Immunodetection of Human LINE-1 Expression in Cultured Cells and Human Tissues

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2016 Feb 20

PMID 26895059

Citations 14

Authors

Reema Sharma

Nemanja Rodic

Kathleen H Burns

Martin S Taylor

Affiliations

Soon will be listed here.

Abstract

Long interspersed element-1 (LINE-1) is the only active protein-coding retrotransposon in humans. It is not expressed in somatic tissue but is aberrantly expressed in a wide variety of human cancers. ORF1p protein is the most robust indicator of LINE-1 expression; the protein accumulates in large quantities in cellular cytoplasm. Recently, monoclonal antibodies have allowed more complete characterizations of ORF1p expression and indicated potential for developing ORF1p as a clinical biomarker. Here, we describe a mouse monoclonal antibody specific for human LINE-1 ORF1p and its application in immunofluorescence and immunohistochemistry of both cells and human tissues. We also describe detection of tagged LINE-1 ORF2p via immunofluorescence. These general methods may be readily adapted to use with many other proteins and antibodies.

Citing Articles

Elevated expression of the retrotransposon LINE-1 drives Alzheimer's disease-associated microglial dysfunction.

Roy N, Haq I, Ngo J, Bennett D, Teich A, De Jager P Acta Neuropathol. 2024; 148(1):75.

PMID: 39604588 PMC: 11602836. DOI: 10.1007/s00401-024-02835-6.

Subcellular location of L1 retrotransposon-encoded ORF1p, reverse transcription products, and DNA sensors in lupus granulocytes.

Moadab F, Sohrabi S, Wang X, Najjar R, Wolters J, Jiang H Mob DNA. 2024; 15(1):14.

PMID: 38937837 PMC: 11212426. DOI: 10.1186/s13100-024-00324-x.

The Ultraviolet Irradiation of Keratinocytes Induces Ectopic Expression of LINE-1 Retrotransposon Machinery and Leads to Cellular Senescence.

Touma F, Lambert M, Martinez Villarreal A, Gantchev J, Ramchatesingh B, Litvinov I Biomedicines. 2023; 11(11).

PMID: 38002016 PMC: 10669206. DOI: 10.3390/biomedicines11113017.

Condensation of LINE-1 is critical for retrotransposition.

Sil S, Keegan S, Ettefa F, Denes L, Boeke J, Holt L Elife. 2023; 12.

PMID: 37114770 PMC: 10202459. DOI: 10.7554/eLife.82991.

Revealing cell populations catching the early stages of human embryo development in naive pluripotent stem cell cultures.

Moya-Jodar M, Ullate-Agote A, Barlabe P, Rodriguez-Madoz J, Abizanda G, Barreda C Stem Cell Reports. 2022; 18(1):64-80.

PMID: 36563688 PMC: 9860119. DOI: 10.1016/j.stemcr.2022.11.015.

References

Feng Q, Moran J, Kazazian Jr H, Boeke J . Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition. Cell. 1996; 87(5):905-16. DOI: 10.1016/s0092-8674(00)81997-2. View

Burns K, Boeke J . Human transposon tectonics. Cell. 2012; 149(4):740-52. PMC: 3370394. DOI: 10.1016/j.cell.2012.04.019. View

Alisch R, Garcia-Perez J, Muotri A, Gage F, Moran J . Unconventional translation of mammalian LINE-1 retrotransposons. Genes Dev. 2006; 20(2):210-24. PMC: 1356112. DOI: 10.1101/gad.1380406. View

Brown J, Wimberly H, Lannin D, Nixon C, Rimm D, Bossuyt V . Multiplexed quantitative analysis of CD3, CD8, and CD20 predicts response to neoadjuvant chemotherapy in breast cancer. Clin Cancer Res. 2014; 20(23):5995-6005. PMC: 4252785. DOI: 10.1158/1078-0432.CCR-14-1622. View

Ostertag E, Kazazian Jr H . Biology of mammalian L1 retrotransposons. Annu Rev Genet. 2001; 35:501-38. DOI: 10.1146/annurev.genet.35.102401.091032. View

Martin S, Cruceanu M, Branciforte D, Li P, Kwok S, Hodges R . LINE-1 retrotransposition requires the nucleic acid chaperone activity of the ORF1 protein. J Mol Biol. 2005; 348(3):549-61. DOI: 10.1016/j.jmb.2005.03.003. View

Kolosha V, Martin S . High-affinity, non-sequence-specific RNA binding by the open reading frame 1 (ORF1) protein from long interspersed nuclear element 1 (LINE-1). J Biol Chem. 2002; 278(10):8112-7. DOI: 10.1074/jbc.M210487200. View

Shukla R, Upton K, Munoz-Lopez M, Gerhardt D, Fisher M, Nguyen T . Endogenous retrotransposition activates oncogenic pathways in hepatocellular carcinoma. Cell. 2013; 153(1):101-11. PMC: 3898742. DOI: 10.1016/j.cell.2013.02.032. View

Taylor M, LaCava J, Mita P, Molloy K, Huang C, Li D . Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition. Cell. 2013; 155(5):1034-48. PMC: 3904357. DOI: 10.1016/j.cell.2013.10.021. View

10.

Martin S, Branciforte D, Keller D, Bain D . Trimeric structure for an essential protein in L1 retrotransposition. Proc Natl Acad Sci U S A. 2003; 100(24):13815-20. PMC: 283504. DOI: 10.1073/pnas.2336221100. View

11.

Mathias S, Scott A, Kazazian Jr H, Boeke J, Gabriel A . Reverse transcriptase encoded by a human transposable element. Science. 1991; 254(5039):1808-10. DOI: 10.1126/science.1722352. View

12.

Martin S, Bushman F . Nucleic acid chaperone activity of the ORF1 protein from the mouse LINE-1 retrotransposon. Mol Cell Biol. 2001; 21(2):467-75. PMC: 86601. DOI: 10.1128/MCB.21.2.467-475.2001. View

13.

Giloh H, Sedat J . Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science. 1982; 217(4566):1252-5. DOI: 10.1126/science.7112126. View

14.

Callahan K, Hickman A, Jones C, Ghirlando R, Furano A . Polymerization and nucleic acid-binding properties of human L1 ORF1 protein. Nucleic Acids Res. 2011; 40(2):813-27. PMC: 3258132. DOI: 10.1093/nar/gkr728. View

15.

Beck C, Garcia-Perez J, Badge R, Moran J . LINE-1 elements in structural variation and disease. Annu Rev Genomics Hum Genet. 2011; 12:187-215. PMC: 4124830. DOI: 10.1146/annurev-genom-082509-141802. View

16.

Hancks D, Kazazian Jr H . Active human retrotransposons: variation and disease. Curr Opin Genet Dev. 2012; 22(3):191-203. PMC: 3376660. DOI: 10.1016/j.gde.2012.02.006. View

17.

Rodic N, Sharma R, Sharma R, Zampella J, Dai L, Taylor M . Long interspersed element-1 protein expression is a hallmark of many human cancers. Am J Pathol. 2014; 184(5):1280-6. PMC: 4005969. DOI: 10.1016/j.ajpath.2014.01.007. View

18.

Khazina E, Truffault V, Buttner R, Schmidt S, Coles M, Weichenrieder O . Trimeric structure and flexibility of the L1ORF1 protein in human L1 retrotransposition. Nat Struct Mol Biol. 2011; 18(9):1006-14. DOI: 10.1038/nsmb.2097. View

19.

Hicks S, Machamer C . The NH2-terminal domain of Golgin-160 contains both Golgi and nuclear targeting information. J Biol Chem. 2002; 277(39):35833-9. DOI: 10.1074/jbc.M206280200. View

20.

Lander E, Linton L, Birren B, Nusbaum C, Zody M, Baldwin J . Initial sequencing and analysis of the human genome. Nature. 2001; 409(6822):860-921. DOI: 10.1038/35057062. View