RNA interference is ineffective as a routine method for gene silencing in chick embryos as monitored by fgf8 silencing.

The in vivo accessibility of the chick embryo makes it a favoured model system for experimental developmental biology. Although the range of available techniques now extends to miss-expression of genes through in ovo electroporation, it remains difficult to knock out individual gene expression. Recently, the possibility of silencing gene expression by RNAi in chick embryos has been reported. However, published studies show only discrete quantitative differences in the expression of the endogenous targeted genes and unclear morphological alterations. To elucidate whether the tools currently available are adequate to silence gene expression sufficiently to produce a clear and specific null-like mutant phenotype, we have performed several experiments with different molecules that trigger RNAi: dsRNA, siRNA, and shRNA produced from a plasmid coexpressing green fluorescent protein as an internal marker. Focussing on fgf8 expression in the developing isthmus, we show that no morphological defects are observed, and that fgf8 expression is neither silenced in embryos microinjected with dsRNA nor in embryos microinjected and electroporated with a pool of siRNAs. Moreover, fgf8 expression was not significantly silenced in most isthmic cells transformed with a plasmid producing engineered shRNAs to fgf8. We also show that siRNA molecules do not spread significantly from cell to cell as reported for invertebrates, suggesting the existence of molecular differences between different model systems that may explain the different responses to RNAi. Although our results are basically in agreement with previously reported studies, we suggest, in contrast to them, that with currently available tools and techniques the number of cells in which fgf8 gene expression is decreased, if any, is not sufficient to generate a detectable mutant phenotype, thus making RNAi useless as a routine method for functional gene analysis in chick embryos

Manipulation of gene expression in early mouse embryo

Maternal (oocyte) and embryonic programmes of hypoxanthine phosphoribosyl transferase (HPRT) and adenine phosphoribosyl transferase (APRT) gene expression have been investigated in mouse oocytes and during preimplantation development. The onset of the embryonic HPRT gene occurs following fertilization, or parthenogenetic activation, before the 4-cell stage. The oocyte-determined increase in HPRT activity due to preformed mRNA continues in aging oocytes, as well as in fertilized or activated eggs, i.e. irrespective of the initiation of embryonic development. Attempts were made to determine whether there is a detectable time difference in the onset of maternal and paternal genomes by assaying the onset of embryo- coded HPRT activity in embryos of different maternal and paternal X-chromosome constitution, but due to difficulties in comparable staging of embryos, no definite conclusion could be drawn. The expression of an exogenously introduced HPRT minigene has been monitored throughout preimplantation development. The embryos injected with supercoiled HPRT minigene showed an approximately twofold increase in HPRT activity at the 2-cell stage compared with control uninjected embryos. Linear minigene DNA was less efficient in giving active enzyme. The efficacy of three different promoters were studied in 2-cell mouse embryos using the expression of the HPRT minigene as a reporter function. The mouse HPRT promoter and the uninduced mouse metallothionein-1 (MT-1) promoter functioned equally well whereas the viral SV40 promoter did not allow HPRT expression. The mouse MT-1 promoter linked to the HPRT minigene allowed induction of HPRT gene expression in mouse embryos cultured in the presence of cadmium. The inhibition of enzyme expression from injected minigene DNA is mediated by simultaneous injection of a fivefold molar excess of HPRT antisense DNA. The same negation of exogenous HPRT activity was observed with simultaneous injection of HPRT exon-1 antisense DNA. The use of an inducible HPRT antisense construct achieved repression of gene activity with equivalent molar amounts of antisense to the sense molecules. Transgenic mice were produced with an antisense HPRT minigene construct attached to the inducible mouse MT-l promoter. The prospect of "cancelling" the endogenous "sense" gene activity in these mice at a specific stage of development by applying the induction stimulus is discussed

Application of the dual-luciferase reporter assay to the analysis of promoter activity in Zebrafish embryos

<p>Abstract</p> <p>Background</p> <p>The dual-luciferase assay has been widely used in cell lines to determine rapidly but accurately the activity of a given promoter. Although this strategy has proved very useful, it does not allow the promoter and gene function to be analyzed in the context of the whole organism.</p> <p>Results</p> <p>Here, we present a rapid and sensitive assay based on the classical dual-luciferase reporter technique which can be used as a new tool to characterize the minimum promoter region of a gene as well as the <it>in vivo </it>response of inducible promoters to different stimuli. We illustrate the usefulness of this system for studying both constitutive (telomerase) and inducible (NF-κB-dependent) promoters. The flexibility of this assay is demonstrated by induction of the NF-κB-dependent promoters using simultaneous microinjection of different pathogen-associated molecular patterns as well as with the use of morpholino-gene mediated knockdown.</p> <p>Conclusion</p> <p>This assay has several advantages compared with the classical <it>in vitro </it>(cell lines) and <it>in vivo </it>(transgenic mice) approaches. Among others, the assay allows a rapid and quantitative measurement of the effects of particular genes or drugs in a given promoter in the context of a whole organism and it can also be used in high throughput screening experiments.</p

Hydroxyproline-based DNA mimics provide an efficient gene silencing in vitro and in vivo

To be effective, antisense molecules should be stable in biological fluids, non-toxic, form stable and specific duplexes with target RNAs and readily penetrate through cell membranes without non-specific effects on cell function. We report herein that negatively charged DNA mimics representing chiral analogues of peptide nucleic acids with a constrained trans-4-hydroxy-N-acetylpyrrolidine-2-phosphonate backbone (pHypNAs) meet these criteria. To demonstrate this, we compared silencing potency of these compounds with that of previously evaluated as efficient gene knockdown molecules hetero-oligomers consisting of alternating phosphono-PNA monomers and PNA-like monomers based on trans-4-hydroxy-L-proline (HypNA-pPNAs). Antisense potential of pHypNA mimics was confirmed in a cell-free translation assay with firefly luciferase as well as in a living cell assay with green fluorescent protein. In both cases, the pHypNA antisense oligomers provided a specific knockdown of a target protein production. Confocal microscopy showed that pHypNAs, when transfected into living cells, demonstrated efficient cellular uptake with distribution in the cytosol and nucleus. Also, the high potency of pHypNAs for down-regulation of Ras-like GTPase Ras-dva in Xenopus embryos was demonstrated in comparison with phosphorodiamidate morpholino oligomers. Therefore, our data suggest that pHypNAs are novel antisense agents with potential widespread in vitro and in vivo applications in basic research involving live cells and intact organisms

RNA interference is ineffective as a routine method for gene silencing in chick embryos as monitored by fgf8 silencing

The in vivo accessibility of the chick embryo makes it a favoured model system for experimental developmental biology. Although the range of available techniques now extends to miss-expression of genes through in ovo electroporation, it remains difficult to knock out individual gene expression. Recently, the possibility of silencing gene expression by RNAi in chick embryos has been reported. However, published studies show only discrete quantitative differences in the expression of the endogenous targeted genes and unclear morphological alterations. To elucidate whether the tools currently available are adequate to silence gene expression sufficiently to produce a clear and specific null-like mutant phenotype, we have performed several experiments with different molecules that trigger RNAi: dsRNA, siRNA, and shRNA produced from a plasmid coexpressing green fluorescent protein as an internal marker. Focussing on fgf8 expression in the developing isthmus, we show that no morphological defects are observed, and that fgf8 expression is neither silenced in embryos microinjected with dsRNA nor in embryos microinjected and electroporated with a pool of siRNAs. Moreover, fgf8 expression was not significantly silenced in most isthmic cells transformed with a plasmid producing engineered shRNAs to fgf8. We also show that siRNA molecules do not spread significantly from cell to cell as reported for invertebrates, suggesting the existence of molecular differences between different model systems that may explain the different responses to RNAi. Although our results are basically in agreement with previously reported studies, we suggest, in contrast to them, that with currently available tools and techniques the number of cells in which fgf8 gene expression is decreased, if any, is not sufficient to generate a detectable mutant phenotype, thus making RNAi useless as a routine method for functional gene analysis in chick embryos

p53 as a regulator of cell differentiation

The present invention involves the role of p53 in the differentiation of embryonic tissues. More particularly, the present invention provides methods of the blocking of p53 function in embryonic tissues, and the use of these tissues as screening tools for substances that are capable of overcoming the p53-related block in differentation, both in vitro and in vivo. The similarities between undifferentiated embryonic cells and tumor cells is evident, and thus these assays serve as a model for possible cancer therapeutics. In addition, methods for identifying additional cellular components that interact p53 or p53-related pathways are provided.Board of Regents, University of Texas Syste

Antibody Genes, Oncogenes and Antisense Genes

There are a number of mechanisms involved in producing a diversity of antibodies including multiple germline genes, somatic gene rearrangement, somatic hypermutation and combinatorial association. By the process of somatic hypermutation, one immunoglobulin gene in the germline can be mutated to produce many different genes in B cells. In chapter 2, this process is characterized. It was found that phosphorylcholine binding antibodies are encoded by one germline VH gene segment. In B cells, this VH gene segment may have extensive point mutations, many of which are silent, indicating the presence of some somatic hypermutational mechanism. Only the VH gene was found to be mutated indicating that the mutational mechanism was specific for VH genes. One way to study somatic immunoglobulin gene rearrangements, presented in chapter 3, might be to characterize rearrangements which are not easily explained. Immunoglobulin gene rearrangement was thought to exclusively involve immunoglobulin genes. However, some immunoglobulin genes can reproducibly rearrange with other DNA sequences. Insight into the basis of these rearrangements was uncovered by identifying the chromosomal origin of the nonimmunoglobulin rearranging DNA. This DNA originated on chromosome 15 whereas the immunoglobulin gene originated on chromosome 12. The juxtaposition of these sequences is common in plasmacytomas but rare or absent in normal B cells suggesting that it is involved in tumorigenesis. For example, it may be that aberrant immunoglobulin rearrangements can activate a cellular oncogene resulting in a plasmacytoma. This possibility was supported by results from other laboratories when it was found that the non-immunoglobulin rearranging DNA contained the cellular homologue of the myc oncogene. To understand lymphocyte tumorigenesis, it would be useful to understand the function of the c-myc gene product in normal and transformed cells. One way to begin is to determine which types of cells express the c-myc gene. This approach was employed in chapter 5 and it was found that the c-myc gene is expressed in dividing, but not resting, lymphocytes. One possible function for the c-myc gene product is that it functions in cellular proliferation. Another way to study the function of the c-myc gene product would be to prevent expression of the rearranged c-myc gene in plasmacytomas. For example, it may be possible to inhibit the synthesis of the c-myc gene product by antisense c-myc RNA. If the antisense RNA can hybridize to the c-myc RNA in vivo, synthesis of myc protein may be prevented. A test case, in which antisense TK RNA is used to inhibit TK expression, is presented in chapter 6. In L cells, high levels of antisense TK RNA expression were capable of inhibiting TK activity. The mechanism of inhibition involves RNA:RNA hybridization since double stranded RNA was formed. If this test case can be applied to other instances, it may be possible to use antisense RNA to inhibit the synthesis of a particular gene product and thus study its cellular function.</p

Transcriptional and Post-Transcriptional Regulation of Histone Variant H2A.Z During Sea Urchin Development

Histone variant H2A.Z promotes chromatin accessibility at transcriptional regulatory elements and is developmentally regulated in metazoans. We characterize the transcriptional and post-transcriptional regulation of H2A.Z in the purple sea urchin Strongylocentrotus purpuratus. H2A.Z depletion by antisense translation-blocking morpholino oligonucleotides during early development causes developmental collapse, in agreement with its previously demonstrated general role in transcriptional multipotency. During H2A.Z peak expression in 24-h embryos, endogenous H2A.Z 3’ UTR sequences stabilize GFP mRNAs relative to those with SV40 3’ UTR sequences, although the 3’UTR of H2A.Z does not determine the spatial distribution of H2A.Z transcripts during embryonic and postembryonic development. We elaborated an H2A.Z::GFP BAC reporter that reproduces embryonic H2A.Z expression. Genome-wide chromatin accessibility analysis using ATAC-seq revealed a cis- regulatory module (CRM) that, when deleted, causes a significant decline of the H2A.Z reporter expression. In addition, the mutation of a Sox transcription factor binding site motif and, more strongly, of a Myb motif cause significant decline of reporter gene expression. Our results suggest that an undetermined Myb-family transcription factor controls the transcriptional regulation of H2A.Z

Identification and characterization of Risp

The aim of this study was to characterize the role of Rev-interacting cellular proteins in controlling the function of Rev in the host cell. The HIV-1 protein Rev plays an essential role in the temporal regulation of the virus gene expression by stimulating the expression of viral structural proteins. Rev enhances the nucleocytoplasmic transport and the translation of unspliced and single spliced viral mRNAs by binding with high affinity to a specific target element on the HIV-RNA. It was assumed that interaction with cellular factors is essential for Rev function. At the onset of this study, only a few potential cofactors were known with no clearly defined functional relevance. So we decided to search for new Rev-interacting factors using the yeast two-hybrid system. In this work a new Rev-interacting protein has been identified, by screening a Jurkat T cell cDNA library. The protein was termed Risp (Rev-interacting shuttle protein), because it shuttles between the nuclear and the cytoplasmic compartments. The Risp gene is widely expressed in human cells and conserved among various species, most probably as part of a larger gene. High amino acid homology (99%) with the C-terminal part of a large brain cDNA clone for KIAA0592 protein has been found, whereas no obvious homology to proteins with known function was observed. However, a weak and partial similarity appeared with several RNA-/DNA-binding and shuttle proteins. This might indicate that the Risp protein - or the larger protein containing it - could be a member of a new family of nucleocytoplasmic shuttle proteins with RNA-/DNA-binding function. Next, the intracellular localization and shuttling of Risp was investigated. In HeLa cells Risp- GFP localized in both nuclear and cytoplasmic compartments, but clearly accumulated in the cytoplasm, indicating the presence of a strong nuclear export signal (NES). The identification of a NES sequence was confirmed by deletion analysis of Risp and by nuclear microinjection of BSA-fusion proteins conjugated to peptides from the C-terminal part of Risp. Treatment with leptomycin B, a drug which has been shown to specifically block Crm1 (exportin) mediated export, resulted in nuclear accumulation of Risp-GFP, showing that the nuclear export of Risp, like that of Rev, is Crm1-dependent. Using bioinformatic tools able to detect weak homologies with high specificity, sequence comparisons between Risp and all currently known Rev interacting factors were performed. This analysis for the first time revealed a common motif shared between Rev and Rev-interacting cellular factors, termed RIP. The region of Risp harboring the RIP motif was neither essential nor sufficient for the Rev-binding in the yeast two hybrid system, suggesting no direct correlation between RIP and the Rev-binding ability. Preliminary experiments suggested, that Risp, as a Rev-interacting protein, is able to inhibit Revtrans- activation, while Risp does not interfere with Tat in a Tat-trans-activation assay. The overexpression of Risp-GFP reduced the production of the Rev-dependent structural viral protein p24gag up to 70%. In addition a previously unrecognized sequence motif in the activation domain of Rev with intrinsic nuclear import activity was found and tested in transfection and microinjection assays. This motif (“PPXXR”) is conserved in various RNA-binding proteins and was proposed to mediate nuclear translocation of the cellular functional homologue of HIV-1 Rev Sam68