Name: analysis of line-1 retrotransposition at the single nucleus level
Uploaded: 2016-04-23T14:00:00
Description: Watch this Scientific Journal Video about Analysis of LINE-1 Retrotransposition at the Single Nucleus Level at JoVE.com

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 &#...

<ol>
	<li>Mathias, S. L., Scott, A. F., Kazazian, H. H., Boeke, J. D., Gabriel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+transcriptase+encoded+by+a+human+transposable+element.">Reverse transcriptase encoded by a human transposable element.</a> Science. 254, 1808-1810 (1991).</li><li>Feng, Q., Moran, J. V., Kazazian, H. H., Boeke, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+L1+retrotransposon+encodes+a+conserved+endonuclease+required+for+retrotransposition.">Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition.</a> Cell. 87, 905-916 (1996).</li><li>Clements, A. P., Singer, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+LINE-1+reverse+transcriptase:effect+of+deletions+outside+the+common+reverse+transcriptase+domain.">The human LINE-1 reverse transcriptase:effect of deletions outside the common reverse transcriptase domain.</a> Nucl Acids Res. 26, 3528-3535 (1998).</li><li>Martin, S. L., Branciforte, D., Keller, D., Bain, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimeric+structure+for+an+essential+protein+in+L1+retrotransposition.">Trimeric structure for an essential protein in L1 retrotransposition.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 13815-13820 (2003).</li><li>Khazina, 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=Trimeric+structure+and+flexibility+of+the+L1ORF1+protein+in+human+L1+retrotransposition.">Trimeric structure and flexibility of the L1ORF1 protein in human L1 retrotransposition.</a> Nat Struct Mol Biol. 18, 1006-1014 (2011).</li><li>Martin, S. L., Li, J., Weisz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+analysis+defines+distinct+functional+domains+for+protein-protein+and+nucleic+acid+interactions+in+the+ORF1+protein+of+mouse+LINE-1.">Deletion analysis defines distinct functional domains for protein-protein and nucleic acid interactions in the ORF1 protein of mouse LINE-1.</a> J Mol Biol. 304, 11-20 (2000).</li><li>Martin, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonucleoprotein+particles+with+LINE-1+RNA+in+mouse+embryonal+carcinoma+cells.">Ribonucleoprotein particles with LINE-1 RNA in mouse embryonal carcinoma cells.</a> Mol Cell Biol. 11 (9), 4804-4807 (1991).</li><li>Hohjoh, H., Singer, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+ribonucleoprotein+complexes+containing+human+LINE-1+protein+and+RNA.">Cytoplasmic ribonucleoprotein complexes containing human LINE-1 protein and RNA.</a> EMBO J. 15, 630-639 (1996).</li><li>Cost, G. J., Feng, Q., Jacquier, A., Boeke, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+L1+element+target-primed+reverse+transcription+in+vitro.">Human L1 element target-primed reverse transcription in vitro.</a> EMBO J. 21, 5899-5910 (2002).</li><li>Szak, S. T., 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=Molecular+archeology+of+L1+insertions+in+the+human+genome.">Molecular archeology of L1 insertions in the human genome.</a> Genome Biol. 3, (2002).</li><li>Sen, S. K., Huang, C. T., Han, K., Batzer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endonuclease-independent+insertion+provides+an+alternative+pathway+for+L1+retrotransposition+in+the+human+genome.">Endonuclease-independent insertion provides an alternative pathway for L1 retrotransposition in the human genome.</a> Nucl Acids Res. 35, 3741-3751 (2007).</li><li>Bojang, P., Roberts, R. A., Anderton, M. J., Ramos, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+the+HepG2+genome+by+long+interspersed+nuclear+element-1.">Reprogramming of the HepG2 genome by long interspersed nuclear element-1.</a> Mol Oncol. 7, 812-825 (2013).</li><li>Boeke, J. D., Garfinkel, D. J., Styles, C. A., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ty+elements+transpose+through+an+RNA+intermediate.">Ty elements transpose through an RNA intermediate.</a> Cell. 40, 491-500 (1985).</li><li>Heidmann, T., Heidmann, O., Nicolas, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+indicator+gene+to+demonstrate+intracellular+transposition+of+defective+retroviruses.">An indicator gene to demonstrate intracellular transposition of defective retroviruses.</a> PNAS. 85, 2219-2223 (1988).</li><li>Moran, J. V., 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=High+frequency+retrotransposition+in+cultured+mammalian+cells.">High frequency retrotransposition in cultured mammalian cells.</a> Cell. 87, 917-927 (1996).</li><li>Bojang, P., Anderton, M. J., Roberts, R. A., Ramos, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+LINE-1+retrotransposition+in+HepG2+cells+preferentially+targets+gene+poor+regions+of+chromosome+13.">De novo LINE-1 retrotransposition in HepG2 cells preferentially targets gene poor regions of chromosome 13.</a> Genomics. 104, 96-104 (2014).</li><li>Rozen, S., 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=Abundant+gene+conversion+between+arms+of+palindromes+in+human+and+ape+Y+chromosomes.">Abundant gene conversion between arms of palindromes in human and ape Y chromosomes.</a> Nature. 423, 873-876 (2003).</li><li>Graves, J. A., Koina, E., Sankovic, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+gene+content+of+human+sex+chromosomes+evolved.">How the gene content of human sex chromosomes evolved.</a> Curr Op Gen Develop. 16, 219-224 (2006).</li><li>Leem, Y. 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=Retrotransposon+Tf1+is+targeted+to+Pol+II+promoters+by+transcription+activators.">Retrotransposon Tf1 is targeted to Pol II promoters by transcription activators.</a> Mol Cell. 30, 98-107 (2008).</li><li>Guo, Y., Levin, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+sequencing+of+retrotransposon+integration+provides+a+saturated+profile+of+target+activity+in+Schizosaccharomyces+pombe.">High-throughput sequencing of retrotransposon integration provides a saturated profile of target activity in Schizosaccharomyces pombe.</a> Genome Res. 20, 239-248 (2010).</li><li>Dai, J., Xie, W., Brady, T. L., Gao, J., Voytas, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+regulates+integration+of+the+yeast+Ty5+retrotransposon+into+heterochromatin.">Phosphorylation regulates integration of the yeast Ty5 retrotransposon into heterochromatin.</a> Mol Cell. 27, 289-299 (2007).</li><li>McClintock, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+behavior+of+mutable+loci+in+maize.">The origin and behavior of mutable loci in maize.</a> Proc. Natl. Acad. Sci. U.S.A. 36, 344-355 (1950).</li><li>Fernandez, L., Torregrosa, L., Segura, V., Bouquet, A., Martinez-Zapater, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon-induced+gene+activation+as+a+mechanism+generating+cluster+shape+somatic+variation+in+grapevine.">Transposon-induced gene activation as a mechanism generating cluster shape somatic variation in grapevine.</a> Plant J Cell Mol Biol. 61, 545-557 (2010).</li><li>Garcia-Perez, J. L., 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=Epigenetic+silencing+of+engineered+L1+retrotransposition+events+in+human+embryonic+carcinoma+cells.">Epigenetic silencing of engineered L1 retrotransposition events in human embryonic carcinoma cells.</a> Nature. 466 (7307), 769-773 (2010).</li><li>Rodic, N., 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=Retrotransposon+insertions+in+the+clonal+evolution+of+pancreatic+ductual+adenocarcinoma.">Retrotransposon insertions in the clonal evolution of pancreatic ductual adenocarcinoma.</a> Nat Med. 21 (9), (2015).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Labeling Probes</td>
			<td></td>
			<td></td>
			<td></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 2mM and dTTP to 1.5mM&#160; in nuclease free water.</td>
		</tr>
		<tr>
			<td>&#160;dUTP-16-Biotin (0.5mM)</td>
			<td>Roche</td>
			<td>11093070910</td>
			<td></td>
		</tr>
		<tr>
			<td>dUTP-FITC (0.5mM)</td>
			<td>Thermo Scientific</td>
			<td>R0101</td>
			<td></td>
		</tr>
		<tr>
			<td>dUTP-CY3 (0.5mM)</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&#160;</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Preparing chromosome Spreads.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Colcemid&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>15212-012</td>
			<td>Add Colcemid directly to growth media&#160; to a final concentration of 0.4 &#181;g/mL</td>
		</tr>
		<tr>
			<td>1M KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>Dilute 1M KCl solution to 75 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 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&#160;</td>
			<td>R001100</td>
			<td>To detech cells, incubate cell in Trypsin for 5 mins and inactive with equal volume of complete media</td>
		</tr>
		<tr>
			<td>Cover slides&#160;</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>&#160;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>&#160;Beaker (600ml)</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagents and Materials for FISH</td>
			<td></td>
			<td></td>
			<td></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&#160;</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&#160;</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 mg/mL.</td>
		</tr>
		<tr>
			<td>Rubber Cement/Cytobond Sealant&#160;</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>&#160; Dark Humidified chamber</td>
			<td>Sigma</td>
			<td>CLS2551</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slides&#160;</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>&#160;Fluorescence Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></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 g of trisodium citrate with 800 mL of molecular grade H2O. Stir while adjusting to pH 7. Once the all salts dissolve, adjust the volume to 1 L and filter through 0.22 &#181;m Filter paper. Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>&#160;1mg/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 mg/mL. Make fresh solution every time.</td>
		</tr>
		<tr>
			<td>1% Pepsin</td>
			<td></td>
			<td></td>
			<td>Dissolve&#160; 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 &#176;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 mL, filter through 0.22 &#181;m Filter paper and store 5 mL aliquots at -20 &#176;C. Thaw aliquot of PFA at 37 &#176;C for 10-15 min for subsequent uses.&#160; 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 H2O.</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.&#160; Amount of Formamide determine how focused chromatids are, titrate the amount&#160;</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 ...

Research

JoVE Journal

Biology

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified &#955; DNA at the Single Molecule Level

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â» DNA at the Single Molecule Level

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...