Browsing by Subject "Transgene"

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Entwicklung von „screening“-Methoden zur Analyse von PTGS-basierter Resistenz gegen Nepoviren in Pflanzen
(2006) Winterhagen, Patrick; Reustle, Götz
Nepoviruses are the causal agent of the fanleaf disease which leads to severe loss in viticulture (Raski et al., 1983). To induce virus resistance by post transcriptional gene silencing (PTGS) against Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV) and Raspberry ringspot virus (RpRSV), grapevine rootstocks were transformed with inverted repeat constructs or constructs containing sequences of the target virus and the defective interfering (DI) sequence of Tomato bushy stunt virus (Reustle et al., 2005). The induction und efficiency of PTGS by different constructs were investigated on the model plant Nicotiana benthamiana. Transgene-induced PTGS was demonstrated by the detection of small interfering (si)RNA in N. benthamiana. Using Agrobacterium for infiltration of a GFP-sensor construct, consisting of the GFP expression cassette and the sequence of the target virus, the efficiency of established transgene-induced PTGS was investigated. GFP-expressing plants accumulated mRNA of the sensor construct after the first day post infiltration in infiltrated leaves. After the second day the accumulation of siRNA with GFP- und virus-specific sequences was detected. In plants, which did not show any GFP-fluorescence after infiltration, GFP or viral sequence specific siRNA were not detectable. Generally, in virus resistant plants GFP-fluorescence was absent after infiltration. A correlation of virus resistance und accumulation of virus- or transgene-specific siRNA was not found. Several systems to evaluate PTGS and virus resistance in transgenic grapevine were tested. Transgenic grapevine did not accumulate transgene specific siRNA. An elevated resistance of transgenic grapevine was discovered by grafting experiments onto virus infected rootstocks. Whereas virus infected grapevine accumulated virus- and transgene-specific RNA and siRNA, in the non-infected grafts viral RNA was not detectable. Obviously, degradation of viral RNA in resistant grapevine und N. benthamiana was rapid und highly efficient without leading to accumulation of siRNA. However, due to the high inoculum pressure, grafting experiments are difficult to interprete and a possible field resistance against natural infection by the vector nematodes is probably not detectable. For investigation of PTGS in transgenic grapevine in vivo a system for vacuum infiltration to transfer the GFP-sensor construct into leaf tissue was established. For inoculation of grapevine using the natural GFLV vector nematode Xiphinema index an in vitro dual culture was developed. This space saving system allows analysis of resistance of grapevine under controlled conditions within a short time. An incubation time of about only six weeks was sufficient for the inoculation of control plants.
Regulatory elements controlling the expression of OR37 genes
(2007) Zhang, Yongquan; Breer, Heinz
The genes of the OR37 family are clustered in two loci (cluster I and cluster II) on mouse chromosome 4. These genes encode distinct olfactory receptors (ORs) which are characterised by an insertion of six amino acids in the third extracellular loop and moreover, these receptor types are only expressed in cells which are segregated in a small patch on the central nasal turbinate. As first steps to unravel the molecular basis of this unique topographic expression pattern previous studies have led to the identification of highly conserved sequence motifs including an olf-1 site in the putative promoter region of these genes and subsequently several transcription factors were identified which did bind to these sites. However, it remained elusive if an interaction between the transcription factors and the putative promoter sites may have functional implications. Therefore, a heterologous system was employed to assess the consequence of an interaction between the putative promoters and the transcription factors. HEK 293 cells were cotransfected with a reporter gene under the control of putative mOR37 promoter regions and an expression vector based gene encoding the transcription factor. The expression rate of the reporter gene was monitored by measuring luciferase activity. It was found that the three O/E transcription factors (O/E-1, O/E-2 and O/E-4) induced significant activation of the mOR37 promoters; in addition, it was observed that the putative promoters of other OR genes were also activated, suggesting that the O/E proteins may play a general role in the regulation of OR gene expression. Mutagenesis experiments revealed that the effects of O/E proteins were dependent on the presence of an olf-1 site within the promoter region. For the transcription factor Lhx-2 it was found that not all but only promoters of distinct OR-genes were affected. For the mOR37 promoters a simultaneous action of O/E protein and Lhx-2 elicited an increase of reporter gene expression. The data indicate that the putative mOR37 promoters could drive gene expression in the presence of the crucial transcription factors in this heterologous system. In order to explore to what extent the promoter may contribute to the characteristic topographic expression pattern of the mOR37 genes in vivo, a mOR37C transgene which included the coding exon and the putative promoter, was randomly inserted into the mouse genome. Seven lines were obtained; in all lines the transgene was specifically expressed in olfactory sensory neurons (OSN). In six lines the transgene expression was restricted to the central patch of the olfactory turbinates, typical for the OR37 genes. In one line (line 7) the transgene was also expressed in OSNs ectopically positioned outside the patch within the medial zone. It was found that the transgene was expressed in a mutually exclusive manner and from only one allele. The axons of OSNs expressing the transgene co-converged in the same glomerulus with the axons from neurons expressing the endogenous gene. In line #7 the formation of ectopic glomeruli was observed. The number of OSNs expressing the transgene varied considerably among lines; these differences were independent from the copy number of the transgene. The data indicate that the short putative promoters, most likely the conserved motifs, were sufficient to drive the OR37 gene expression in a tissue specific way and most aspects of the OR37 gene expression were mimicked by the transgene; however, considerable differences between certain lines suggested additional regulatory elements, such as a locus control region (LCR). Since regulatory elements for gene transcription, such as promoters, enhancers and LCRs, appear to be conserved across species, a comparative approach was utilized to search for the LCR-like element for the OR37 locus by sequence alignment across distantly related mammals. A segment of 270 base pairs located 137 Kb upstream of OR37 cluster I was found to be highly conserved between mouse, human, dog and opossum. It was not associated with an exon of any known gene and was highly correlated with OR37 cluster I rather than with the neighboring genes, since the flanking genes did not show syntenic conservation in the opossum genome. A homologous counterpart for this segment was found downstream of the OR37 cluster II locus; an alignment of the cluster II sequence across species identified the conservation of this counterpart. Examination for relevant motifs in this segment and comparison with the conserved H element revealed two common transcription factor binding sites, at least one of them is known to be essential for generating DNase I hypersensitive sites in the LCR of the beta globin gene locus. Further studies are required to evaluate a possible role of this conserved segment in the regulation of the OR37 gene expression.