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Megan K Pugach - One of the best experts on this subject based on the ideXlab platform.
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dose dependent rescue of ko amelogenin enamel by Transgenes in vivo
2017Co-Authors: Felicitas B Bidlack, Yan Xia, Megan K PugachAbstract:Mice lacking amelogenin (KO) have hypoplastic enamel. Overexpression of the most abundant amelogenin splice variant M180 and LRAP Transgenes can substantially improve KO enamel, but only ~40% of the incisor thickness is recovered and the prisms are not as tightly woven as in WT enamel. This implies that the compositional complexity of the enamel matrix is required for different aspects of enamel formation, such as organizational structure and thickness. The question arises, therefore, how important the ratio of different matrix components, and in particular amelogenin splice products, is in enamel formation. Can optimal expression levels of amelogenin Transgenes representing both the most abundant splice variants and cleavage product at protein levels similar to that of WT improve the enamel phenotype of KO mice? Addressing this question, our objective was here to understand dosage effects of amelogenin Transgenes (Tg) representing the major splice variants M180 and LRAP and cleavage product CTRNC on enamel properties. Amelogenin KO mice were mated with M180Tg, CTRNCTg and LRAPTg mice to generate M180Tg and CTRNCTg double transgene and M180Tg, CTRNCTg, LRAPTg triple transgene mice with transgene hemizygosity (on one allelle) or homozygosity (on both alleles). Transgene homo- versus hemizygosity was determined by qPCR and relative transgene expression confirmed by Western blot. Enamel volume and mineral density were analyzed by microCT, thickness and structure by SEM, and mechanical properties by Vickers microhardness testing. There were no differences in incisor enamel thickness between amelogenin KO mice with three or two different Transgenes, but mice homozygous for a given transgene had significantly thinner enamel than mice hemizygous for the transgene (p<0.05).The presence of the LRAPTg did not improve the phenotype of M180Tg/CTRNCTg/KO enamel. In the absence of endogenous amelogenin, the addition of amelogenin Transgenes representing the most abundant splice variants and cleavage product can rescue abnormal enamel properties and structure, but only up to a maximum of ~80% that of molar and ~40% that of incisor wild-type enamel.
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dose dependent rescue of ko amelogenin enamel by Transgenes in vivo
2017Co-Authors: Felicitas B Bidlack, Yan Xia, Megan K PugachAbstract:Mice lacking amelogenin (KO) have hypoplastic enamel. Overexpression of the most abundant amelogenin splice variant M180 and LRAP Transgenes can substantially improve KO enamel, but only ~40% of the incisor thickness is recovered and the prisms are not as tightly woven as in WT enamel. This implies that the compositional complexity of the enamel matrix is required for different aspects of enamel formation, such as organizational structure and thickness. The question arises, therefore, how important the ratio of different matrix components, and in particular amelogenin splice products, is in enamel formation. Can optimal expression levels of amelogenin Transgenes representing both the most abundant splice variants and cleavage product at protein levels similar to that of WT improve the enamel phenotype of KO mice? Addressing this question, our objective was here to understand dosage effects of amelogenin Transgenes (Tg) representing the major splice variants M180 and LRAP and cleavage product CTRNC on enamel properties. Amelogenin KO mice were mated with M180Tg, CTRNCTg and LRAPTg mice to generate M180Tg and CTRNCTg double transgene and M180Tg, CTRNCTg, LRAPTg triple transgene mice with transgene hemizygosity (on one allelle) or homozygosity (on both alleles). Transgene homo- vs. hemizygosity was determined by qPCR and relative transgene expression confirmed by Western blot. Enamel volume and mineral density were analyzed by microCT, thickness and structure by SEM, and mechanical properties by Vickers microhardness testing. There were no differences in incisor enamel thickness between amelogenin KO mice with three or two different Transgenes, but mice homozygous for a given transgene had significantly thinner enamel than mice hemizygous for the transgene (p < 0.05). The presence of the LRAPTg did not improve the phenotype of M180Tg/CTRNCTg/KO enamel. In the absence of endogenous amelogenin, the addition of amelogenin Transgenes representing the most abundant splice variants and cleavage product can rescue abnormal enamel properties and structure, but only up to a maximum of ~80% that of molar and ~40% that of incisor wild-type enamel.
Hervé Vaucheret - One of the best experts on this subject based on the ideXlab platform.
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second site mutagenesis of a hypomorphic argonaute1 allele identifies superkiller3 as an endogenous suppressor of transgene posttranscriptional gene silencing
2015Co-Authors: Agnes Yu, Baptiste Saudemont, Nathalie Bouteiller, Jean Sébastien Parent, Jean-benoit Morel, Emilie Elviramatelot, Gersende Lepère, Taline Elmayan, Hervé VaucheretAbstract:Second-site mutagenesis was performed on the argonaute1-33 (ago1-33) hypomorphic mutant, which exhibits reduced sense transgene posttranscriptional gene silencing (S-PTGS). Mutations in FIERY1, a positive regulator of the cytoplasmic 5′-to-3′ EXORIBONUCLEASE4 (XRN4), and in SUPERKILLER3 (SKI3), a member of the SKI complex that threads RNAs directly to the 3′-to-5′ exoribonuclease of the cytoplasmic exosome, compensated AGO1 partial deficiency and restored S-PTGS with 100% efficiency. Moreover, xrn4 and ski3 single mutations provoked the entry of nonsilenced Transgenes into S-PTGS and enhanced S-PTGS on partially silenced Transgenes, indicating that cytoplasmic 5′-to-3′ and 3′-to-5′ RNA degradation generally counteract S-PTGS, likely by reducing the amount of transgene aberrant RNAs that are used by the S-PTGS pathway to build up small interfering RNAs that guide transgene RNA cleavage by AGO1. Constructs generating improperly terminated transgene messenger RNAs (mRNAs) were not more sensitive to ski3 or xrn4 than regular constructs, suggesting that improperly terminated transgene mRNAs not only are degraded from both the 3′ end but also from the 5′ end, likely after decapping. The facts that impairment of either 5′-to-3′ or 3′-to-5′ RNA degradation is sufficient to provoke the entry of transgene RNA into the S-PTGS pathway, whereas simultaneous impairment of both pathways is necessary to provoke the entry of endogenous mRNA into the S-PTGS pathway, suggest poor RNA quality upon the transcription of Transgenes integrated at random genomic locations.
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mutations in the arabidopsis h3k4me2 3 demethylase jmj14 suppress posttranscriptional gene silencing by decreasing transgene transcription
2012Co-Authors: Ivan Le Masson, Nathalie Bouteiller, Vincent Jauvion, Maud Rivard, Taline Elmayan, Hervé VaucheretAbstract:Posttranscriptional gene silencing (PTGS) mediated by sense Transgenes (S-PTGS) results in RNA degradation and DNA methylation of the transcribed region. Through a forward genetic screen, a mutant defective in the Histone3 Lysine4 di/trimethyl (H3K4me2/3) demethylase Jumonji-C (JmjC) domain-containing protein14 (JMJ14) was identified. This mutant reactivates various Transgenes silenced by S-PTGS and shows reduced Histone3 Lysine9 Lysine14 acetylation (H3K9K14Ac) levels, reduced polymerase II occupancy, reduced transgene transcription, and increased DNA methylation in the promoter region, consistent with the hypothesis that high levels of transcription are required to trigger S-PTGS. The jmj14 mutation also reduces the expression of Transgenes that do not trigger S-PTGS. Moreover, expression of Transgenes that undergo S-PTGS in a wild-type background is reduced in jmj14 sgs3 double mutants compared with PTGS-deficient sgs3 mutants, indicating that JMJ14 is required for high levels of transcription in a PTGS-independent manner. Whereas endogenous loci regulated by JMJ14 exhibit increased H3K4me2 and H3K4me3 levels in the jmj14 mutant, transgene loci exhibit unchanged H3K4me2 and decreased H3K4me3 levels. Because jmj14 mutations impair PTGS of Transgenes expressed under various plant or viral promoters, we hypothesize that JMJ14 demethylation activity is prevented by antagonistic epigenetic marks specifically imposed at transgene loci. Removing JMJ14 likely allows other H3K4 demethylases encoded by the Arabidopsis thaliana genome to act on Transgenes and reduce transcription levels, thus preventing the triggering of S-PTGS.
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systemic acquired silencing transgene specific post transcriptional silencing is transmitted by grafting from silenced stocks to non silenced scions
1997Co-Authors: Jeanchristophe Palauqui, Taline Elmayan, Jeanmarie Pollien, Hervé VaucheretAbstract:Using grafting procedures, we investigated the transmission of co-suppression of nitrate reductase and nitrite reductase host genes and Transgenes and of post-transcriptional silencing of a uidA transgene encoding glucuronidase in tobacco. We demonstrate that silencing is transmitted with 100% efficiency from silenced stocks to non-silenced scions expressing the corresponding transgene. Transmission is unidirectional from stock to scion, transgene specific, locus independent and requires the presence of a transcriptionally active transgene in the target scion. The transmission of co-suppression occurs when silenced stocks and non-silenced target scions are physically separated by up to 30 cm of stem of a non-target wild-type plant. Taken together, these results suggest that a non-metabolic, transgene-specific, diffusable messenger mediates the propagation of de novo post-transcriptional silencing through the plant.
Kristine J Kines - One of the best experts on this subject based on the ideXlab platform.
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germline Transgenesis and insertional mutagenesis in schistosoma mansoni mediated by murine leukemia virus
2012Co-Authors: Gabriel Rinaldi, Sabine E Eckert, Isheng J Tsai, Suttas Suttiprapa, Kristine J Kines, Jose F Tort, Victoria H Mann, Daniel J TurnerAbstract:Functional studies will facilitate characterization of role and essentiality of newly available genome sequences of the human schistosomes, Schistosoma mansoni ,S .japonicum and S. haematobium. To develop Transgenesis as a functional approach for these pathogens, we previously demonstrated that pseudotyped murine leukemia virus (MLV) can transduce schistosomes leading to chromosomal integration of reporter Transgenes and short hairpin RNA cassettes. Here we investigated vertical transmission of Transgenes through the developmental cycle of S. mansoni after introducing Transgenes into eggs. Although MLV infection of schistosome eggs from mouse livers was efficient in terms of snail infectivity, .10-fold higher transgene copy numbers were detected in cercariae derived from in vitro laid eggs (IVLE). After infecting snails with miracidia from eggs transduced by MLV, sequencing of genomic DNA from cercariae released from the snails also revealed the presence of Transgenes, demonstrating that Transgenes had been transmitted through the asexual developmental cycle, and thereby confirming germline Transgenesis. High-throughput sequencing of genomic DNA from schistosome populations exposed to MLV mapped widespread and random insertion of Transgenes throughout the genome, along each of the autosomes and sex chromosomes, validating the utility of this approach for insertional mutagenesis. In addition, the germline-transmitted transgene encoding neomycin phosphotransferase rescued cultured schistosomules from toxicity of the antibiotic G418, and PCR analysis of eggs resulting from sexual reproduction of the transgenic worms in mice confirmed that retroviral Transgenes were transmitted to the next (F1) generation. These findings provide the first description of wide-scale, random insertional mutagenesis of chromosomes and of germline transmission of a transgene in schistosomes. Transgenic lines of schistosomes expressing antibiotic resistance could advance functional genomics for these significant human pathogens. Database accession: Sequence data from this study have been submitted to the European Nucleotide Archive (http://www. ebi.ac.uk/embl) under accession number ERP000379.
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integration of reporter Transgenes into schistosoma mansoni chromosomes mediated by pseudotyped murine leukemia virus
2008Co-Authors: Kristine J Kines, Victoria H Mann, Maria E Morales, Geoffrey N Gobert, Paul J BrindleyAbstract:The recent release of draft genome sequences of two of the major human schistosomes has underscored the pressing need to develop functional genomics approaches for these significant pathogens. The sequence information also makes feasible genome-scale investigation of transgene integration into schistosome chromosomes. Retrovirus-mediated transduction offers a means to establish transgenic lines of schistosomes, to elucidate schistosome gene function and expression, and to advance functional genomics approaches for these parasites. We investigated the utility of the Moloney murine leukemia retrovirus (MLV) pseudotyped with vesicular stomatitis virus glycoprotein (VSVG) for the transduction of Schistosoma mansoni and delivery of reporter Transgenes into schistosome chromosomes. Schistosomula were exposed to virions of VSVG-pseudotyped MLV, after which genomic DNA was extracted from the transduced schistosomes. Southern hybridization analysis indicated the presence of proviral MLV retrovirus in the transduced schistosomes. Fragments of the MLV transgene and flanking schistosome sequences recovered using an anchored PCR-based approach demonstrated definitively that somatic Transgenesis of schistosome chromosomes had taken place and, moreover, revealed widespread retrovirus integration into schistosome chromosomes. More specifically, MLV Transgenes had inserted in the vicinity of genes encoding immunophilin, zinc finger protein Sma-Zic, and others, as well as near the endogenous schistosome retrotransposons, the fugitive and SR1. Proviral integration of the MLV transgene appeared to exhibit primary sequence site specificity, targeting a gGATcc-like motif. Reporter luciferase transgene activity driven by the schistosome actin gene promoter was expressed in the tissues of transduced schistosomula and adult schistosomes. Luciferase activity appeared to be developmentally expressed in schistosomula with increased activity observed after 1 to 2 wk in culture. These findings indicate the utility of VSVG-pseudotyped MLV for Transgenesis of S. mansoni, herald a tractable pathway forward toward germline Transgenesis and functional genomics of parasitic helminths, and provide the basis for comparative molecular pathogenesis studies of chromosomal lesions arising from retroviral integration into human compared with schistosome chromosomes.—Kines, K. J., Morales, M. E., Mann, V. H., Gobert, G. N., Brindley, P. J. Integration of reporter Transgenes into Schistosoma mansoni chromosomes mediated by pseudotyped murine leukemia virus.
Erik M Jorgensen - One of the best experts on this subject based on the ideXlab platform.
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single copy insertion of Transgenes in caenorhabditis elegans
2008Co-Authors: Christian Frokjaerjensen, Wayne M Davis, Chris E Hopkins, Blake J Newman, Jason M Thummel, Sorenpeter Olesen, Morten Grunnet, Erik M JorgensenAbstract:Erik Jorgensen and colleagues report a highly efficient method for generating single-copy transgene insertions in C. elegans. Notably, these single-copy Transgenes are expressed at endogenous levels and can be expressed in the female and male germlines.
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single copy insertion of Transgenes in caenorhabditis elegans
2008Co-Authors: Christian Frokjaerjensen, Wayne M Davis, Chris E Hopkins, Blake J Newman, Jason M Thummel, Sorenpeter Olesen, Morten Grunnet, Erik M JorgensenAbstract:At present, Transgenes in Caenorhabditis elegans are generated by injecting DNA into the germline. The DNA assembles into a semistable extrachromosomal array composed of many copies of injected DNA. These Transgenes are typically overexpressed in somatic cells and silenced in the germline. We have developed a method that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double-strand break in noncoding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single-copy insertions can be obtained in less than 2 weeks by injecting approximately 20 worms. We have successfully inserted Transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single-copy Transgenes are expressed at endogenous levels and can be expressed in the female and male germlines.
Felicitas B Bidlack - One of the best experts on this subject based on the ideXlab platform.
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dose dependent rescue of ko amelogenin enamel by Transgenes in vivo
2017Co-Authors: Felicitas B Bidlack, Yan Xia, Megan K PugachAbstract:Mice lacking amelogenin (KO) have hypoplastic enamel. Overexpression of the most abundant amelogenin splice variant M180 and LRAP Transgenes can substantially improve KO enamel, but only ~40% of the incisor thickness is recovered and the prisms are not as tightly woven as in WT enamel. This implies that the compositional complexity of the enamel matrix is required for different aspects of enamel formation, such as organizational structure and thickness. The question arises, therefore, how important the ratio of different matrix components, and in particular amelogenin splice products, is in enamel formation. Can optimal expression levels of amelogenin Transgenes representing both the most abundant splice variants and cleavage product at protein levels similar to that of WT improve the enamel phenotype of KO mice? Addressing this question, our objective was here to understand dosage effects of amelogenin Transgenes (Tg) representing the major splice variants M180 and LRAP and cleavage product CTRNC on enamel properties. Amelogenin KO mice were mated with M180Tg, CTRNCTg and LRAPTg mice to generate M180Tg and CTRNCTg double transgene and M180Tg, CTRNCTg, LRAPTg triple transgene mice with transgene hemizygosity (on one allelle) or homozygosity (on both alleles). Transgene homo- versus hemizygosity was determined by qPCR and relative transgene expression confirmed by Western blot. Enamel volume and mineral density were analyzed by microCT, thickness and structure by SEM, and mechanical properties by Vickers microhardness testing. There were no differences in incisor enamel thickness between amelogenin KO mice with three or two different Transgenes, but mice homozygous for a given transgene had significantly thinner enamel than mice hemizygous for the transgene (p<0.05).The presence of the LRAPTg did not improve the phenotype of M180Tg/CTRNCTg/KO enamel. In the absence of endogenous amelogenin, the addition of amelogenin Transgenes representing the most abundant splice variants and cleavage product can rescue abnormal enamel properties and structure, but only up to a maximum of ~80% that of molar and ~40% that of incisor wild-type enamel.
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dose dependent rescue of ko amelogenin enamel by Transgenes in vivo
2017Co-Authors: Felicitas B Bidlack, Yan Xia, Megan K PugachAbstract:Mice lacking amelogenin (KO) have hypoplastic enamel. Overexpression of the most abundant amelogenin splice variant M180 and LRAP Transgenes can substantially improve KO enamel, but only ~40% of the incisor thickness is recovered and the prisms are not as tightly woven as in WT enamel. This implies that the compositional complexity of the enamel matrix is required for different aspects of enamel formation, such as organizational structure and thickness. The question arises, therefore, how important the ratio of different matrix components, and in particular amelogenin splice products, is in enamel formation. Can optimal expression levels of amelogenin Transgenes representing both the most abundant splice variants and cleavage product at protein levels similar to that of WT improve the enamel phenotype of KO mice? Addressing this question, our objective was here to understand dosage effects of amelogenin Transgenes (Tg) representing the major splice variants M180 and LRAP and cleavage product CTRNC on enamel properties. Amelogenin KO mice were mated with M180Tg, CTRNCTg and LRAPTg mice to generate M180Tg and CTRNCTg double transgene and M180Tg, CTRNCTg, LRAPTg triple transgene mice with transgene hemizygosity (on one allelle) or homozygosity (on both alleles). Transgene homo- vs. hemizygosity was determined by qPCR and relative transgene expression confirmed by Western blot. Enamel volume and mineral density were analyzed by microCT, thickness and structure by SEM, and mechanical properties by Vickers microhardness testing. There were no differences in incisor enamel thickness between amelogenin KO mice with three or two different Transgenes, but mice homozygous for a given transgene had significantly thinner enamel than mice hemizygous for the transgene (p < 0.05). The presence of the LRAPTg did not improve the phenotype of M180Tg/CTRNCTg/KO enamel. In the absence of endogenous amelogenin, the addition of amelogenin Transgenes representing the most abundant splice variants and cleavage product can rescue abnormal enamel properties and structure, but only up to a maximum of ~80% that of molar and ~40% that of incisor wild-type enamel.
