyadenylated transcripts, and utilize strand-specific RNA-Seq analyses to uniquely map reads to L1 loci in the human reference genome. We visually curate each L1 locus with uniquely mapped reads to confirm transcription from its own promoter and adjust mapped transcript reads to account for mappability of each individual L1 locus. This approach was applied to a prostate tumor cell line, DU145, to demonstrate the ability of this protocol to detect expression from a small number of the full-length L1 elements.

Here, we present a bioinformatic approach and analyses to identify LINE-1 expression at the locus specific level.

Long INterspersed Elements-1 (LINEs/L1s) are repetitive elements that can copy and randomly insert in the genome resulting in genomic instability and ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

2D Gel Electrophoresis for Analyzing DNA Replication and Fork Stalling Events

Electrophoretic Analysis of Replication Through Structure-Prone DNA Repeats Within the SV40-Based Human Episome

Two-dimensional neutral/neutral gel-electrophoresis (2DGE) emerged as a benchmark technique to analyze DNA replication through natural impediments. This protocol describes how to analyze replication fork progression through structure-prone, expandable DNA repeats within the simian virus 40 (SV40)-based episome in human cells. In brief, upon plasmid transfection into human cells, replication intermediates are isolated by the modified Hirt protocol and treated with the DpnI restriction enzyme to remove non-replicated DNA. Intermediates are then digested by appropriate restriction enzymes to place the repeat of interest within the origin-distal half of a 3-5 kb-long DNA fragment. The replication intermediates are separated into two perpendicular dimensions, first by size and then by shape. Following Southern blot hybridization, this approach allows researchers to observe fork stalling at various structure-forming repeats on the descending half of the replication Y-arc. Furthermore, this positioning of the stall site allows the visualization of various outcomes of repeat-mediated fork stalling, such as fork reversal, the advent of a converging fork, and recombinational fork restart.

Author Spotlight: Characterizing DNA Replication of Pathogenic Repeats to Uncover Mechanisms of Replication Fork Stalling and Expansion

Here, we outline the procedure for analyzing replication progression through pathogenic, structure-prone repeats using 2-dimensional gel electrophoresis.

Two-dimensional neutral/neutral gel-electrophoresis (2DGE) emerged as a benchmark technique to analyze DNA replication through natural impediments. This ...

Nuclei Isolation from Mouse Cardiac Progenitor Cells at Single-Cell Resolution

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

Author Spotlight: Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution  

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every eukaryotic genome. This process is an important mechanism for generating diversity between and within organisms1-3. The human genome consists of approximately 40% repetitive sequence of retrotransposon origin, including a variety of LINEs and SINEs4. Exchange events between these repetitive elements can lead to genome rearrangements, including translocations, that can disrupt gene dosage and expression that can result in autoimmune and cardiovascular diseases5, as well as cancer in humans6-9.
Exchange between repetitive elements occurs in a variety of ways. Exchange between sequences that share perfect (or near-perfect) homology occurs by a process called homologous recombination (HR). By contrast, non-homologous end joining (NHEJ) uses little-or-no sequence homology for exchange10,11. The primary purpose of HR, in mitotic cells, is to repair double-strand breaks (DSBs) generated endogenously by aberrant DNA replication and oxidative lesions, or by exposure to ionizing radiation (IR), and other exogenous DNA damaging agents. 
In the assay described here, DSBs are simultaneously created bordering recombination substrates at two different chromosomal loci in diploid cells by a galactose-inducible HO-endonuclease (Figure 1). The repair of the broken chromosomes generates chromosomal translocations by single strand annealing (SSA), a process where homologous sequences adjacent to the chromosome ends are covalently joined subsequent to annealing. One of the substrates, his3-&Delta;3', contains a 3' truncated HIS3 allele and is located on one copy of chromosome XV at the native HIS3 locus. The second substrate, his3-&Delta;5', is located at the LEU2 locus on one copy of chromosome III, and contains a 5' truncated HIS3 allele. Both substrates are flanked by a HO endonuclease recognition site that can be targeted for incision by HO-endonuclease. HO endonuclease recognition sites native to the MAT locus, on both copies of chromosome III, have been deleted in all strains. This prevents interaction between the recombination substrates and other broken chromosome ends from interfering in the assay. The KAN-MX-marked galactose-inducible HO endonuclease expression cassette is inserted at the TRP1 locus on chromosome IV. The substrates share 311 bp or 60 bp of the HIS3 coding sequence that can be used by the HR machinery for repair by SSA. Cells that use these substrates to repair broken chromosomes by HR form an intact HIS3 allele and a tXV::III chromosomal translocation that can be selected for by the ability to grow on medium lacking histidine (Figure 2A). Translocation frequency by HR is calculated by dividing the number of histidine prototrophic colonies that arise on selective medium by the total number of viable cells that arise after plating appropriate dilutions onto non-selective medium (Figure 2B). A variety of DNA repair mutants have been used to study the genetic control of translocation formation by SSA using this system12-14.

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

The HO-stimulated translocation assay monitors single-strand annealing following the creation of DNA double-strand breaks at multiple loci in diploid Saccharomyces cerevisiae. This mechanism may model genome rearrangements in somatic cells of higher eukaryotes following exposure to high doses of ionizing radiation.

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every ...

LTR Retrotransposons

LTR retrotransposons are class I transposable elements with long terminal repeats flanking an internal coding region. These elements are less abundant in mammals compared to other class I transposable elements. About 8 percent of human genomic DNA comprises LTR retrotransposons. Some of the common examples of LTR retrotransposons are Ty elements in yeast and Copia elements in Drosophila.
The internal coding region of LTR retrotransposons and their mechanism of transposition closely resembles a retroviral genome.&#160; They can encode enzymes required for their mobilization as well as synthesize structural proteins to form a virus-like particle. But most LTR retrotransposons lack the genes to synthesize a viral envelope. Hence, in contrast to the retroviruses that can form infectious virions and move horizontally from one cell to another, LTR retrotransposons are only allowed to move from one locus to another within the genome of a single cell.
However, some LTR retroelements have been found to have an extra ORF in the same position as the &#8220;env&#8221; gene found in retrovirus genomes, for example, gypsy elements in Drosophila. These elements can encode for three putative proteins, one of which resembles a retroviral envelope protein that results in an infectious form of the element.
In humans, the most common LTR retrotransposons are called endogenous retroviruses or ERVs. These ERVs are products of ancestral exogenous viral infections in the germline cells, which lead to their vertical transmission in humans. However, because of co-evolution spanning millions of years, these ERVs now play an important role in human physiology, including maintaining certain regulatory networks.

Long Terminal Repeats LTR Retrotransposons

LTR retrotransposons are class I transposable elements with long terminal repeats flanking an internal coding region. These elements are less abundant in ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

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