Repeat-induced Gene Silencing Research Articles

Concatenation of Transgenic DNA: Random or Orchestrated?

Generation of transgenic organisms by pronuclear microinjection has become a routine procedure. However, while the process of DNA integration in the genome is well understood, we still do not know much about the recombination between transgene molecules that happens in the first moments after DNA injection. Most of the time, injected molecules are joined together in head-to-tail tandem repeats—the so-called concatemers. In this review, we focused on the possible concatenation mechanisms and how they could be studied with genetic reporters tracking individual copies in concatemers. We also discuss various features of concatemers, including palindromic junctions and repeat-induced gene silencing (RIGS). Finally, we speculate how cooperation of DNA repair pathways creates a multicopy concatenated insert.

SIRT1 stabilizes extrachromosomal gene amplification and contributes to repeat-induced gene silencing

Sirtuin 1 (SIRT1) is a protein deacetylase that maintains genome stability by preventing the activation of latent replication origins. Amplified genes in cancer cells localize on either extrachromosomal double minutes (DMs) or the chromosomal homogeneously staining region. Previously, we found that a plasmid with a mammalian replication initiation region and a matrix attachment region spontaneously mimics gene amplification in cultured animal cells and efficiently generates DMs and/or an homogeneously staining region. Here, we addressed the possibility that SIRT1 might be involved in initiation region/matrix attachment region–mediated gene amplification using SIRT1-knockout human COLO 320DM cells. Consequently, we found that extrachromosomal amplification was infrequent in SIRT1-deficient cells, suggesting that DNA breakage caused by latent origin activation prevented the formation of stable extrachromosomal amplicons. Moreover, we serendipitously found that reporter gene expression from the amplified repeats, which is commonly silenced by repeat-induced gene silencing (RIGS) in SIRT1-proficient cells, was strikingly higher in SIRT1-deficient cells, especially in the culture treated with the histone deacetylase inhibitor butyrate. Compared with the SIRT1-proficient cells, the gene expression per copy was up to thousand-fold higher in the sorter-isolated highest 10% cells among the SIRT1-deficient cells. These observations suggest that SIRT1 depletion alleviates RIGS. Thus, SIRT1 may stabilize extrachromosomal amplicons and facilitate RIGS. This result could have implications in cancer malignancy and protein expression.

Fluorescence-assisted sequential insertion of transgenes (FASIT): an approach for increasing specific productivity in mammalian cells

Currently, the generation of cell lines for the production of recombinant proteins has the limitation of unstable gene expression due to the repeat-induced gene silencing or the loss of transgene copies resulting from recombination events. In this work, we developed a new strategy based on the sequential insertion of transgenes for generating stable clones producing high levels of a chimeric human follicle-stimulating hormone (hscFSH). Gene insertion was done by transducing HEK-293 cells with a lentiviral vector containing a bicistronic transcriptional unit for expressing hscFSH and GFP genes. Clone selection was performed by flow cytometry coupled to cell sorting, and the GFP gene was further removed by CRE-mediated site-specific recombination. High-producing clones of hscFSH were obtained after three rounds of lentiviral transduction. Expression levels increased in a step-wise manner from 7 to 23 pg/cell/day, with a relatively constant rate of 7 pg/cell/day in each round of transduction. The GFP gene was successfully removed from the cell genome without disturbing the hscFSH gene expression. Clones generated using this approach showed stable expression levels for more than two years. This is the first report describing the sequential insertion of transgenes as an alternative for increasing the expression levels of transformed cell lines. The methodology described here could notably impact on biotechnological industry by improving the capacity of mammalian cells to produce biopharmaceuticals.

Repeat induces not only gene silencing, but also gene activation in mammalian cells.

Repeat-induced gene silencing (RIGS) establishes the centromere structure, prevents the spread of transposons and silences transgenes, thereby limiting recombinant protein production. We previously isolated a sequence (B-3-31) that alleviates RIGS from the human genome. Here, we developed an assay system for evaluating the influence of repeat sequences on gene expression, based on in vitro ligation followed by our original gene amplification technology in animal cells. Using this assay, we found that the repeat of B-3-31, three core sequences of replication initiation regions (G5, C12, and D8) and two matrix attachment regions (AR1 and 32-3), activated the co-amplified plasmid-encoded d2EGFP gene in both human and hamster cell lines. This upregulation effect persisted for up to 82 days, which was confirmed to be repeat-induced, and was thus designated as a repeat-induced gene activation (RIGA). In clear contrast, the repeat of three bacterial sequences (lambda-phage, Amp, and ColE1) and three human retroposon sequences (Alu, 5'-untranslated region, and ORF1 of a long interspersed nuclear element) suppressed gene expression, thus reflecting RIGS. RIGS was CpG-independent. We suggest that RIGA might be associated with replication initiation. The discovery of RIGS and RIGA has implications for the repeat in mammalian genome, as well as practical value in recombinant production.

Protein 2b of Cucumber mosaic virus strains IA and SD preferentially suppresses RDR6-dependent silencing pathway

The 2b protein of Cucumber mosaic virus (CMV) subgroup IB strain IA (IA2b) suppresses the sense transgene-induced post-transcriptional gene silencing (S-PTGS) but not the inverted repeat-induced post-transcriptional gene silencing (IR-PTGS) of a tobacco microsome-localized α-linolenate synthase gene (NtFAD3). In contrast, the 2b protein of CMV subgroup IB strain SD (SD2b) has been reported to suppress IR-PTGS. We overexpressed the SD2b gene in tobacco, and this transgenic line was crossed with the transgenic plants showing S-PTGS and IR-PTGS of the NtFAD3 gene. The phenotype of offspring showed that SD2b inhibited S-PTGS but not IR-PTGS. Next, we determined the suppressor activity of IA2b and SD2b proteins in a transient IR-PTGS assay. The transient expression of firefly luciferase (LUC) gene was efficiently decreased by IR-PTGS. Co-infiltration of SD2b and IA2b gene partially suppressed IR-PTGS of the LUC gene in the wild-type plants; however, these 2b proteins did not suppress IR-PTGS in the RNA-dependent RNA polymerase6 (RDR6) knockdown plants. It has been reported that RDR6-dependent secondary small interfering RNA synthesis does not occur in the IR-PTGS, when targeting endogenous genes such as NtFAD3, but is efficiently induced in the IR-PTGS of the reporter transgenes. These results indicate that the SD2b and IA2b preferentially suppress the RDR6-dependent silencing pathway but do not suppress the RDR6-independent IR-PTGS pathway.

Effects of the 2b Protein of Cucumber mosaic virus Subgroup IB Strain IA on Different Transgene-Induced RNA Silencing Pathways

The Cucumber mosaic virus (CMV) 2b protein has multiple activities as a viral suppressor of RNA silencing (VSR). Here, we characterized the 2b protein (IA2b) of a CMV-IA isolate. This IA2b protein has two nuclear localization signals, and when it was fused with GFP, the protein was found to be preferentially localized in the nucleus. A transgenic tobacco line, 2b8, expressing the IA2b gene showed an enhanced expression of an agroinfiltrated sense transgene, which indicated that IA2b has VSR activity. When 2b8 plants were crossed with plants showing sense-transgene-induced post-transcriptional gene silencing (S-PTGS), the IA2b protein caused the disappearance of small interfering RNAs (siRNAs) and the S-PTGS phenotype was converted to the overexpression phenotype. When 2b8 plants were crossed with plants showing inverted repeat-induced post-transcriptional gene silencing (IR-PTGS), the IA2b protein did not suppress the accumulation of siRNAs processed from hairpin RNAs and the silencing phenotype was maintained. When a tobacco plant showing an overexpressed phenotype of a sense transgene was crossed with IR-PTGS plants targeting the same transgene, the primary siRNAs that were produced by the IR-PTGS construct triggered the production of the secondary siRNAs through the RNA-dependent RNA polymerase6 (RDR6)-dependent pathway. The IA2b protein suppressed the secondary siRNA formation but not the primary siRNA production from hairpin RNAs, resulting in the silencing phenotype from IR-PTGS being maintained. These results indicate that IA2b suppresses RDR6-dependent siRNA production and does not interfere with the silencing step by siRNA-guided RNA degradation.

Epigenetic Repeat-Induced Gene Silencing in the Chromosomal and Extrachromosomal Contexts in Human Cells.

A plasmid bearing both a replication initiation region and a matrix attachment region is spontaneously amplified in transfected mammalian cells and generates plasmid repeats in the extrachromosomal double minutes (DMs) or the chromosomal homogeneously staining region (HSR). Generally, the repeat sequences are subject to repeat-induced gene silencing, the mechanism of which remains to be elucidated. Previous research showed that gene expression from the same plasmid repeat was higher from repeats located at DMs than at the HSR, which may reflect the extrachromosomal environment of the DMs. In the current study, plasmid repeats in both DMs and HSR were associated with repressive histone modifications (H3K9me3, H3K9me2), and the levels of repressive chromatin markers were higher in HSR than in DMs. Inactive chromatin is known to spread to neighboring regions in chromosome arm. Here, we found that such spreading also occurs in extrachromosomal DMs. Higher levels of active histone modifications (H3K9Ac, H3K4me3, and H3K79me2) were detected at plasmid repeats in DMs than in HSR. The level of DNA CpG methylation was generally low in both DMs and HSR; however, there were some hypermethylated copies within the population of repeated sequences, and the frequency of such copies was higher in DMs than in HSR. Together, these data suggest a “DNA methylation-core and chromatin-spread” model for repeat-induced gene silencing. The unique histone modifications at the extrachromosomal context are discussed with regard to the model.

Cloning and Characterization of a Human Genomic Sequence that Alleviates Repeat-Induced Gene Silencing.

Plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) are spontaneously amplified in transfected mammalian cells, and such amplification generates chromosomal homogeneously staining regions (HSRs) or extrachromosomal double minutes (DMs). This method provides a novel, efficient, and rapid way to establish cells that stably produce high levels of recombinant proteins. However, because IR/MAR plasmids are amplified as repeats, they are frequently targeted by repeat-induced gene silencing (RIGS), which silences a variety of repeated sequences in transgenes and the genome. To address this problem, we developed a novel screening system using the IR/MAR plasmid to isolate human genome sequences that alleviate RIGS. The screen identified a 3,271 bp sequence (B-3-31) that elevated transgene expression without affecting the amplification process. Neither non-B structure (i.e., the inverted repeats or bending) nor known epigenetic modifier elements such as MARs, insulators, UCOEs, or STARs could explain the anti-silencing activity of B-3-31. Instead, the activity was distributed throughout the entire B-3-31 sequence, which was extremely A/T-rich and CpG-poor. Because B-3-31 effectively and reproducibly alleviated RIGS of repeated genes, it could be used to increase recombinant protein production.

Drosophila oncogene Gas41 is an RNA interference modulator that intersects heterochromatin and the small interfering RNA pathway.

Glioma amplified sequence41 (Gas41) is a highly conserved putative transcription factor that is frequently abundant in human gliomas. Gas41 shows oncogenic activity by promoting cell growth and viability. In the present study, we show that Gas41 is required for proper functioning of RNA interference (RNAi) machinery in the nuclei, although three basic structural domains of RNAi components PAZ, PIWI and dsRNA with respect to binding are absent in the structural sequences. Variations of structural domains are highly conserved among prokaryotes and eukaryotes. Gas41 interacts with cytological RNase III enzyme Dicer1 both biochemically and genetically. However, Drosophila Gas41 functions as chromatin remodeler and interacts with different heterochromatin markers and repeat-induced transgene silencing by modulating position effect variegation. We also show that transcriptional inactive Gas41 mutant interferes with the functional assembly of heterochromatin-associated proteins, dimethylated lysine 9 of histone H3 and heterochromatic protein 1 in developing embryos. A reduction of heterochromatic markers is accompanied by the mini-w promoter sequence in Gas41 mutants. These findings suggest that Drosophila Gas41 guides the repeat associated gene silencing and the Dicer1 interaction, thereby depicting a new role for Gas41. Gas41 is a critical RNAi component. In Drosophila, Gas41 plays a dual role. On the one hand, it appears to participate with Dicer 1 in the RNAi pathway and, alternatively, it also participates in repeat-induced gene silencing by accumulating heterochromatin proteins at the mini-w array promoters. Therefore, it represents an intriguing and apparently paradoxical new finding in RNA technology with respect to the process of heterochromatin gene silencing.

Digital Genotyping of Macrosatellites and Multicopy Genes Reveals Novel Biological Functions Associated with Copy Number Variation of Large Tandem Repeats

Tandem repeats are common in eukaryotic genomes, but due to difficulties in assaying them remain poorly studied. Here, we demonstrate the utility of Nanostring technology as a targeted approach to perform accurate measurement of tandem repeats even at extremely high copy number, and apply this technology to genotype 165 HapMap samples from three different populations and five species of non-human primates. We observed extreme variability in copy number of tandemly repeated genes, with many loci showing 5–10 fold variation in copy number among humans. Many of these loci show hallmarks of genome assembly errors, and the true copy number of many large tandem repeats is significantly under-represented even in the high quality ‘finished’ human reference assembly. Importantly, we demonstrate that most large tandem repeat variations are not tagged by nearby SNPs, and are therefore essentially invisible to SNP-based GWAS approaches. Using association analysis we identify many cis correlations of large tandem repeat variants with nearby gene expression and DNA methylation levels, indicating that variations of tandem repeat length are associated with functional effects on the local genomic environment. This includes an example where expansion of a macrosatellite repeat is associated with increased DNA methylation and suppression of nearby gene expression, suggesting a mechanism termed “repeat induced gene silencing”, which has previously been observed only in transgenic organisms. We also observed multiple signatures consistent with altered selective pressures at tandemly repeated loci, suggesting important biological functions. Our studies show that tandemly repeated loci represent a highly variable fraction of the genome that have been systematically ignored by most previous studies, copy number variation of which can exert functionally significant effects. We suggest that future studies of tandem repeat loci will lead to many novel insights into their role in modulating both genomic and phenotypic diversity.

Development of pronuclear injection-based targeted transgenesis in mice through Cre-loxP site-specific recombination.

Microinjection of DNA into the pronuclei of zygotes is the simplest and most widely used method for generating transgenic (Tg) mice. However, it is always associated with random integration of multiple copies of the transgene, resulting in unstable, low, or no transgene expression due to positional effects and/or repeat-induced gene silencing. In addition, random integration sometimes disrupts an endogenous gene that can affect the phenotypes of Tg mice. Our recently developed pronuclear injection-based targeted transgenesis (PITT) method enables the integration of a single-copy transgene into a predetermined genomic locus through Cre-loxP site-specific recombination. The PITT method enables stable and reliable transgene expression in Tg mice and is also applicable for generating knockdown mice. Therefore, the PITT method could represent next-generation transgenesis that overcomes the pitfalls of conventional transgenesis.

PITT: Pronuclear Injection-Based Targeted Transgenesis, a Reliable Transgene Expression Method in Mice

Transgenic (Tg) mice have been extensively used as valuable tools for analyses of gene function and have also served as models for many human diseases. Typically, a transgenic mouse is created by microinjection of DNA into pronuclei in which the DNA gets integrated at random locations in the genome. Frequently however, the random integration of multiple copies of a transgene results in transgene silencing, probably because of a positional effect and/or repeat-induced gene silencing. The transgene silencing issue has been overcome by single-copy transgene integration into a predetermined locus through ES cell-mediated transgenesis, despite it being expensive and more time-consuming compared with pronuclear injection (PI)-mediated transgenesis. Recently, several groups have reported novel approaches that employ PI for targeted transgenesis. They are based on site-specific recombination catalyzed by a recombinase or an integrase or homologous recombination enhanced by a zinc-finger nuclease via PI. These next-generation transgenesis methods, which we termed as PI-based Targeted Transgenesis (PITT), are more convenient and faster than ES cell-based transgenesis. Furthermore, the Tg mice generated by these newer methods contain a single-copy transgene and exhibit reliable expression of the transgene. The objective of this review is to present the recent progress in mouse targeted transgenesis.

Repeat-induced gene silencing of L1 transgenes is correlated with differential promoter methylation

Recent transgenic studies on L1 retrotransposons have afforded exciting insights into L1 biology, and a unique opportunity to model their function and regulation in vivo. Thus far, the majority of the transgenic L1 mouse lines are constructed via pronuclear microinjection, a procedure that typically results in the integration of tandem arrayed transgenes. Transgene arrays are susceptible to repeat-induced gene silencing (RIGS) in both plants and animals. In order to examine the potential impact of RIGS on L1 retrotransposition, we derived a cohort of animals carrying reduced copies of ORFeus transgene at the same genomic locus by Cre-mediated recombination. The copy number reduction of ORFeus transgenes did not decrease the overall retrotransposition activity. Using a sensitive and reproducible quantitative PCR assay, an average frequency of 0.45 insertions per cell was observed for animals carrying the donor transgene at a single copy, representing a 9-fold increase of retrotransposition frequency on a per-copy basis. DNA methylation analyses revealed that the observed retrotransposition activity was correlated with differential CpG methylation at the heterologous promoter: the promoter region was largely methylated in animals with the high-copy array but significantly hypomethylated in animals with the single-copy counterpart. In contrast, the ORF2 region, which represents the body of the ORFeus transgene, and the 3′ end of the transgene showed high level of methylation in both high-copy and single-copy samples. The observed methylation patterns were metastable across generations. In summary, our data suggest that tandem arrayed L1 transgenes are subject to RIGS, and transgenes present at a single copy in the genome are thus recommended for modeling L1 in animals.

High frequency, cell type-specific visualization of fluorescent-tagged genomic sites in interphase and mitotic cells of living Arabidopsis plants

BackgroundInterphase chromosome organization and dynamics can be studied in living cells using fluorescent tagging techniques that exploit bacterial operator/repressor systems and auto-fluorescent proteins. A nuclear-localized Repressor Protein-Fluorescent Protein (RP-FP) fusion protein binds to operator repeats integrated as transgene arrays at defined locations in the genome. Under a fluorescence microscope, the tagged sites appear as bright fluorescent dots in living cells. This technique has been used successfully in plants, but is often hampered by low expression of genes encoding RP-FP fusion proteins, perhaps owing to one or more gene silencing mechanisms that are prevalent in plant cells.ResultsWe used two approaches to overcome this problem. First, we tested mutations in four factors involved in different types of gene silencing and/or epigenetic modifications for their effects on nuclear fluorescence. Only mutations in DDM1, a chromatin remodelling ATPase involved in repeat-induced heterochromatin formation and DNA methylation, released silencing of the RP-FP fusion protein. This result suggested that the operator repeats can trigger silencing of the adjacent gene encoding the RP-FP fusion protein. In the second approach, we transformed the tagged lines with a second T-DNA encoding the RP-FP fusion protein but lacking operator repeats. This strategy avoided operator repeat-induced gene silencing and increased the number of interphase nuclei displaying fluorescent dots. In a further extension of the technique, we show that green fluorescent-tagged sites can be visualized on moving mitotic chromosomes stained with red fluorescent-labelled histone H2B.ConclusionsThe results illustrate the propensity of operator repeat arrays to form heterochromatin that can silence the neighbouring gene encoding the RP-FP fusion protein. Supplying the RP-FP fusion protein in trans from a second T-DNA largely alleviates this problem. Depending on the promoter used to drive expression of the RP-FP fusion protein gene, the fluorescent tagged sites can be visualized at high frequency in different cell types. The ability to observe fluorescent dots on both interphase and mitotic chromosomes allows tagged sites to be tracked throughout the cell cycle. These improvements enhance the versatility of the fluorescent tagging technique for future studies of chromosome arrangement and dynamics in living plants.

A study of monoclonal antibody‐producing CHO cell lines: What makes a stable high producer?

Generating stable, high-producing cell lines for recombinant protein production requires an understanding of the potential limitations in the cellular machinery for protein expression. In order to increase our understanding of what makes a stable high producer, we have generated a panel of 17 recombinant monoclonal antibody expressing Chinese hamster ovary subclones (CHO-mAb) with specific productivities ranging between 3 and 75 pg cell(-1) day(-1) using the dihydrofolate reductase (dhfr) expression system and compared the molecular features of these high- and low-producer clones. The relative heavy chain (HC) and light chain (LC) transgene copy numbers and mRNA levels were determined using real-time quantitative PCR (RT qPCR). We observed that not only higher transgene copy numbers and mRNA levels of both HC and LC were characteristic for the high-producer clones as compared to the low-producer clones but also a more favorable HC to LC transgene copy numbers ratio. By studying the long-term stability of the CHO-mAb subclones in the absence of methotrexate (MTX) selective pressure over 36 passages we observed a 35-92% decrease in volumetric productivity, primarily caused by a significant decrease in HC and LC mRNA levels with little change in the transgene copy numbers. Using Southern blot hybridization we analyzed the HC and LC transgene integration patterns in the host chromosome and their changes in course of gene amplification and long-term culturing. We observed that MTX-induced gene amplification caused chromosomal rearrangements resulting in clonal variability in regards to growth, productivity, and stability. No further obvious DNA rearrangements occurred during long-term culturing in the absence of MTX, indicating that other mechanisms were responsible for the decreased transcription efficiency. Our results implicate that the amplified transgene sequences were arranged in tandem repeats potentially triggering repeat-induced gene silencing. We hypothesize that the decline in transgene mRNA levels upon long-term culturing without MTX was mainly caused by transgene silencing consequently leading to a loss in mAb productivity. The exact molecular mechanisms causing production instability are not yet fully understood. The herein described extensive characterization studies could help understand the limitations to high-level, stable recombinant protein production and find ways to improving and accelerating the process for high-producer cell line generation and selection.

Position effect variegation and imprinting of transgenes in lymphocytes

Sequences proximal to transgene integration sites are able to deregulate transgene expression resulting in complex position effect phenotypes. In addition, transgenes integrated as repeated arrays are susceptible to repeat-induced gene silencing. Using a Cre recombinase-based system we have addressed the influence of transgene copy number (CN) on expression of hCD2 transgenes. CN reduction resulted in a decrease, increase or no effect on variegation depending upon the site of integration. This finding argues that repeat-induced gene silencing is not the principle cause of hCD2 transgene variegation. These results also suggest that having more transgene copies can be beneficial at some integration sites. The transgenic lines examined in this report also exhibited a form of imprinting, which was manifested by decreased levels of expression and increased levels of variegation, upon maternal transmission; and this correlated with DNA hypermethylation and a reduction in epigenetic chromatin modifications normally associated with active genes.

Repeat Organization and Epigenetic Regulation of the DH-Cμ Domain of the Immunoglobulin Heavy-Chain Gene Locus

The first steps of murine immunoglobulin heavy-chain (IgH) gene recombination take place within a chromosomal domain that contains diversity (D(H)) and joining (J(H)) gene segments, but not variable (V(H)) gene segments. Here we show that the chromatin state of this domain is markedly heterogeneous. Specifically, only 5'- and 3'-most D(H) gene segments carry active chromatin modifications, whereas intervening D(H)s are associated with heterochromatic marks that are maintained by ongoing histone deacetylation. The intervening D(H)s form part of a tandemly repeated sequence that expresses tissue-specific, antisense oriented transcripts. We propose that the intervening D(H) genes are actively suppressed by repeat-induced epigenetic silencing, which is reflected in their infrequent representation in DJ(H) junctions compared to the flanking D(H) genes.

Agrobacterium proteins VirD2 and VirE2 mediate precise integration of synthetic T-DNA complexes in mammalian cells.

Agrobacterium tumefaciens-mediated plant transformation, a unique example of interkingdom gene transfer, has been widely adopted for the generation of transgenic plants. In vitro synthesized transferred DNA (T-DNA) complexes comprising single-stranded DNA and Agrobacterium virulence proteins VirD2 and VirE2, essential for plant transformation, were used to stably transfect HeLa cells. Both proteins positively influenced efficiency and precision of transgene integration by increasing overall transformation rates and by promoting full-length single-copy integration events. These findings demonstrate that the virulence proteins are sufficient for the integration of a T-DNA into a eukaryotic genome in the absence of other bacterial or plant factors. Synthetic T-DNA complexes are therefore unique protein:DNA delivery vectors with potential applications in the field of mammalian transgenesis.

Embryo culture-based generation of enhanced green fluorescent protein-transgenic mice

One of the limitations of transgenesis is low efficiency. In this study, we generated transgenic mice harboring the enhanced green fluorescent protein (EGFP) gene, under the control of chicken-β-actin promoter and cytomegalovirus enhancer, using two approaches and compared their efficiencies. One involved culture of EGFP-injected embryos developing through EGFP-expressing “green” blastocysts, followed by their transfer to uterus. The second was oviductal-transfer of EGFP-injected-eggs. Embryo culture-based-transgenesis (ECBT) produced 100% transgenic mice, unlike the second approach. Moreover, ECBT required reduced number of recipients and markedly increased pregnancy rates. Of the nine founders, seven exhibited ubiquitous EGFP-expression, one (GU1) was a mosaic and the other (G18) was non-expressing. The molecular basis for this was attributed to repeat-induced gene silencing, since the G18 had a high copy number (≃99/genome) of the non-mutated and non-rearranged EGFP-transgene integrated at a single site. Our results show the superiority of ECBT over the conventional oviductal approach for generating transgenic “green” mice.

P element-encoded regulatory products enhance Repeat-Induced Gene Silencing (RIGS) of P-lacZ-white clusters in Drosophila melanogaster.

In Drosophila melanogaster, some clusters of P transgenes ( P-lacZ-white) display a variegating phenotype for the white marker in the eye, a phenomenon termed "Repeat-Induced Gene Silencing" (RIGS). We have tested the influence of the P element repression state (P cytotype) on the eye phenotype of several P-lac-w clusters that differ in transgene copy number or genomic insertion site. P element-encoded regulatory products strongly enhance RIGS. The effect occurs in both sexes, is detectable with clusters having at least three copies and is observed at both genomic locations tested (cytogenetic regions 50C and 92E). Single variegating P-lac-w transgenes located in pericentromeric heterochromatin are not affected by P regulatory products. All P strain backgrounds tested enhance RIGS, including chromosomes bearing a single P element encoding a truncated P transposase or carrying a single internally deleted KP element. Therefore, clusters are highly sensitive to different types of P repressors. Finally, a chimeric gene in which the 5' portion of the P element is fused to the polyhomeotic coding sequence (ph(p1)) also strongly enhances silencing of P-lac-w clusters. These results have implications for the mechanism of action of the P repressors and show that P transgene clusters represent a new class of P-sensitive alleles, providing a simple assay for somatic P repression that can be completed in one generation.