A highly active synthetic mammalian retrotransposon

Han, Jeffrey S.; Boeke, Jef D.

doi:10.1038/nature02535

Cited by 173 publications

(218 citation statements)

References 23 publications

Supporting

Mentioning

209

Contrasting

Unclassified

Order By: Relevance

“…The endogenous L1 5′UTR promoter is derived from L1spa (34), an active Tf family element that inserted within intron 6 of the glycine receptor β-subunit, causing muscular spasticity in the spastic mouse. The codonoptimized ORF1 and ORF2 sequences are from ORFeus-Mm, which supports efficient transcription elongation (35) and consequently higher retrotransposition both in vitro (36) and in vivo (25). The intron-disrupted EGFP cassette has been used to report retrotransposition in vivo (26,28).…”

Section: Developing An L1 Transgene Regulated By the Endogenous Mouse L1mentioning

confidence: 99%

See 1 more Smart Citation

Intact piRNA pathway prevents L1 mobilization in male meiosis

Newkirk

Lee

Grandi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

The PIWI-interacting RNA (piRNA) pathway is essential for retrotransposon silencing. In piRNA-deficient mice, L1-overexpressing male germ cells exhibit excessive DNA damage and meiotic defects. It remains unknown whether L1 expression simply highlights piRNA deficiency or actually drives the germ-cell demise. Specifically, the sheer abundance of genomic L1 copies prevents reliable quantification of new insertions. Here, we developed a codon-optimized L1 transgene that is controlled by an endogenous mouse L1 promoter. Importantly, DNA methylation dynamics of a single-copy transgene were indistinguishable from those of endogenous L1s. Analysis of Mov10l1 −/− testes established that de novo methylation of the L1 transgene required the intact piRNA pathway. Consistent with loss of DNA methylation and programmed reduction of H3K9me2 at meiotic onset, the transgene showed 1,400-fold increase in RNA expression and consequently 70-fold increase in retrotransposition in postnatal day 14 Mov10l1 −/− germ cells compared with the wildtype. Analysis of adult Mov10l1 −/− germ-cell fractions indicated a stage-specific increase of retrotransposition in the early meiotic prophase. However, extrapolation of the transgene data to endogenous L1s suggests that it is unlikely insertional mutagenesis alone accounts for the Mov10l1 −/− phenotype. Indeed, pharmacological inhibition of reverse transcription did not rescue the meiotic defect. Cumulatively, these results establish the occurrence of productive L1 mobilization in the absence of an intact piRNA pathway but leave open the possibility of processes preceding L1 integration in triggering meiotic checkpoints and germ-cell death. Additionally, our data suggest that many heritable L1 insertions originate from individuals with partially compromised piRNA defense.LINE-1 reporter transgene | meiotic arrest | PIWI-interacting RNA | retrotransposition | spermatogenesis T he bulk of mammalian genomes are made up of transposable elements, the majority of which are retrotransposons (1). Retrotransposons are classified into long-interspersed elements (LINEs), short-interspersed elements (SINEs), and LTR retrotransposons, which collectively account for 43% and 37% of the human and mouse genomes, respectively (2). Retrotransposons amplify in the genome through an RNA intermediate, a process termed retrotransposition. LINEs are autonomous elements. An intact, full-length LINE-1 (L1) encodes two ORFs (i.e., ORF1 and ORF2); both are required for L1 mobilization (3). SINEs are nonautonomous elements and rely on L1's proteins for propagation in the genome (4). LTR retrotransposons are autonomous but appear to be inactivated in the human genome (5). Retrotransposition endangers the integrity of both somatic and germline genomes through insertional mutagenesis. In somatic tissues, both elevated L1 expression and retrotransposition have been strongly associated with many types of human cancers (6, 7). In a few cases, specific retrotransposition events (i.e., insertions) have been determined to drive t...

show abstract

Section: Developing An L1 Transgene Regulated By the Endogenous Mouse L1mentioning

confidence: 99%

“…The second parameter was the ratio of insertion frequencies between the transgene and endogenous L1s. It was set to 200:1 based on retrotransposition assays in cell culture (36). The third parameter was the calculated number of insertions per cell, representing the average mutational burden.…”

Section: Inhibition Of Retrotransposition Does Not Rescue the Meioticmentioning

confidence: 99%

Intact piRNA pathway prevents L1 mobilization in male meiosis

Newkirk

Lee

Grandi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…pBudORF2 syn and pBudORF1 syn contain the L1 spa codon optimized sequences from plasmid psmL1 (a kind gift from Dr. Jef Boeke) (Han and Boeke 2004).…”

Section: Plasmidsmentioning

confidence: 99%

LINE-1 ORF1 protein enhances Alu SINE retrotransposition

Wallace

Wagstaff

Deininger

et al. 2008

Gene

View full text Add to dashboard Cite

Retroelements have contributed over one third of the human genome mass. The currently active LINE-1 (L1) codes for two proteins (ORF1p and ORF2p), both strictly required for retrotransposition. In contrast, the non-coding parasitic SINE (Alu) only appears to need the L1 ORF2p for its own amplification. This requirement was previously determined using a tissue culture assay system in human cells (HeLa). Because HeLa are likely to express functional L1 proteins, it is possible that low levels of endogenous ORF1p are necessary for the observed tagged Alu mobilization. By individually expressing ORF1 and ORF2 proteins from both human (L1 RP and LRE3) and rodent (L1 A102 and L1 spa ) L1 sources, we demonstrate that increasing amounts of ORF1 expressing vector enhances tagged Alu mobilization in HeLa cells. In addition, using chicken fibroblast cells as an alternate cell culture source, we confirmed that ORF1p is not strictly required for Alu mobilization in our assay. Supporting our observations in HeLa cells, we find that tagged Alu retrotransposition is improved by supplementation of ORF1p in the cultured chicken cells. We postulate that L1 ORF1p plays either a direct or indirect role in enhancing the interaction between the Alu RNA and the required factors needed for its retrotransposition.

show abstract

“…However, the low frequency of such germline transposition events (around 1%) is probably not sufficient for this to be used as a germline mutagen. Recent increases in transposition frequency up to 200-fold were observed upon reengineering the L1 open reading frame with synthetic codons (Han and Boeke, 2004). These improvements show promise for using this system as an in vivo insertional mutagen.…”

Section: Transposon-mediated Mutagenesis In Vertebratesmentioning

confidence: 96%

Engineering mutations: Deconstructing the mouse gene by gene

Raymond¹,

Soriano²

2006

Developmental Dynamics

View full text Add to dashboard Cite

Over the past years new vectors and methodologies have been developed to carry out large-scale genomewide insertional mutagenesis screens in the mouse. Gene trapping, the most commonly used technique, is based on the insertion of a retroviral-or plasmid-based vector into a gene, resulting in a loss-of-function mutation, while simultaneously reporting its expression pattern and providing a molecular tag to facilitate cloning. The discovery of vertebrate DNA transposons in the mouse and recent improvements has also led to their increased use in insertional mutagenesis screens. Several public resources have been set-up recently by the academic community to distribute information and materials generated from these largescale screens. These new resources should accelerate the study and understanding of biological and developmental processes. Developmental Dynamics 235:2424 -2436, 2006.

show abstract

A highly active synthetic mammalian retrotransposon

Cited by 173 publications

References 23 publications

Intact piRNA pathway prevents L1 mobilization in male meiosis

Intact piRNA pathway prevents L1 mobilization in male meiosis

LINE-1 ORF1 protein enhances Alu SINE retrotransposition

Engineering mutations: Deconstructing the mouse gene by gene

Contact Info

Product

Resources

About