A ribozyme gene and an antisense gene are equally effective in conferring resistance to tobacco mosaic virus on transgenic tobacco

Ribozymes of the hammerhead class can be designed to cleave a target RNA in a sequence-specific manner and can potentially be used to specifically modulate gene activity. We have targeted the tobacco mosaic virus (TMV) genome with a ribozyme containing three catalytic hammerhead domains embedded within a 1 kb antisense RNA. The ribozyme was able to cleave TMV RNA at all three target sites in vitro at 25°C. Transgenic tobacco plants were generated which expressed the ribozyme or the corresponding antisense constructs directed at the TMV genome. Six of 38 independent transgenic plant lines expressing the ribozyme and 6 of 39 plant lines expressing the antisense gene showed some level of protection against TMV infection. Homozygous progeny of some lines were highly resistant to TMV; at least 50% of the plants remained asymptomatic even when challenged with high levels of TMV. These plants also displayed resistance to infection with TMV RNA or the related tomato mosaic virus (ToMV). In contrast, hemizygous plants of the same lines displayed only very weak resistance when inoculated with low amounts of TMV and no resistance against high inoculation levels. Resistance in homozygous plants was not overcome by a TMV strain which was altered at the three target sites to abolish ribozyme-mediated cleavage, suggesting that the ribozyme conferred resistance primarily by an antisense mechanism.     

Strategies for the suppression of peroxidase gene expression in tobacco. II.In vivo suppression of peroxidase activity in transgenic tobacco using ribozyme and antisense constructs

Several strategies involving the use of antisense and ribozyme constructs in different expression vectors were investigated as methods of suppressing gene expressionin planta. We had previously identified an efficiently cleaving ribozyme (Rz), with two catalytic units and 60 nucleotide (nt) of complementary sequence, to the ligninforming peroxidase of tobacco (TPX). This Rz was cloned behind the 35S CaMV (35S) and nopaline synthase (NOS) promoters, and into a vector utilising the tobacco tyrosine tRNA for expression. For comparison with more traditional antisense strategies, full-length TPX antisense (AS) constructs were also constructed behind the NOS and 35S promoters. Populations of transgenic tobacco containing these constructs were produced and compared to control plants transformed with the vector only. Significant suppression of peroxidase expression in the range of 40–80% was seen in the T₀ and T₁ populations carrying 35S-AS, 35S-Rz and tRNA-Rz constructs. Co-segregation of the suppressed peroxidase phenotype and the tRNA-Rz transgenes was demonstrated. Northern blot analysis indicated that levels of TPX mRNA were lower in the Rz plants. No evidence of mRNA cleavage was observed and thus it was unclear if the Rz constructs were acting as Rzsin vivo. Transgenic plants containing the tRNA-Rz construct had significantly lower levels of peroxidase than the other transgenic plants. There was no significant difference in levels of suppression of TPX between the short Rz in the 35S vector and the full-length AS constructs. Although peroxidase levels were significantly reduced in transgenic plants carrying 35S-AS, 35S-Rz and tRNA-Rz constructs, no significant difference in lignin levels was observed.     

Targeted Expression of the only Zinc Finger Gene in Transgenic Mice is Associated with Impaired Mammary Development

The only zinc finger (OZF) gene encodes a protein consisting mainly of 10 zinc finger motifs of the Krüppel type of yet unknown function. To potentially assess its in vivo role, mammary targeted deregulation of the expression of the murine gene was performed in transgenic mice using a goat -casein-based transgene. Mammary expression of the transgene was observed in the 11 lines obtained. In three expressing lines, this expression was tissue-specific and developmentally regulated. Further analysis of mice from two expressing lines revealed that transgene-homozygous females could not sustain full growth of their pups. This phenotype was associated with an impaired mammary gland development noticeable only after mid-gestation. It was characterised by an increase of the adipocyte to acini ratio and low or absence of fat globules within these acini compared to non-transgenic control animals. These transgenic observations strongly suggest that OZF is active in the mammary gland, interfering with the lactation process and thus that the described transgenic mice could be useful models to search for the cellular partner(s) of this protein.     

Thein vivo pattern of firefly luciferase expression in transgenic plants

Expression of the firefly luciferase gene in transgenic plants produces light emission patterns when the plants are supplied with luciferin. We explored whether inin vivo pattern of light emission truly reveals the pattern of luciferase gene expression or whether it reflects other parameters such as the availability of the substrate, luciferin, or the tissue-specific distribution of organelles in which luciferase was localized. The tissue-specific distribution of luciferase activity and thein vivo pattern of light were examined when the luciferase gene was driven by different promoters and when luciferase was redirected from the peroxisome, where it is normally targeted, to the chloroplast compartment. It was found that the distribution of luciferase activity closely correlated with the tissue-specific pattern of luciferase mRNA. However, thein vivo light pattern appeared to reflect not only tissue-specific distribution of luciferase activity, but also the pattern of luciferin uptake.     

A two unit antisense RNA cassette test system for silencing of target genes.

This communication describes a two unit antisense RNA cassette system for use in gene silencing. Cassettes consist of a recognition unit and an inhibitory unit which are transcribed into a single RNA that carries sequences of non-contiguous complementarity to the chosen target RNA. The recognition unit is designed as a stem-loop for rapid formation of long- lived binding intermediates with target sequences and resembles the major stem-loop of a naturally occurring antisense RNA, CopA. The inhibitory unit consists of either a sequence complementary to a ribosome binding site or of a hairpin ribozyme targeted at a site within the chosen mRNA. The contributions of the individual units to inhibition was assessed using the lacI gene as a target. All possible combinations of recognition and inhibitory units were tested in either orientation. In general, inhibition of lacI expression was relatively low. Fifty per cent inhibition was obtained with the most effective of the constructs, carrying the recognition stem-loop in the antisense orientation and the inhibitory unit with an anti-RBS sequence. Several experiments were performed to assess activities of the RNAs in vitro and in vivo : antisense RNA binding assays, cleavage assays, secondary structure analysis as well as Northern blotting and primer extension analysis of antisense and target RNAs. The problems associated with this antisense RNA approach as well as its potential are discussed with respect to possible optimization strategies.     

Expression of a reporter gene is reduced by a ribozyme in transgenic plants

A chimeric gene encoding a ribozyme under the control of the cauliflower mosaic virus (CaMV) 35S promoter was introduced into transgenic tobacco plants. In vivo activity of this ribozyme, which was designed to cleave npt mRNA, was previously demonstrated by transient expression assays in plant protoplasts. The ribozyme gene was transferred into transgenic tobacco plants expressing an rbcS-npt chimeric gene as an indicator. Five double transformants out of sixteen exhibited a reduction in the amount of active NPT enzyme. To measure the amount of ribozyme produced, in the absence of its target, the ribozyme and target genes were separated by genetic segregation. The steady-state concentrations of ribozyme and target RNA were shown to be similar in the resulting single transformants. Direct evidence for a correlation between reduced npt gene expression and ribozyme expression was provided by crossing a plant containing only the ribozyme gene with a transgenic plant expressing the npt gene under control of the 35S promoter, i.e. the same promoter used to direct ribozyme expression. The expression of npt was reduced in all progeny containing both transgenes. Both steady-state levels of npt mRNA and amounts of active NPT enzyme are decreased. In addition, our data indicate that, at least in stable transformants, a large excess of ribozyme over target is not a prerequisite for achieving a significant reduction in target gene expression.     

Construction of adenovirus for high level expression of small RNAs in mammalian cells

A series of plasmid vectors have been generated to allow the rapid construction of adenoviral vectors designed to express small RNA sequences. A truncated human U6 gene containing convenient restriction sites has been shown to be expressed at high levels following electroporation into a series of human cell lines. This gene was ligated into a promoterless adenoviral plasmid, and we have generated high titer virus by homologous recombination with adenoviral Addl327 DNA in 293 cells. Recombinant adenovirus containing a hammerhead ribozyme sequence targeted toward the Bcl-2 mRNA has been used to transduce a panel of human tumor cell lines. We have demonstrated high level expression of the recombinant U6 gene containing the ribozyme and reduction of Bcl-2 protein in transduced cells. These plasmids are suitable for the development of adenoviral vectors designed to express both ribozymes and antisense RNA in human cells.     

Evaluation of an antisense RNA transgene for inhibiting growth hormone gene expression in transgenic rats

We compared the levels of growth hormone (GH) mRNA in the pituitary, plasma GH concentration, and altered phenotype in rats heterozygous and homozygous for an antisense RNA transgene targeted to the rat GH gene, with those in nontransgenic rats. We initially investigated whether the transgene promoter, which is connected to four copies of a thyroid hormone response element (TRE) that increases promoter activity, affected in vivo transgene expression in the pituitary of the transgenic rats. Plasma GH concentration correlated negatively with T, injection in surgically thyroidectomized heterozygous transgenic rats. There was a reduction of about ?35–40% in GH mRNA levels in the pituitary of homozygous animals compared with those in non-transgenic rats. Plasma GH concentration was significantly ?25–32 and ?29–41% lower in heterozygous and homozygous transgenic rats, respectively, compared with that in nontransgenic animals. Furthermore, the growth rates in homozygous transgenic rats were reduced by ?72–81 and ?51–70% compared with those of their heterozygous and nontransgenic littermates, respectively. The results of these studies suggested that the biological effect of GH in vivo is modulated dose-dependently by the antisense RNA transgene. The rat GH gene can therefore be targeted by antisense RNA produced from a transgene, as reflected in the protein and RNA levels. © 1995 Wiley-Liss, Inc.     

反义RNA寡核苷酸及其与脂质体的复合物对于乙型肝炎病毒的抑制

我们设计合成了特异性靶向乙型肝炎病毒(HBV)mRNA的反义RNA寡核苷酸P-2987、X-60和X-519.在瞬时转染pHBV1.3质粒(含有1.3拷贝的HBV基因组)的HepG2细胞和整合了HBV基因组的HepG2.2.15细胞中,转染2μmol/L的反义RNA寡核苷酸,ELISA和实时定量PCR结果表明,这3条寡核苷酸可以明显抑制HBV的复制和抗原表达.在HBV转基因鼠中,尾静脉注射反义RNA寡核苷酸,结果表明,肝脏中HBV的复制得到了抑制,但是血清中抗原含量和HBV DNA拷贝数没有明显变化.反义RNA寡核苷酸X-519与脂质体的复合物可以增强其对于HBV在肝脏中复制的抑制作用.在通过高压尾静脉注射pHBV1.3质粒建立的HBV急性感染模型中,反义RNA寡核苷酸X-519可以显著地抑制HBV在肝脏中的复制以及降低血清中病毒抗原水平和DNA拷贝数.上述实验结果说明,X-519及其与脂质体的复合物对于HBV的复制和抗原表达起到明显的抑制作用,可能作为一种潜在的针对HBV的基因治疗药物.     

An upstream regulatory region mediates high-level, tissue-specific expression of the human alpha 1(I) collagen gene in transgenic mice.

Studies in vitro have not adequately resolved the role of intronic and upstream elements in regulating expression of the alpha 1(I) collagen gene. To address this issue, we generated 12 separate lines of transgenic mice with alpha 1(I) collagen-human growth hormone (hGH) constructs containing different amounts of 5'-flanking sequence, with or without most of the first intron. Transgenes driven by 2.3 kb of alpha 1(I) 5'-flanking sequence, whether or not they contained the first intron, were expressed at a high level and in a tissue-specific manner in seven out of seven independent lines of transgenic mice. In most tissues, the transgene was expressed at levels approaching that of the endogenous alpha 1(I) gene and was regulated identically with the endogenous gene as animals aged. However, in lung, expression of the transgene was anomalously high, and in muscle, expression was lower than that of the endogenous gene, suggesting that in these tissues other regions of the gene may participate in directing appropriate expression. Five lines of mice were generated containing transgenes driven by 0.44 kb of alpha 1(I) 5'-flanking sequence (with or without the first intron), and expression was detected in four out of five of these lines. The level of expression of the 0.44-kb constructs in the major collagen-producing tissues was 15- to 500-fold lower than that observed with the longer 2.3-kb promoter. While transgenes containing the 0.44-kb promoter and the first intron retained a modest degree of tissue-specific expression, those without the first intron lacked tissue specificity and were poorly expressed in all tissues except lung. These results contribute to our understanding of the role of the first intron in regulating alpha1(I) gene expression and identify a region, upstream of the basal alpha1(I) promotor, which is necessary for full tissue-specific, developmentally regulated expression of the alpha1(I) collagen gene.