Transgene mRNA Levels Research Articles

Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle.

Although the transient nature of transgene expression using first-generation adenovirus (Ad) vectors is well known, the exact mechanisms responsible for this phenomenon are uncertain. Rats were given intramuscular (i.m.) injections of a first-generation Ad containing the human fibroblast growth factor 4 (hFGF-4) gene driven by the cytomegalovirus (CMV) promoter and enhancer (CMV-PE). The copy number of hFGF-4 mRNA and viral DNA was measured in the same muscles by quantitative RT-PCR and quantitative PCR at times between 1 h and 84 days after virus injection. Quantitative Southern blot analysis for the intact hFGF-4 transcription unit DNA was also performed, and the methylation status of the CMV-PE DNA in the muscle was determined using bisulfite sequencing. The copy number of hFGF-4 mRNA peaked at 6 h then decreased 56-fold by 24 h, and a further 240-fold between days 3 and 28. Although the viral DNA copy number also decreased 23-fold between 6 h and 28 days, the ratio of copies of hFGF-4 mRNA per copy of viral DNA decreased 385-fold over this period. Methylation of the CMV-PE DNA in the muscle at both CpG and non-CpG sites was observed 24 h after virus administration and had increased at day 7. Decreased transcription associated with extensive methylation of the CMV-PE was the major mechanism responsible for the decrease in transgene mRNA levels. Strategies for preventing transcriptional silencing will be valuable for improving the duration of transgene expression from adenoviral vectors.

High Incidence of Thymic Epithelial Tumors in E2F2 Transgenic Mice

In virtually all human tumors, genetic and epigenetic alterations have been found which affect the INK4/-CYCLIN D/RB pathway, which regulates cell cycle entry and exit in normal cells. E2F transcription factors are important downstream components of this pathway, which act by controlling the expression of genes involved in DNA replication and cell cycle progression. To determine whether E2F2 deregulation promotes proliferation and tumorigenesis in vivo, we generated E2F2 transgenic mice, in which the Emu and murine pim1 promoter (pp) direct high expression of E2F2 in thymic epithelial cells. Emu-pp-E2F2 mice start to develop cytokeratin- and ER-TR4-positive cortical thymomas from the age of 20 weeks, and within 1 year, nearly all mice succumb to gross thymic epithelial tumors. General thymic morphology is largely maintained, but T cell development is perturbed in thymomas, with proportionately less CD4(+)CD8(+) double-positive thymocytes. In the first 3 months, E2F2 transgenic thymi exhibit only mild epithelial hyperplasia, and thereafter thymomas arise stochastically, probably following additional mutations. Interestingly, Emu-pp-E2F1 mice do not display cortical thymomas. These data argue that E2F2 promotes unscheduled cell division and oncogenic transformation of thymic epithelial cells.

Cucumber mosaic virus infection transiently breaks dsRNA-induced transgenic immunity to Potato virus Y in tobacco.

Post-transcriptional gene silencing (PTGS), an intrinsic plant defense mechanism, can be efficiently triggered by double stranded (ds)RNA-producing transgenes and can provide high level virus resistance by specific targeting of cognate viral RNA. The discovery of virus-encoded suppressors of PTGS led to concerns about the stability of such resistance. Here, we show that Cucumber mosaic virus (CMV) is able to suppress dsRNA-induced PTGS and the associated Potato virus Y (PVY) immunity in tobacco. CMV suppression supported only a transient PVY accumulation and did not prevent recovery of the transgenic plants from PVY infection. CMV inoculation resulted in strongly increased transgene mRNA levels due to suppression of PTGS, but accumulation of PVY-specific small interfering (si)RNA was unaffected. However, PVY accumulation in previously immune plants resulted in increased PVY siRNA levels and transgene mRNA was no longer detected, despite the presence of CMV. Transgene mRNA returned to high levels once PVY was no longer detected in CMV-infected plants. Recovered and chronically CMV-infected tissues were immune to further PVY infection.

Increased in vitro and in vivo transgene expression levels mediated through cis‐acting elements

Gene therapy for neurodegenerative diseases depends critically on the vector system to direct sustained and stable expression of the transgene. It is, however, a commonly observed phenomenon that transgene expression from currently available vectors is down-regulated following ex vivo gene transfer to the central nervous system (CNS). In an attempt to circumvent this problem, we have systematically evaluated the potential of different cis-acting elements to increase and stabilize transgene expression in vitro and after grafting of engineered cell lines to the CNS. Plasmid vector constructs incorporating Woodchuck hepatitis post-transcriptional regulatory element (WPRE), cHS4 insulator elements and/or the translational enhancer element SP163 were produced. Stable, polyclonal cultures of HiB5 cells were generated by transfection with reporter constructs, and in vitro transgene mRNA and protein levels were determined. Finally, HiB5 clones engineered to express the enhanced green fluorescent protein (EGFP) were grafted to the rat striatum and expression levels were evaluated. Inserting the WPRE element downstream of the open reading frame (ORF) of a reporter gene and flanking the transcriptional unit with cHS4 insulator elements significantly increased protein and mRNA expression levels. Surprisingly, the SP163 element, previously reported to be a translational enhancer, apparently did not promote any translational enhancing activity. Furthermore, the SP163 element exerted a negative effect on transcription. The ability of cHS4 and WPRE elements to stabilize in vivo transgene expression was demonstrated by transplantation of HiB5 clones containing expression constructs into the rat striatum. The data suggest that incorporating cis-acting elements in gene therapy vectors may result in improvements to currently available therapeutic vectors.

Expression of a Fungal Glucose Oxidase Gene in Three Potato Cultivars with Different Susceptibility to Late Blight (Phytophthora infestans Mont. deBary)

Research was done to determine if enhanced resistance to potato (Solanum tuberosum L.) late blight could be obtained by combining host plant resistance and engineered resistance. Late blight susceptible cultivars, Atlantic, and Spunta and the partially resistant cultivar Libertas were transformed with a fungal glucose oxidase gene, resulting in lines which ranged in transgene copy number from 1 to 8. Glucose oxidase enzyme activity ranged from 0.00 to 96.74×10-5 units/mg plant tissue. There was no correlation between copy number and level of transgene mRNA, level of transgene mRNA and enzyme activity, or between level of enzyme activity and disease resistance. Field and growth chamber evaluation of late blight response demonstrated little to no effect of the glucose oxidase transgene in either late blight susceptible or partially late blight resistant cultivars. However, enzyme activity levels were much lower than levels reported in previous research, which may account for the lack of effect of glucose oxidase against Phytophthora infestans. Twenty-one percent of the transgenic lines were phenotypically off-type compared to nontransgenic controls. Most of the off-type transgenic lines (four out of seven) were derived from `Libertas'. Because several off-type lines did not express the glucose oxidase protein, this phenomenon could not be attributed solely to the glucose oxidase transgene. Based on these results, transgenic lines produced for this study do not increase resistance to P. infestans even in combination with moderate host plant resistance. However, production of greater numbers of transgenic lines with the current construct or, production of transgenic lines in which a different constitutive promoter drives the expression of the glucose oxidase gene might result in greater disease resistance. However, the usefulness of any small increase in resistance would need to be evaluated against the time and cost required for development of transgenic potato cultivars and the potential for off-type tubers and plants.

TBX5 overexpression stimulates differentiation of chamber myocardium in P19C16 embryonic carcinoma cells.

In vitro differentiation of pluripotent embryonic cells is becoming a model system to study factors and genes involved in early developmental processes including cardiogenesis. An additional application involves the development of donor cells for treatment of diseases among which cardiac infarction. For this purpose differentiated cells should meet the functional characteristics of chamber myocardium, a requirement not convincingly reached as yet. The T-box transcription factor Tbx5 has been demonstrated to be crucial for heart formation. Using stably transfected clones of the P19C16 embryonic carcinoma cell line, reported to differentiate efficiently into the cardiac lineage, we investigated whether Tbx5 is sufficient to enhance cardiogenesis and differentiation of chamber myocardium. TBX5-transfected clones started to beat earlier, however, a relation between transgenic TBX5 mRNA levels and the number of beating foci or levels of Serca2a mRNA, a myocardial marker, could not be observed. However, TBX5-transfected clones displayed significantly higher levels of atrial natriuretic factor (Anf) and Connexin (Cx)40 mRNAs, which are associated with the formation of chamber myocardium. This indicates that Tbx5 enhances cardiac maturation within this system rather than cardiogenesis.

Silencing of a viral RNA silencing suppressor in transgenic plants.

High expression levels of the helper component proteinase (HC(pro)), a known virus suppressor of RNA silencing, were attained in Nicotiana benthamiana transformed with the HC(pro) cistron of Potato virus A (PVA, genus Potyvirus). No spontaneous silencing of the HC(pro) transgene was observed, in contrast to the PVA coat protein (CP)-encoding transgene in other transgenic lines. HC(pro)-transgenic plants were initially susceptible to PVA and were systemically infected by 14 days post-inoculation (p.i.) but, 1 to 2 weeks later, the new expanding leaves at positions +6 and +7 above the inoculated leaf showed a peculiar recovery phenotype. Leaf tips (the oldest part of the leaf) were chlorotic and contained high titres of PVA, whereas the rest of the leaf was symptomless and contained greatly reduced or non-detectable levels of viral RNA, CP and transgene mRNA. The spatial recovery phenotype suggests that RNA silencing is initiated in close proximity to meristematic tissues. Leaves at position +8 and higher were symptomless and virus-free but not completely resistant to mechanical inoculation with PVA. However, they were not infected with the virus systemically transported from the lower infected leaves, suggesting a vascular tissue-based resistance mechanism. Recovery of the HC(pro)-transgenic plants from infection with different PVA isolates was dependent on the level of sequence homology with the transgene. Methylation of the HC(pro) transgene followed recovery. These data show that the transgene mRNA for a silencing suppressor can be silenced by a presumably 'strong' silencing inducer (replicating homologous virus).

Retinoid regulation of the phosphoenolpyruvate carboxykinase gene in liver

The cytosolic PEPCK gene is a model gene for assessing retinoid regulation of liver-specific genes encoding enzymes of carbohydrate metabolism. In vivo, we have demonstrated that the PEPCK gene is inhibited by vitamin A deficiency. Specifically, under conditions of food deprivation, induction of the PEPCK gene is inhibited in the vitamin A deficient mouse. Inhibition of the PEPCK gene by vitamin A deficiency is reversed by all- trans or 9- cis retinoic acid (RA) treatment. In a transgenic mouse model, a −460 and −355 bp PEPCK promoter fragment confers susceptibility to inhibition by vitamin A deficiency and responsiveness to all- trans RA treatment. However, there is a differential effect of 9- cis RA on the PEPCK promoter; the −460 fragment confers responsiveness to 9- cis RA, but the −355 fragment does not. Taken together, these results indicate that the PEPCK retinoic acid response element (RARE)1 is required for 9- cis RA induction—but not all- trans RA induction—of the PEPCK gene. In order to determine if vitamin A deficiency alters specific localized expression of the PEPCK gene in the periportal cells of the liver, the effect of vitamin A status on PEPCK localization in the liver was also measured. The PEPCK transgenes were expressed specifically in the periportal region of the liver acinus and although vitamin A deficiency caused a decrease in PEPCK transgene mRNA levels in periportal cells, it did not alter the periportal cell-specific pattern of expression. Retinoid treatment induced PEPCK transgene mRNA levels in the same population of cells, however, the −355 bp PEPCK promoter fragment did not respond to 9- cis RA treatment. In order to determine the nuclear transcription factor(s) responsible for retinoid regulation of the PEPCK gene in the liver, we investigated retinoic acid receptor (RAR)α and β and the retinoid X receptor (RXR)α—the major retinoid receptors in liver—in terms of expression and the ability of the receptors to bind the PEPCK RAREs. Vitamin A deficiency significantly decreased hepatic RARβ, but not RARα or RXRα mRNA levels. In situ hybridization showed that RARα, RARβ and RXRα mRNAs were localized in the periportal region, however, immunohistochemistry showed that RARα and RXRα were distributed evenly across the liver acinus, whereas only RARβ levels were higher in periportal cells. The binding of nuclear receptors to PEPCK RARE1, RARE2 and RARE3 indicates a complex pattern of retinoid receptor and orphan nuclear receptor binding.

Accumulation of proteolytic fragments of mutant presenilin 1 and accelerated amyloid deposition are co-regulated in transgenic mice.

The activities of presenilin 1 (PS1) and 2 modulate the proteolytic processing of amyloid precursor proteins to produce Abeta1-42, and mutations in these proteins are associated with an accelerated rate of Abeta deposition. PS1 and PS2 themselves are subject to a highly-regulated endoproteolytic cleavage to generate stable 27 kDa N-terminal and 17 kDa C-terminal fragments. Here, we examined the relationship between the regulated cleavage of PS1 and the acceleration of Abeta deposition in transgenic mice that co-express Mo/Hu APPswe and varied levels mutant PS1 (A246E variant). The steady-state levels of the N- and C-terminal fragments of mutant PS1 in mice expressing low levels of mRNA were similar to that of mice expressing high levels of mRNA. Only mice expressing high levels of transgene mRNA accumulated uncleaved full-length protein. In mice co-expressing low levels of PS1A246E mRNA with Mo/Hu APPswe the age of appearance of Abeta deposits was similar to that of mice co-expressing expressing Mo/Hu APPswe with very high levels of mutant PS1. Our findings demonstrate that the levels of accumulated human PS1 N- and C-terminal fragments do not increase in proportion to the level of transgene mRNA and that similarly, the magnitude by which mutant PS1 accelerates the deposition of beta-amyloid is not proportional to the level of transgene expression.

Overexpression of the 3'-untranslated region of myelin proteolipid protein mRNA leads to reduced expression of endogenous proteolipid mRNA.

The current studies focus on what mechanisms regulate the concentration of PLP mRNA in cells. The PLP mRNA is very stable and these studies suggest that its stability is regulated by a trans-acting factor specific to oligodendrocytes. In order to test whether the 3'untranslated region (3'UTR) of the PLP mRNA might regulate PLP RNA stability, C6 cells were transfected with cDNAs that expressed either luciferase or luciferase fused to the 3'UTR of PLP. Although transgene expression was low, in cells transfected with the PLP 3'UTR, there was a significant decrease in the endogenous PLP mRNA. These cells showed a distinct change in morphology and in adhesion properties. Thus, there may be a role for plp gene products in cell adhesion, which was downregulated in these cells, or an unknown function may be encoded by the PLP 3'UTR. Transgenic mice that overexpress enhanced green fluorescent protein fused to the PLP 3'UTR under control of PLP regulatory sequences were tested for the expression of the endogenous PLP mRNA. Three of four lines of transgenic mice had decreased endogenous PLP mRNA, relative to their non-transgenic littermates; the EGFP-PLP 3'UTR mouse line that expressed the highest level of transgene mRNA had a 54% reduction in PLP mRNA. We hypothesize that the PLP mRNA is regulated by elements in the 3'UTR and stabilizing proteins specific to oligodendrocytes, and that in cells that overexpress the PLP 3'UTR, these stabilizing proteins may be insufficient to maintain the normal level of the endogenous PLP mRNA.

New molecular phenotypes in the dst mutants of Arabidopsis revealed by DNA microarray analysis.

In this study, DNA microarray analysis was used to expand our understanding of the dst1 mutant of Arabidopsis. The dst (downstream) mutants were isolated originally as specifically increasing the steady state level and the half-life of DST-containing transcripts. As such, txhey offer a unique opportunity to study rapid sequence-specific mRNA decay pathways in eukaryotes. These mutants show a threefold to fourfold increase in mRNA abundance for two transgenes and an endogenous gene, all containing DST elements, when examined by RNA gel blot analysis; however, they show no visible aberrant phenotype. Here, we use DNA microarrays to identify genes with altered expression levels in dst1 compared with the parental plants. In addition to verifying the increase in the transgene mRNA levels, which were used to isolate these mutants, we were able to identify new genes with altered mRNA abundance in dst1. RNA gel blot analysis confirmed the microarray data for all genes tested and also was used to catalog the first molecular differences in gene expression between the dst1 and dst2 mutants. These differences revealed previously unknown molecular phenotypes for the dst mutants that will be helpful in future analyses. Cluster analysis of genes altered in dst1 revealed new coexpression patterns that prompt new hypotheses regarding the nature of the dst1 mutation and a possible role of the DST-mediated mRNA decay pathway in plants.

A Cucumber Mosaic Virus (CMV) RNA 1 Transgene Mediates Suppression of the Homologous Viral RNA 1 Constitutively and Prevents CMV Entry into the Phloem

Resistance to Cucumber mosaic virus (CMV) in tobacco lines transformed with CMV RNA 1 is characterized by reduced virus accumulation in the inoculated leaf, with specific suppression of accumulation of the homologous viral RNA 1, and by the absence of systemic infection. We show that the suppression of viral RNA 1 occurs in protoplasts from resistant transgenic plants and therefore is not due to a host response activated by the cell-to-cell spread of virus. In contrast, suppression of Tobacco rattle virus vectors carrying CMV RNA 1 sequences did not occur in protoplasts from resistant plants. Furthermore, steady-state levels of transgene mRNA 1 were higher in resistant than in susceptible lines. Thus, the data indicate that sequence homology is not sufficient to induce suppression. Grafting experiments using transgenic resistant or susceptible rootstocks and scions demonstrated that the resistance mechanism exhibited an additional barrier to phloem entry, preventing CMV from moving a long distance in resistant plants. On the other hand, virus from susceptible rootstocks could systemically infect grafted resistant scions via the phloem. Analysis of viral RNA accumulation in the infected scions showed that the mechanism that suppresses the accumulation of viral RNA 1 at the single-cell level was overcome. The data indicate that this transgene-mediated systemic resistance probably is not based on a posttranscriptional gene-silencing mechanism.

Post-transcriptional gene silencing in plum pox virus resistant transgenic European plum containing the plum pox potyvirus coat protein gene.

Transgenic plums containing the plum pox potyvirus coat protein (PPV-CP) gene were inoculated with PPV. Infection was monitored by evaluating symptoms, ELISA, and IC-RT-PCR. Transgenic clone C5 was highly resistant to PPV during four years of testing and displayed characteristics typical of post-transcriptional gene silencing (PTGS), including a high level of transgene transcription in the nucleus, low levels of transgene mRNA in the cytoplasm, a complex multicopy transgene insertion with aberrant copies, and methylation of the silenced PPV-CP transgene. The PPV-CP transgene was also methylated in seedlings of C5 and these seedlings were resistant to PPV. Our results show, for the first time, that PTGS functions as a mechanism for virus resistance in a woody perennial species.

Transgene translatability increases effectiveness of replicase-mediated resistance to cucumber mosaic virus.

Transgenic tobacco plants expressing an altered form of the 2a replicase gene from the Fny strain of Cucumber mosaic virus (CMV) exhibit suppressed virus replication and restricted virus movement when inoculated mechanically or by aphid vectors. Additional transformants have been generated which contain replicase gene constructs designed to determine the role(s) of transgene mRNA and/or protein in resistance. Resistance to systemic disease caused by CMV, as well as delayed infection, was observed in several lines of transgenic plants which were capable of expressing either full-length or truncated replicase proteins. In contrast, among plants which contained nontranslatable transgene constructs, only one of 61 lines examined exhibited delays or resistance. Once infected, plants never recovered, regardless of transgene translatability. Transgenic plants exhibiting a range of resistance levels were examined for transgene copy number, mRNA and protein levels. Although ribonuclease protection assays demonstrated that transgene mRNA levels were very low, resistant lines had consistently more steady-state transgene mRNA than susceptible lines. Furthermore, chlorotic or necrotic local lesions developed on the inoculated leaves of transgenic lines containing translatable transgenes, but not on inoculated leaves of lines containing nontranslatable transgenes. These results demonstrate that translatability of the transgene and possibly expression of the transgene protein itself facilitates replicase-mediated resistance to CMV in tobacco.

Production of Bean yellow mosaic virus resistant subterranean clover (Trifolium subterraneum) plants by transformation with the virus coat protein gene

SUMMARYTransgenic lines of subterranean clover were constructed that contained three different Bean yellow mosaic virus (BYMV) coat protein (CP) gene constructs; full‐length CP, the core region of the CP, and full‐length CP plus the 3′ untranslated region of the viral genome. Transgenic plants containing the full‐length and core CP gene constructs showed high and moderate levels of BYMV resistance. Resistance was measured as a lack or amelioration of viral disease symptoms, which was correlated with a reduction in virus levels and yield loss. A range of different resistance phenotypes was observed. They included reduced infection rates, delay and reduction in local lesion development, and delay and reduction in severity of systemic symptom development. Resistance levels were not correlated with transgene mRNA levels and no transgene‐encoded protein was detected in any of the transgenic lines. This is the first example of genetically engineered virus resistance in a clover.

Highly sensitive and species-specific assay for quantification of human transgene expression levels.

During the past few years great efforts have been made to construct and to test human factor VIII (hFVIII) and IX (hFIX) vectors suitable for haemophilia gene therapy in vivo. However, little is known about the molecular mechanisms of persistence and shut-off of transgene expression in the target organs after gene transfer using recombinant adenoviral vectors. To evaluate low transgene mRNA levels in different tissues, especially at long times after the gene transfer, the common northern blot method is often not sensitive enough. For this reason we developed a new, highly sensitive and species-specific method for hFIX mRNA quantification and employed it in mice treated with an adenoviral vector (Ad5CMVFIX) expressing human FIX. In addition to its very high sensitivity (lowest detection level=1 fg RNA), the method was shown to be strictly species-specific, since hFIX mRNA signals were never detected in untreated mice. In a long-term study of 18 vector-treated mice we compared the human FIX:Ag levels in the mouse plasma, the human FIX mRNA levels and human FIX vector DNA concentrations in the mouse liver. We found that a slow but continuous decrease of hFIX:Ag levels in mouse plasma was associated with a corresponding decrease of hFIX mRNA levels in the liver. However, the Ad5CMVFIX vector DNA levels did not decrease to a comparable degree, suggesting that the decrease of human FIX:Ag levels in mouse plasma is, to a significant extent, also caused by CMV promotor shut-off and only to a minor degree by loss of vector DNA.

Analysis of a 762-bp Proximal Leptin Promoter to Drive and Control Regulation of Transgene Expression of Growth Hormone Receptor in Mice

Transgenic (TG) mice expressing porcine GH receptor (pGHR) directed by a 762-bp proximal leptin promoter were used to analyze the capability of the promoter to drive and regulate pGHR expression in vivo. Transgene expression occurred in inguinal, retroperitoneal, and epididymal/parametrial fat depots in both male and female TG mice, but not in wild type (WT) mice. pGHR transgene was also expressed in liver, heart, kidney, muscle, lung, and brain. Levels of pGHR transgene mRNA were higher in tissues other than adipose tissue. Fasting reduced leptin mRNA levels in adipose; however, pGHR transgene expression was not affected in either adipose or muscle. These results suggest (1) the region between +3 and −759 bp of the leptin promoter is able to drive gene expression in vivo, (2) this region may not be responsible for adipose tissue specificity of leptin expression, and (3) this region may not be responsible for negative regulation of leptin gene expression during fasting.

A 3'-flanking NF-kappaB site mediates developmental silencing of the human zeta-globin gene.

The central developmental event in the human (h)alpha-globin gene cluster is selective silencing of the zeta-globin gene as erythropoiesis shifts from primitive erythroblasts in the embryonic yolk sac to definitive erythroblasts in the fetal liver. Previous studies have demonstrated that full developmental silencing of the hzeta-globin gene in transgenic mice requires the proximal 2.1 kb of its 3'-flanking region. In the current report, we localize this silencing activity to a 108 bp segment located 1.2 kb 3' to the zeta-globin gene. Protein(s) in nuclear extracts from cell lines representing the fetal/adult erythroid stage bind specifically to an NF-kappaB motif located at this site. In contrast, this binding activity is lacking in the nuclear extract of an embryonic-stage erythroid line expressing zeta-globin. This complex is quantitatively recognized by antisera to the NF-kappaB p50 and to a lesser extent to p65 subunits. A two-base substitution that disrupts NF-kappaB site protein binding in vitro also results in the loss of the developmental silencing activity in vivo. The data suggest that NF-kappaB complex formation is a crucial component of hzeta-globin gene silencing. This finding expands the roles of this widely distributed transcriptional complex to include negative regulation in mammalian development.

Posttranscriptional gene silencing in transgenic sugarcane. Dissection Of homology-dependent virus resistance in a monocot that has a complex polyploid genome

RNA-mediated, posttranscriptional gene silencing has been determined as the molecular mechanism underlying transgenic virus resistance in many plant virus-dicot host plant systems. In this paper we show that transgenic virus resistance in sugarcane (Saccharum spp. hybrid) is based on posttranscriptional gene silencing. The resistance is derived from an untranslatable form of the sorghum mosaic potyvirus strain SCH coat protein (CP) gene. Transgenic sugarcane plants challenged with sorghum mosaic potyvirus strain SCH had phenotypes that ranged from fully susceptible to completely resistant, and a recovery phenotype was also observed. Clones derived from the same transformation event or obtained after vegetative propagation could display different levels of virus resistance, suggesting the involvement of a quantitative component in the resistance response. Most resistant plants displayed low or undetectable steady-state CP transgene mRNA levels, although nuclear transcription rates were high. Increased DNA methylation was observed in the transcribed region of the CP transgenes in most of these plants. Collectively, these characteristics indicate that an RNA-mediated, homology-dependent mechanism is at the base of the virus resistance. This work extends posttranscriptional gene silencing and homology-dependent virus resistance, so far observed only in dicots, to an agronomically important, polyploid monocot.

The role of TGFβ1 in initiating hepatic stellate cell activation in vivo

Background/Aims: The activation of hepatic stellate cells is a key initiating event in hepatic fibrogenesis. Although TGFβ1 is a potent inducer of collagen α1(I) expression in vitro and elevated levels of TGFβ1 are found in patients and experimental animals with hepatic fibrosis and cirrhosis, the role of increased TGFβ1 in the initiation of hepatic stellate cell activation in vivo is unknown. We used two experimental approaches to study this relationship: 1) Induction of an acute liver injury with carbon tetrachloride (CCl 4) in normal and TGFβ1-knockout (ko) mice, and 2) overexpression of TGFβ1 in the liver of wild-type mice using a recombinant replication-deficient adenovirus encoding human TGFβ1 (Ad-TGFβ1). Methods: TGFβ1-ko mice ( n=6) and normal mice ( n=6) were injected once intraperitoneally (ip) with CCl 4 (1 μl/g BW) or mineral oil. Wild-type mice ( n=3) were injected intravenously with Ad-TGFβ1 (10 10 pfu) or a control virus expressing β-galactosidase (Ad-LacZ, 10 10 pfu). Animals were sacrificed after 3 days and total liver RNA was prepared. The expression of collagen α1(I) mRNA normalized to GAPDH mRNA was measured by RNase protection assay, α-smooth muscle actin (α-sma) protein expression was analyzed by Western blotting. The expression of TGFβ1, TGFβ2, and TGFβ3 mRNAs were determined semi-quantitatively with RT-PCR. Results: The collagen α1(I) mRNA was increased 10-fold in CCl 4-treated wild-type mice compared to the controls. This increase was reduced about 80% in the TGFβ1-ko mice. The TGFβ1 mRNA levels in the wild-type mice were proportional to the collagen α1(I) mRNA levels. α-sma, a marker of hepatic stellate cell activation, was expressed earlier and at a higher level in wild-type mice than TGFβ-ko mice after CCl 4 treatment. The Ad-TGFβ1 infected mice had 14-fold higher hepatic TGFβ protein levels and 15-fold higher collagen α1(I) mRNA levels than the Ad-LacZ-infected control mice. Collagen α1(I) mRNA levels were proportional to the transgenic TGFβ1 mRNA levels, while the endogenous TGFβ1 was only slightly higher than in the controls. TGFβ2 and TGFβ3 mRNA levels were elevated in CCl 4-treated wild-type and TGFβ1-ko mice and in Ad-TGFβ1-infected mice compared to the controls. Conclusions: Absence of TGFβ1 inhibits hepatic collagen α1(I) mRNA and α-sma protein expression by the toxic stimulus CCl 4, and targeted TGFβ1 overexpression increases collagen α1(I) mRNA and α-sma protein levels in the liver in vivo. Other TGFβ family members do not compensate for the TGFβ1 deficiency. This indicates that TGFβ1 accelerates, but is not absolutely required, for the activation of hepatic stellate cells.