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PubMed1996

Hepatocyte growth factor/scatter factor overexpression induces growth, abnormal development, and tumor formation in transgenic mouse livers.

To investigate the in vivo role of hepatocyte growth factor/scatter factor (HGF/SF) in liver function, we generated transgenic mice using a mouse HGF/SF cDNA under the control of the mouse metallothionein gene promoter and 5'/3' flanking sequences. In adult HGF/SF transgenic mice, liver weight as a percentage of total body weight was at least twice that of wild-type mice. Comparison of transgenic and control liver morphology revealed dramatic heterogeneity in the size and appearance of hepatocytes as a distinctive feature of HGF/SF overexpression. Transgenic livers exhibited a significant increase in the number of small hepatocytes with a 2N DNA content, accounting for the observed increase in liver mass. The DNA labeling index of hepatocytes increased 11-fold at 4 weeks of age, when liver enlargement first became apparent, and was still elevated about 5-fold in adult HGF/SF transgenic mice. Moreover, hepatocytes isolated by perfusion of transgenic livers doubled every 2 days in culture, whereas little or no growth was observed with isolated control hepatocytes. The mechanistic basis of hepatocyte proliferation was elucidated as the chronic activation of the c-met proto-oncogene product. Met and substrates such as phosphatidylinositol 3-kinase, Src homology and collagen-like, pp60c-src, focal adhesion kinase p125FAK, and paxillin were associated with tyrosine-phosphorylated complexes in a hepatocyte cell line established from the transgenic liver. This proliferative stimulus triggered the formation of hepatocellular adenomas and/or carcinomas in most transgenic mice > or = 1.5 years of age. Finally, the rate of transgenic mouse liver regeneration was increased 3-fold over control livers following partial hepatectomy.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Xenopus tropicalis transgenic lines and their use in the study of embryonic induction.

For over a century, amphibian embryos have been a source of significant insight into developmental mechanisms, including fundamental discoveries about the process of induction. The recently developed transgenesis for Xenopus offers new approaches to these poorly understood processes, particularly when undertaken in the quickly maturing species Xenopus tropicalis, which greatly facilitates establishment of permanent transgenic lines. Several X. tropicalis transgenic lines have now been generated, and experiments demonstrating the value of these lines to study induction in embryonic tissue recombinants and explants are presented here. A revised protocol for transgenesis in X. tropicalis resulting in a significant increase in the percentage of transgenic animals that reach adulthood is presented, as well as improvements in tadpole and froglet husbandry, which have facilitated the raising of large numbers of adults. Working transgenic populations have been rapidly expanded, and some transgenes have been bred to homozygosity. Established lines include those bearing the promoter regions of Pax-6, Otx-2, Rx, and EF1alpha coupled to fluorescent reporter genes. Multireporter lines combining, in a single animal, up to three gene promoters coupled to different fluorescent reporters have also been established. The value of X. tropicalis transgenic lines for the study of induction is demonstrated by showing activation of Pax-6 by noggin treatment of Pax-6/GFP transgenic animal caps, illustrating how reporter lines allow a rapid, in vivo assay for an inductive response. An experiment showing lens induction in gamma-crystallin/GFP transgenic lens ectoderm when it is recombined with mouse optic vesicle demonstrates conservation of inducing signals from amphibians and mammals. It also shows how the warmer culture temperatures tolerated by X. tropicalis embryos can be used in assays of factors produced by mammalian cells and tissues. The many applications of transgenic reporter lines and other lines designed to target gene expression in particular tissues promise to bring significant new insights to the classic issues first defined in amphibian systems.

Animals↗
PubMed2004

Cryopreservation of Xenopus transgenic lines.

Xenopus laevis has been widely used for molecular, cellular, and developmental studies. With the development of the sperm-mediated transgenic method, it is now possible to study gene function during vertebrate development by using this popular model. On the other hand, like other animal species, it is labor intensive, and the maintenance of transgenic lines is expensive. In this article, we investigated the possibility of using sperm-cryopreservation as a means to preserve transgenic frog lines. We demonstrated that cryopreserved sperms are viable but not fertile under our in vitro fertilization (IVF) conditions. However, by microinjecting cryopreserved sperm nuclei, we successfully regenerated a transgenic line carrying a double promoter transgene construct, where the marker gene encoding the green fluorescent protein (GFP) is driven by the gamma-crystallin gene promoter and a gene of interest, encoding a fusion protein of GFP with the matrix metalloproteinase stromelysin-3 (ST3-GFP), is driven by a heat shock-inducible promoter. We demonstrated the functional transmission of the ST3-GFP transgene by analyzing the phenotype of the F1 animals after heat-shock to induce its expression. Our method thus provides an inexpensive means to preserve transgenic frog lines and a convenient way for distribution of transgenic lines. Furthermore, the ease with which to microinject nuclei compared to the technically demanding transgenesis procedure with variable outcome should facilitate more laboratories to use transgenic Xenopus laevis for functional studies in vivo. Mol. Reprod. Dev. 67: 65-69, 2004.

Animals↗
PubMed1994

Development of transgenic sheep that express the visna virus envelope gene.

The ovine lentiviruses cause encephalitis, pneumonia, and arthritis in sheep worldwide. Visna virus is a prototype of this family and the pathogenesis and molecular biology of the virus has been well characterized. The envelope proteins of visna virus are responsible for binding of virus to host cells and for causing cell fusion. The surface glycoprotein also elicits cellular and humoral immune responses to the virus, the former being thought to be responsible for eliminating infected cells as well as causing inflammatory lesions. In this study, transgenic sheep were constructed that expressed the envelope genes of visna virus under the control of the visna LTR to investigate the role of the env gene in the pathogenesis of lentiviral disease in its natural host. Three transgenic lambs were identified that contain the env transgene and express the envelope glycoproteins. These transgenic animals have remained healthy and expression of the viral gene has had no obvious deleterious effect. Expression of the visna envelope protein was demonstrated by cell fusion mediated by the envelope gene as well as by immunoprecipitation of the envelope proteins with monoclonal antibodies and immunofluorescence analyses of Env protein in cells. The target cell for visna virus replication in infected animals is the monocyte/macrophage. In natural infection, the level of viral gene expression in these cells increases with cell maturation. In the transgenic sheep, monocytes did not express the envelope glycoproteins until they differentiated into macrophages in vitro. Expression of the env mRNA in macrophages was quantitated by an RNase protection assay. In addition to expression in macrophages, the transgene was expressed by fibroblasts isolated from skin of the transgenic sheep. Expression of both the Env and Rev proteins was detected by immunoprecipitation and immunofluorescence. Two of the three lambs responded immunologically to the expression of the transgene by producing binding antibodies to the envelope glycoproteins. Thus, these transgenic sheep provide a model to study whether a lentivirus glycoprotein will prevent infection or modulate disease in its natural host after virus challenge.

Animals↗
PubMed1995

Characterization of resistance to cymbidium ringspot virus in transgenic plants expressing a full-length viral replicase gene.

Transgenic Nicotiana benthamiana plants expressing the replicase gene of cymbidium ringspot tombusvirus (CymRSV) were tested for the biological activity of the transgene. Protoplasts from one resistant and three susceptible transgenic lines were transfected with in vitro synthesized defective interfering (DI) RNA, a small subviral molecule which requires only the virus replicase for replication. Only protoplasts of susceptible transgenic lines were able to sustain the replication of DI RNA, whereas protoplasts of the resistant line were not. The transgene of the resistant line was recovered from transgenic cells, cloned, and shown to be intact and functional, which suggests that this is not a case of dysfunctional replicase conferring resistance to transgenic plants as a "dominant negative mutant." Plants of the transgenic line resistant to CymRSV were shown to be susceptible to two other tombusviruses, artichoke mottled crinkle and carnation Italian ringspot virus. Since the resistance is specific and inversely correlated to the level of expression of the transgene, it appears to be a form of RNA-mediated resistance.

Base Sequence↗
PubMed1994

Zona glomerulosa of the adrenal gland in a transgenic strain of rat: a morphologic and functional study.

Transgenic rats for the murine Ren-2 gene display high blood pressure, low circulating levels of angiotensin II, and high renin content in the adrenal glands. Moreover, transgenic rats possess an increased aldosterone secretion (maximal from 6 to 18 weeks of age), paralleling the development of hypertension. To investigate further the cytophysiology of the adrenal glands of this strain of rats, we performed a combined morphometric and functional study of the zona glomerulosa of 10-week-old female transgenic rats. Morphometry did not reveal notable differences between zona glomerulosa cells of transgenic and age- and sex-matched Sprague-Dawley rats, with the exception of a marked accumulation of lipid droplets, in which cholesterol and cholesterol esters are stored. The volume of the lipid-droplet compartment underwent a significant decrease when transgenic rats were previously injected with angiotensin II or ACTH. Dispersed zona glomerulosa cells of transgenic rats showed a significantly higher basal aldosterone secretion, but their response to angiotensin II and ACTH was similar to that of Sprague-Dawley animals. Angiotensin II-receptor number and affinity were not dissimilar in zona glomerulosa cells of transgenic and Sprague-Dawley rats. These data suggest that the sustained stimulation of the adrenal renin-angiotensin system in transgenic animals causes an increase in the accumulation in zona glomerulosa cells of cholesterol available for steroidogenesis, as indicated by the expanded volume of the lipid-droplet compartment and the elevated basal steroidogenesis. However, the basal hyperfunction of the zona glomerulosa in transgenic animals does not appear to be coupled with an enhanced responsivity to its main secretagogues, at least in terms of aldosterone secretion.

Adrenocorticotropic Hormone↗
PubMed1996

The renin-angiotensin system in transgenic rats.

Transgenic animals are used to study the function, regulation and in vivo expression of genes. The effects of the genes of the renin-angiotensin system on blood pressure regulation and hypertension were invested in transgenic rats. The role of the renin-angiotensin system in the development of the cardiovascular hypertrophy or hypertensive renal damage was analysed, as well as its interaction with other hormonal systems, i.e., adrenal steroids. The development of a transgenic rat strain carrying the mouse REN-2 gene has provided a new model of hypertension with systolic blood pressure values of 200 mmHg. This model is characterised by low active plasma renin, hyperproreninaemia and high expression of renin in the adrenal gland and other external tissues. Transgenic rats with the human components of the renin-angiotensin system expressed the human renin and angiotensinogen proteins which interacted species-specifically in transgenic rats. These transgenic models demonstrate the feasibility of studying the function of candidate hypertension genes in transgenic animals. In the future, further refinements in transgene construction, mutation, and modification can be tested in such transgenic animal models.

Animals↗
PubMed1994

Liver, renal and subcutaneous histopathology in PEPCK-bGH transgenic pigs.

Transgenic pigs were created that harboured a phosphoenolpyruvate carboxykinase-bovine growth hormone construct (PEPCK-bGH). Four founder animals and two transgenic offspring from one line were evaluated between 6 1/2 and 12 months of age. There was no evidence of severe hepatic or renal lesions in these pigs, which characterised transgenic PEPCK-bGH mice previously described. While glomerular and tubular lesions in kidney sections were not identified in the transgenic pigs, mesangial cell proliferation was observed in two transgenic offspring from a single line. Additionally, glomerular size was significantly increased in four of four puberal transgenic swine when compared to age- and sex-matched controls (28.30 +/- 4.1 vs. 14.2 +/- 2.7 x 10(5) microns 3; representing 3 transgenic lines, p < 0.05). Surprisingly, no mature adipocytes were observed in subcutaneous sections obtained in transgenic GH pigs. Histological evaluation of these transgenic pigs further illustrates the requirement for precise control of growth-related genes and their protein products.

Adipocytes↗
PubMed1999

Growth Performance and Gonadal Development of Growth Enhanced Transgenic Tilapia Oreochromis niloticus (L.) Following Heat-Shock-Induced Triploidy.

Triploid induction offers a way of considerably reducing fertility in fish, and could therefore be employed to help ensure that any adverse environmental impact of transgenic fish was markedly less. In order to produce sterile growth-enhanced transgenic fish, we have induced triploidy in two lines of transgenic tilapia. Growth performance and gonadal development were analyzed following triploidization by heat shock. Ploidy status was confirmed by nuclear size measurement of erythrocytes. Erythrocytes of triploids were found to be 1.5 times larger than diploids. Observations of growth enhancement and gonadal development were made on diploids and triploids from both transgenic and nontransgenic full sibling batches. In both lines, transgenic diploids were superior in growth performance, followed by transgenic triploids, nontransgenic diploids, and nontransgenic triploids. Although the testes of transgenic triploids were significantly smaller than those of nontransgenic triploids and nontransgenic diploids, histologically they did not show signs of gross deformation. There were also some spermatozoa present in the testes of some triploids, which could be indicative of reproductive functionality. However, the ovaries were devoid of oocytes, underdeveloped, and deformed in all triploids and were completely nonfunctional. Although the best growth performance was shown by the fertile diploid transgenics, the triploid transgenic females could offer a good option for aquaculture purposes because they showed superior growth performance over the normal wild-type tilapias with the advantage of sterility to ensure nonhybridization and noncontamination with the local gene pool. However, careful monitoring of potential gene flow would be required prior to commercial use.

Journal Article↗
PubMed1999

Transgenic rats expressing the human ET-2 gene: a model for the study of endothelin actions in vivo.

Previous studies have characterized the endothelin peptides (ET-1, ET-2, ET-3) as strong vasoconstrictors which are possibly involved in the pathogenesis of cardiovascular disease. Whereas ET-1 and ET-3 have been characterized using a number of approaches, little is known about the function of ET-2. The aim of this study was to define the role of ET-2 in physiology and pathophysiology using a transgenic approach. Transgenic rats expressing a genomic construct of the human ET-2 gene were generated by microinjection of fertilized oocytes from Sprague-Dawley rats. Two transgenic lines were generated, and one line was further characterized in detail. Studies on mRNA expression demonstrated that the transgene is expressed predominantly in kidney, gastrointestinal tract, adrenal gland, lung, and brain. Plasma endothelin levels were elevated 2-fold, and big-endothelin levels were elevated 2.5-fold. Despite these alterations blood pressure in transgenic rats remained normal. Further analysis of transgenic animals revealed that endothelin receptors were not downregulated, and that infusion of exogenous human ET-2 results in an enhanced blood pressure response. These observations suggest the presence of counterregulatory mechanisms influencing the effects of endothelin on blood pressure. One of these mechanisms may involve the nitric oxide system since infusion of an inhibitor of nitric oxide synthase resulted in a greater blood pressure response than in non-transgenic littermates. Despite unchanged blood pressure, alterations were observed in organ development and function, namely of hearts and kidneys, indicating an interference between transgene expression and growth processes. Male rats seem to be more susceptible to endothelin actions. These data show that the elevation in endothelin-2 expression in this transgenic model does not induce hypertension but leads to changes at the end-organ level. Normotension is most likely due to compensatory mechanisms such as increased nitric oxide formation.

Animals↗
PubMed2002

Destiny of a transgene escape from Brassica napus into Brassica rapa.

Transgenic Brassica napus can be easily crossed with wild Brassica rapa. The spread of the transgene to wild species has aroused the general concern about its effect on ecological and agricultural systems. This paper was designated, by means of population genetics, to study the fate of a transgene escape from B. napus to B. rapa. Three models were proposed to survey the change in gene frequency during successive backcross processes by considering selection pressures against aneuploids, against herbicide-susceptible individuals, and by considering A-C intergenomic recombination and the effect of genetic drift. The transmission rate of an A-chromosome gene through an individual to the next generation was 50%, irrespective of the chromosome number; while that of a C-chromosome transgene varied from 8.7% to 39.9%, depending on the chromosome number of the individual used in the backcross. Without spraying herbicide, the frequency of an A-chromosome gene was 50% in the BC(1) generation, and decreased by 50% with the advance of each backcross generation; that of a C-chromosome gene was around 39.9% in BC(1), 7.7% in BC(2), 1.2% in BC(3) and 0.1% in the BC(4) generation. Under the selection pressure against herbicide-susceptible individuals, the frequency of a transgene reached a stable value of about 5.5% within six generations of successive backcrossings. The effect of genetic drift and intergenomic exchange on gene transmission rate was discussed. It is suggested that the transgene integrated on a C-chromosome (or better on a cytoplasm genome) is safer than that on an A-chromosome. The transgenic cultivars should be cultivated rotationally by year(s) with other non-transgenic varieties in order to reduce the transfer of the transgene to wild B. rapa species.

Journal Article↗
PubMed2002

Field evaluation and agronomic performance of transgenic wheat.

Seven transgenic lines of wheat have been evaluated under field conditions during 2 agonomic years. Four lines contained the transgenes for beta-glucuronidase ( uidA), herbicide resistance ( bar) and for one high-molecular-weight (HMW) subunit, and three lines contained only one transgene for one HMW glutenin subunit and no marker genes. Agronomic traits and yield components were studied in transgenic lines and compared with the non-transgenic parent and null segregant lines. Although phenotypic differences for many traits have been found, only heading date and the number of spikelets per spike showed clear genotypic differences for both field trials. All transgenic lines had a longer heading date than parent lines whereas the number of spikelets per spike in transgenic lines was around that for L88-31 and higher for L88-6 than the corresponding parent lines. No differences were found between lines constitutively expressing the uidA and bar genes from those which only expressed the HMW genes. We conclude that differences between transgenic lines and their parents are small, and could be eliminated by backcrossing transgenic lines with their parents and selecting for the wanted genotype.

Journal Article↗
PubMed2003

Field performance of transgenic tall fescue (Festuca arundinacea Schreb.) plants and their progenies.

Tall fescue (Festuca arundinacea Schreb.) is a hexaploid, outcrossing grass species widely used for forage and turf purposes. Transgenic tall fescue plants were generated by biolistic transformation of embryogenic cell suspension cultures that were derived from single genotypes of widely used cultivar Kentucky-31. Primary transgenics from two genotypes, their corresponding regenerants from the same genotypes and control seed-derived plants were transferred to the field and evaluated for 2 years. Progenies of these three classes of plants were obtained and evaluated together with seed-derived plants in a second field experiment. The agronomic characteristics evaluated were: heading date, anthesis date, height, growth habit, number of reproductive tillers, seed yield and biomass. The agronomic performance of the primary transgenics and regenerants was generally inferior to that of the seed-derived plants, with primary transgenics having fewer tillers and a lower seed yield. However, no major differences between the progenies of transgenics and the progenies of seed-derived plants were found for the agronomic traits evaluated. Primary transgenics and regenerants from the same genotype were more uniform than plants from seeds. Progenies of transgenics performed similarly to progenies of the regenerants. The addition of a selectable marker gene in the plant genome seems to have had little effect on the agronomic performance of the regenerated plants. No indication of weediness of the transgenic tall fescue plants was observed. Our results indicate that outcrossing grass plants generated through transgenic approaches can be incorporated into forage breeding programs.

Biolistics↗
PubMed2004

Transgenic rice plants expressing the snowdrop lectin gene (gna) exhibit high-level resistance to the whitebacked planthopper (Sogatella furcifera).

Transgenic rice plants, expressing snowdrop lectin [Galanthus nivalis agglutinin (GNA)], obtained by Agrobacterium-mediated genetic transformation, were evaluated for resistance against the insect, the whitebacked planthopper (WBPH). The transgene gna was driven by the phloem-specific, rice-sucrose synthase promoter RSs1, and the bar was driven by the CaMV 35S promoter. In our previous study, the transgenic status of these lines was confirmed by Southern, Northern and Western blot analyses. Both the transgenes, gna and bar, were stably inherited and co-segregated into progenies in T1 to T5 generations. Insect bioassays on transgenic plants revealed the potent entomotoxic effects of GNA on the WBPH. Also, significant decreases were observed in the survival, development and fecundity of the insects fed on transgenic plants. Furthermore, intact GNA was detected in the total proteins of WBPHs fed on these plants. Western blot analysis revealed stable and consistent expression of GNA throughout the growth and development of transgenic plants. Transgenic lines expressing GNA exhibited high-level resistance against the WBPH. As reported earlier, these transgenics also showed substantial resistance against the brown planthopper and green leafhopper.

Animals↗
PubMed2006

Establishment and characterization of CAG/EGFP transgenic rabbit line.

Cell marking is a very important procedure for identifying donor cells after cell and/or organ transplantation in vivo. Transgenic animals expressing marker proteins such as enhanced green fluorescent protein (EGFP) in their tissues are a powerful tool for research in fields of tissue engineering and regenerative medicine. The purpose of this study was to establish transgenic rabbit lines that ubiquitously express EGFP under the control of the cytomegalovirus immediate early enhancer/beta-actin promoter (CAG) to provide a fluorescent transgenic animal as a bioresource. We microinjected the EGFP expression vector into 945 rabbit eggs and 4 independent transgenic candidate pups were obtained. Two of them died before sexual maturation and one was infertile. One transgenic male candidate founder rabbit was obtained and could be bred by artificial insemination. The rabbit transmitted the transgene in a Mendelian manner. Using fluorescence in situ hybridization analysis, we detected the transgene at 7q11 on chromosome 7 as a large centromeric region in two F1 offspring (one female and one male). Eventually, one transgenic line was established. Ubiquitous EGFP fluorescence was confirmed in all examined organs. There were no gender-related differences in fluorescence. The established CAG/EGFP transgenic rabbit will be an important bioresource and a useful tool for various studies in tissue engineering and regenerative medicine.

Animals↗
PubMed2005

Generation of lentiviral transgenic rats expressing glutamate receptor interacting protein 1 (GRIP1) in brain, spinal cord and testis.

In neuroscience, rats have several advantages over mice as a model organism. For instance, behavioral experiments are more advanced and the larger size of the brain is better suited for surgical manipulation and biochemistry. Furthermore, the vascular physiology of rats is considered closer to human, providing clinical relevance. Because transgenesis rates achieved by conventional pronuclear injection are extremely low (0.2-3.5%), the availability of transgenic rats in neuroscience is limited. Lentivirus infection is an efficient way to integrate exogenous genes into the genome of a one-cell embryo to generate transgenic animals. We report here the generation of synapsin I promoter driven GRIP1-transgenic rats using lentiviral transgenesis. GRIP1 was chosen as a transgene because it interacts with AMPA receptors and is involved in glutamate receptor signaling. From a single infection experiment, 45% of the offspring carried the transgene and 40% achieved germ-line transmission. The expression of GRIP1 was observed at low levels in brain, spinal cord and testis. Interestingly, one transgenic copy lacked a 147 bp fragment in the GRIP1 coding region most likely caused by alternative splicing of genomic lentiviral RNA. Co-immunoprecipitation from rat brains showed that transgenic GRIP1 is in complex with the endogenous GluR2 subunit of AMPA receptors. These results indicate that functional transgenic GRIP1 protein is expressed in rat brain using lentiviral vectors containing a human synapsin I promoter. Tissue specific lentiviral transgenic rats will be a powerful tool for various applications in modern neuroscience.

Amino Acid Sequence↗
PubMed2005

Secretion of bacterial xenobiotic-degrading enzymes from transgenic plants by an apoplastic expressional system: an applicability for phytoremediation.

In search of an effective method for phytoremediation of wastewater contaminated with organic compounds, we investigated the application of an apoplastic expressional system that secretes useful bacterial enzymes from transgenic plants into hydroponic media through the addition of a targeting signal. We constructed transgenic Arabidopsis expressing the aromatic-cleaving extradiol dioxygenase (DbfB), which degrades 2,3-dihydroxybiphenyl (2,3-DHB), and transgenic tobacco expressing haloalkane dehalogenase (DhaA), which catalyzes hydrolytic dechlorination of 1-chlorobutane (1-CB). Although crude leaf extracts of transgenic plants expressing cytoplasm-targeted degradative enzymes showed higher activity than did those from transgenic plants expressing apoplast-targeted enzymes, the hydroponic media of the latter showed 23.2 times (DbfB) and 76.4 times (DhaA) higher activity than plants containing the cytoplasm-targeted enzymes. Addition of crystalline 2,3-DHB to 100 mL of the hydroponic medium of transgenic or wild-type seedlings revealed that only medium from the transgenic Arabidopsis expressing apoplast-targeted DbfB showed rapid ring cleavage of 2,3-DHB. Transgenic tobacco expressing apoplast-targeted DhaA also resulted in the accumulation of the dehalogenation product 1-butanol in the hydroponic medium and showed a higher tolerance to 1-CB than wild-type or transgenic plants expressing cytoplasm-targeted DhaA. These results demonstrate the usefulness of the apoplastic expression of bacterial recombinant proteins in phytoremediation.

Actinomycetales↗
PubMed2003

Stable transgene expression and random gene silencing in wheat.

Wheat genes for pathogenesis-related (PR-)proteins, chitinase and beta-1,3-glucanase, under the control of maize ubiquitin promoter-intron were used for transforming the spring wheat 'Bobwhite', using a biolistic approach. Twenty of the 24 primary transgenic lines expressing the PR-protein genes in the T0 generation were silenced in either the T1 or T2 generations. Two apparently genetically identical regenerants arising from a single callus co-bombarded with chitinase and beta-1,3-glucanase transgene combinations, but differing in the expression of the transgenes were selected for further characterization. In one homozygous line, transgene silencing was observed in the T3 plants, while the other line homozygous for the transgene loci stably expressed and inherited the transgenes to at least the T4 generation. Southern blot analyses of genomic DNA from the two lines using the isoschizomeric methylation-sensitive enzymes, MspI and HpaII, revealed a higher degree of methylation of CCGG sequences in the line with the silenced transgene locus. Analysis by reverse transcriptase-polymerase chain reaction, Northern blotting and Western blotting detected stable expression of the transgenes in the line with a lesser extent of methylation, whereas the line with a higher level of CCGG methylation had no transgene expression by the T3 generation. The germination of seeds from the silenced plants in the presence of a cytidine analogue, 5-azacytidine (azaC), did not lead to a reversion of this phenotype.

Journal Article↗