LINE-1 Cultured Cell Retrotransposition Assay

Kopera, Huira C.; Larson, Peter A.; Moldovan, John B.; Richardson, Sandra R.; Liu, Ying; Moran, John V.

doi:10.1007/978-1-4939-3372-3_10

Cited by 53 publications

(91 citation statements)

References 81 publications

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“…At 48 h post-transfection, cells were subjected to flow cytometry on a CytoFLEX flow cytometer (Beckman-Coulter) at the Translational Research Institute Flow Cytometry Core. The percentage of EGFP positive cells for each L1 reporter construct was used to normalize the G418-resistant colony counts obtained in the retrotransposition assay (Wei et al 2000;Kopera et al 2016).…”

Section: Cultured Cell Retrotransposition Assaymentioning

confidence: 99%

L1 retrotransposition is a common feature of mammalian hepatocarcinogenesis

et al. 2018

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The retrotransposon Long Interspersed Element 1 (LINE-1 or L1) is a continuing source of germline and somatic mutagenesis in mammals. Deregulated L1 activity is a hallmark of cancer, and L1 mutagenesis has been described in numerous human malignancies. We previously employed retrotransposon capture sequencing (RC-seq) to analyze hepatocellular carcinoma (HCC) samples from patients infected with hepatitis B or hepatitis C virus and identified L1 variants responsible for activating oncogenic pathways. Here, we have applied RC-seq and whole-genome sequencing (WGS) to an Abcb4 (Mdr2)−/− mouse model of hepatic carcinogenesis and demonstrated for the first time that L1 mobilization occurs in murine tumors. In 12 HCC nodules obtained from 10 animals, we validated four somatic L1 insertions by PCR and capillary sequencing, including T F subfamily elements, and one G F subfamily example. One of the T F insertions carried a 3 ′ transduction, allowing us to identify its donor L1 and to demonstrate that this full-length T F element retained retrotransposition capacity in cultured cancer cells. Using RC-seq, we also identified eight tumor-specific L1 insertions from 25 HCC patients with a history of alcohol abuse. Finally, we used RC-seq and WGS to identify three tumor-specific L1 insertions among 10 intra-hepatic cholangiocarcinoma (ICC) patients, including one insertion traced to a donor L1 on Chromosome 22 known to be highly active in other cancers. This study reveals L1 mobilization as a common feature of hepatocarcinogenesis in mammals, demonstrating that the phenomenon is not restricted to human viral HCC etiologies and is encountered in murine liver tumors.

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Section: Cultured Cell Retrotransposition Assaymentioning

confidence: 99%

L1 retrotransposition is a common feature of mammalian hepatocarcinogenesis

et al. 2018

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“…At 48 h post-transfection, cells were subjected to flow cytometry on a Cyan ADP Analyzer (Beckman-Coulter) at the Translational Research Institute Flow Cytometry Core. The percentage of EGFP positive cells for each L1 reporter construct was used to normalize the G418-resistant colony counts obtained in the retrotransposition assay (Wei et al 2000;Kopera et al 2016). Full details of the cultured cell retrotransposition assay can be found in Supplemental Methods.…”

Section: Cultured Cell Retrotransposition Assaymentioning

confidence: 99%

Heritable L1 retrotransposition in the mouse primordial germline and early embryo

et al. 2017

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LINE-1 (L1) retrotransposons are a noted source of genetic diversity and disease in mammals. To expand its genomic footprint, L1 must mobilize in cells that will contribute their genetic material to subsequent generations. Heritable L1 insertions may therefore arise in germ cells and in pluripotent embryonic cells, prior to germline specification, yet the frequency and predominant developmental timing of such events remain unclear. Here, we applied mouse retrotransposon capture sequencing (mRC-seq) and whole-genome sequencing (WGS) to pedigrees of C57BL/6J animals, and uncovered an L1 insertion rate of ≥1 event per eight births. We traced heritable L1 insertions to pluripotent embryonic cells and, strikingly, to early primordial germ cells (PGCs). New L1 insertions bore structural hallmarks of target-site primed reverse transcription (TPRT) and mobilized efficiently in a cultured cell retrotransposition assay. Together, our results highlight the rate and evolutionary impact of heritable L1 retrotransposition and reveal retrotransposition-mediated genomic diversification as a fundamental property of pluripotent embryonic cells in vivo.

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“…In all retrotransposition assays, we also included a transfection efficiency control to calculate rates of engineered retrotransposition as described (Garcia-Perez et al 2010;Kopera et al 2016). Where indicated, engineered L1 retrotransposons were co-transfected with a second expression plasmid for TEX19 orthologs or controls.…”

Section: Retrotransposition Assaysmentioning

confidence: 99%

“…Where indicated, engineered L1 retrotransposons were co-transfected with a second expression plasmid for TEX19 orthologs or controls. For mneoI and mblastI-based assays, we included a plasmid containing a neomycin or blasticidin resistance expression cassette respectively, to control for cytotoxicity (Kopera et al 2016;Richardson et al 2014b) when over-expressing TEX19 orthologs.…”

Section: Retrotransposition Assaysmentioning

confidence: 99%

Tex19.1Restricts LINE-1 Mobilisation in Mouse Embryonic Stem Cells

MacLennan

García-Cañadas

Reichmann

et al. 2017

Preprint

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Mobilisation of retrotransposons to new genomic locations is a significant driver of mammalian genome evolution. In humans, retrotransposon mobilisation is mediated primarily by proteins encoded by LINE-1 (L1) retrotransposons, which mobilise in pluripotent cells early in development.Here we show that TEX19.1, which is induced by developmentally programmed DNA hypomethylation, can directly interact with the L1-encoded protein L1-ORF1p, stimulate its polyubiquitylation and degradation, and restrict L1 mobilisation. We also show that TEX19.1 likely acts, at least in part, through promoting the activity of the E3 ubiquitin ligase UBR2 towards L1-ORF1p. Moreover, we show that loss of Tex19.1 increases L1-ORF1p levels and mobilisation of L1 reporters in pluripotent mouse embryonic stem cells implying that Tex19.1 prevents new retrotransposition-mediated mutations from arising in the germline genome. These data show that post-translational regulation of L1 retrotransposons plays a key role in maintaining transgenerational genome stability in the epigenetically dynamic developing mammalian germline.

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LINE-1 Cultured Cell Retrotransposition Assay

Cited by 53 publications

References 81 publications

L1 retrotransposition is a common feature of mammalian hepatocarcinogenesis

L1 retrotransposition is a common feature of mammalian hepatocarcinogenesis

Heritable L1 retrotransposition in the mouse primordial germline and early embryo

Tex19.1Restricts LINE-1 Mobilisation in Mouse Embryonic Stem Cells

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