L1 retrotransposon-mediated stable gene silencing

RNA interference (RNAi) is widely used for functional studies and has been proposed as a potential therapeutic agent. Current RNAi systems are largely efficient, but have limitations including transient effect, the need for viral handling and potential insertional mutations. Here, we describe a simple L1 retrotransposon-based system for the delivery of small interfering RNA (siRNA) and stable silencing in human cells. This system demonstrated long-term siRNA expression and significant reduction in both exogenous and endogenous gene expression by up to 90%. Further characterization indicated that retrotransposition occurred in a controlled manner such that essentially only one RNAi-cassette was integrated into the host genome and was sufficient for strong interference. Our system provides a novel strategy for stable gene silencing that is easy and efficient, and it may have potential applications for ex vivo and in vivo molecular therapy

Mobile DNA transposition in somatic cells

It had been long assumed that almost all insertions of mobile DNA elements occurred during germ-cell development rather than in somatic-cell development, but solid evidence for transposition in somatic cells is now accumulating. To add to this evidence, a recent paper in Mobile DNA reports the somatic transposition of a site-specific retrotransposon, R2, into its insertion site in 28S ribosomal DNA in Drosophila embryos

Factor VIII gene inversions causing severe hemophilia A originate almost exclusively in male germ cells

The factor VIII gene, which is defective In hemophilia A, is located in the last megabase of the long arm of the X chromosome. Inversions due to intrachromosomal homologous recombination between mispaired copies of gene A located within intron 22 of the gene and about 500 kb telomeric to it account for nearly half of all cases of severe hemophilia A. We hypothesized that pairing of Xq with its homolog inhibits the Inversion process, and that, therefore, the event originates predominantly in male germ cells. In all 20 informative cases In which the inversion originated in a maternal grandparent, DNA polymorphism analysis determined that it occurred in the male germline. In addition, all but one of 50 mothers of sporadic cases due to an Inversion were carriers. Thus, these data support the hypothesis and Indicate that factor VIII gene inversions leading to severe hemophilia A occur almost exclusively In male germ cell

Widespread somatic L1 retrotransposition occurs early during gastrointestinal cancer evolution

Somatic L1 retrotransposition events have been shown to occur in epithelial cancers. Here, we attempted to determine how early somatic L1 insertions occurred during the development of gastrointestinal (GI) cancers. Using L1-targeted resequencing (L1-seq), we studied different stages of four colorectal cancers arising from colonic polyps, seven pancreatic carcinomas, as well as seven gastric cancers. Surprisingly, we found somatic L1 insertions not only in all cancer types and metastases but also in colonic adenomas, well-known cancer precursors. Some insertions were also present in low quantities in normal GI tissues, occasionally caught in the act of being clonally fixed in the adjacent tumors. Insertions in adenomas and cancers numbered in the hundreds, and many were present in multiple tumor sections, implying clonal distribution. Our results demonstrate that extensive somatic insertional mutagenesis occurs very early during the development of GI tumors, probably before dysplastic growth

Extensive somatic L1 retrotransposition in colorectal tumors

L1 retrotransposons comprise 17% of the human genome and are its only autonomous mobile elements. Although L1-induced insertional mutagenesis causes Mendelian disease, their mutagenic load in cancer has been elusive. Using L1-targeted resequencing of 16 colorectal tumor and matched normal DNAs, we found that certain cancers were excessively mutagenized by human-specific L1s, while no verifiable insertions were present in normal tissues. We confirmed de novo L1 insertions in malignancy by both validating and sequencing 69/107 tumor-specific insertions and retrieving both 5′ and 3′ junctions for 35. In contrast to germline polymorphic L1s, all insertions were severely 5′ truncated. Validated insertion numbers varied from up to 17 in some tumors to none in three others, and correlated with the age of the patients. Numerous genes with a role in tumorigenesis were targeted, including ODZ3, ROBO2, PTPRM, PCM1, and CDH11. Thus, somatic retrotransposition may play an etiologic role in colorectal cancer

Heritable L1 retrotransposition in the mouse primordial germline and early embryo

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

Many LINE1 elements contribute to the transcriptome of human somatic cells

Over 600 LINE 1 elements are shown to be transcribed in humans; 400 of these are full-length elements in the reference genome

Metabolic phenotype of methylmalonic acidemia in mice and humans: the role of skeletal muscle

<p>Abstract</p> <p>Background</p> <p>Mutations in methylmalonyl-CoA mutase cause methylmalonic acidemia, a common organic aciduria. Current treatment regimens rely on dietary management and, in severely affected patients, liver or combined liver-kidney transplantation. For undetermined reasons, transplantation does not correct the biochemical phenotype.</p> <p>Methods</p> <p>To study the metabolic disturbances seen in this disorder, we have created a murine model with a null allele at the methylmalonyl-CoA mutase locus and correlated the results observed in the knock-out mice to patient data. To gain insight into the origin and magnitude of methylmalonic acid (MMA) production in humans with methylmalonyl-CoA mutase deficiency, we evaluated two methylmalonic acidemia patients who had received different variants of combined liver-kidney transplants, one with a complete liver replacement-kidney transplant and the other with an auxiliary liver graft-kidney transplant, and compared their metabolite production to four untransplanted patients with intact renal function.</p> <p>Results</p> <p>Enzymatic, Western and Northern analyses demonstrated that the targeted allele was null and correctable by lentiviral complementation. Metabolite studies defined the magnitude and tempo of plasma MMA concentrations in the mice. Before a fatal metabolic crisis developed in the first 24–48 hours, the methylmalonic acid content per gram wet-weight was massively elevated in the skeletal muscle as well as the kidneys, liver and brain. Near the end of life, extreme elevations in tissue MMA were present primarily in the liver. The transplant patients studied when well and on dietary therapy, displayed massive elevations of MMA in the plasma and urine, comparable to the levels seen in the untransplanted patients with similar enzymatic phenotypes and dietary regimens.</p> <p>Conclusion</p> <p>The combined observations from the murine metabolite studies and patient investigations indicate that during homeostasis, a large portion of circulating MMA has an extra-heptorenal origin and likely derives from the skeletal muscle. Our studies suggest that modulating skeletal muscle metabolism may represent a strategy to increase metabolic capacity in methylmalonic acidemia as well as other organic acidurias. This mouse model will be useful for further investigations exploring disease mechanisms and therapeutic interventions in methylmalonic acidemia, a devastating disorder of intermediary metabolism.</p