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At least 73 records · Page 4Linked to original sources
PubMed1996

Epigenetic modification of transgenes under the control of the mouse mammary tumor virus LTR: tissue-dependent influence on transcription of the transgenes.

Transgenic mice expressing human urokinase, as well as animals expressing human urokinase receptor under the control of the murine mammary tumor virus (MMTV) long terminal repeat, were established. In the vast majority of the founder animals and their descendants, the transgene was completely methylated, corresponding to down-regulation of transgene expression in the mammary gland. Two lineages with human urokinase receptor as the transgene with mixed methylation of the transgenes were analyzed in more detail. We show here for the first time that the methylation status of the transgene is identical in different organs of an animal, but may differ from animal to animal among the descendents. In the mammary gland, complete methylation of the transgene was incompatible with expression; unmethylated and mixed methylation transgenes gave rise to expression at the RNA as well as at the protein level. The methylation observed was not the consequence of an imprinting process. Surprisingly, in organs other than the mammary gland, such as liver, kidney and spleen, weak expression of the transgene was noted independent of the methylation status of the MMTV promoter. With respect to the molecular mechanism it is unresolved whether the human growth hormone sequence of the transgene harbors a methylation inducing element responsible for the observed methylation pattern.

3T3 Cells↗
PubMed1995

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 T3 injection in surgically thyroidectomized heterozygous transgenic rats. There was a reduction of about approximately 35-40% in GH mRNA levels in the pituitary of homozygous animals compared with those in non-transgenic rats. Plasma GH concentration was significantly approximately 25-32 and approximately 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 approximately 72-81 and approximately 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.

Animals↗
PubMed2006

Comparative study of compact bone tissue microstructure between non-transgenic and transgenic rabbits with WAP-hFVIII gene construct.

The aim of this study was to investigate differences in compact bone tissue microstructure between New Zealand White transgenic rabbits with human blood clotting factor VIII gene and the non-transgenic ones. The transgene was under the transcriptional control of the whey acidic protein (WAP) promoter. Altogether, 42 femora were analysed. Specimens were prepared using standard histological equipment, producing thin sections approximately 80-100 microm. Areas, perimeters, minimum and maximum diameters of vascular canals of primary osteons, Haversian canals and secondary osteons were measured. Our results indicate the basic structural pattern of the bone tissue was primary vascular longitudinal in both groups of rabbits. However, a fibrolamellar bone tissue was identified only in the transgenic rabbits. The measured variables of the vascular canals of the primary osteons, the Haversian canals and the secondary osteons were higher in transgenic rabbits in comparison with non-transgenic ones in most cases and the differences were often statistically significant (P < 0.01; P < 0.001). More significant differences were noticed in males (transgenic versus non-transgenic). However, it is not easy to find the real cause of the changes. Despite the fact that no hFVIII mRNA expression was found in the analysed bone of transgenic rabbits, we presume that the observed differences could be associated with transgenesis. In this article, we discuss possible associations between transgene integration as well as aneuploidy and/or observed microstructural changes in compact bone tissue.

Aneuploidy↗
PubMed2006

In situ methods to localize transgenes and transcripts in interphase nuclei: a tool for transgenic plant research.

Genetic engineering of commercially important crops has become routine in many laboratories. However, the inability to predict where a transgene will integrate and to efficiently select plants with stable levels of transgenic expression remains a limitation of this technology. Fluorescence in situ hybridization (FISH) is a powerful technique that can be used to visualize transgene integration sites and provide a better understanding of transgene behavior. Studies using FISH to characterize transgene integration have focused primarily on metaphase chromosomes, because the number and position of integration sites on the chromosomes are more easily determined at this stage. However gene (and transgene) expression occurs mainly during interphase. In order to accurately predict the activity of a transgene, it is critical to understand its location and dynamics in the three-dimensional interphase nucleus. We and others have developed in situ methods to visualize transgenes (including single copy genes) and their transcripts during interphase from different tissues and plant species. These techniques reduce the time necessary for characterization of transgene integration by eliminating the need for time-consuming segregation analysis, and extend characterization to the interphase nucleus, thus increasing the likelihood of accurate prediction of transgene activity. Furthermore, this approach is useful for studying nuclear organization and the dynamics of genes and chromatin.

Journal Article↗
PubMed1998

Frequent occurrence of transgene deletion in transgenic plants.

The status of the transgene in tobacco plants transformed by Agrobacterium was analyzed with PCR. Twelve percent of the transgenic plants with the nptII gene showed different levels of transgene deletion, which was also found in transgenic watermelon (10-30%) and carrot (40-60%). It appeared that the percentage of transgenic plants carrying deleted transgenes depended on both the transgene and the plant. It is suggested that the transgene should be inserted between a right border and a selection marker to reduce the number of transgenic plants containing deleted transgenes after selection.

Agrobacterium tumefaciens↗
PubMed1997

Kinetics of wound-induced v-Ha-ras transgene expression and papilloma development in transgenic Tg.AC mice.

The Tg.AC transgenic mouse, which harbors an activated v-Ha-ras coding region that is fused to an embryonic zeta globin transcriptional control region and a 3' simian virus 40 polyadenylation sequence, rapidly develops epidermal papillomas in response to topical application of chemical carcinogens or tumor promoters or to full-thickness wounding of the dorsal skin. In this report, we investigated the localization and temporal induction of v-Ha-ras transgene expression after full-thickness wounding of Tg.AC mouse skin. Surgically inflicted full-thickness incisions 3 cm long yielded four to six papillomas per Tg.AC mouse by 5 wk after wounding. Similar wounding of the FVB/N isogenic host strain did not produce tumors, which implicates a causal role for the v-Ha-ras transgene. Reverse transcription-polymerase chain reaction assays detected the v-Ha-ras transgene transcript in total RNA samples isolated from wound-associated tissue 3 and 4 wk after wounding. Tissues 1-2 wk after wounding and all non-wound-associated tissues were negative for transgene expression. In situ hybridization experiments using transgene-specific 35S-labeled antisense RNA probes localized transgene expression to the basal epidermal cells in wound-induced papillomas. Adjacent normal and hyperplastic skin tissues were negative for transgene expression by this assay. This work supports the hypothesis that the wound repair response leads to the transcriptional activation and continued expression of the v-Ha-ras transgene in specific cells in the skin, which alters normal epithelial differentiation and ultimately results in neoplastic growth.

Animals↗
PubMed2005

Epigenetic mechanisms affect mutant p53 transgene expression in WAP-mutp53 transgenic mice.

We describe the construction and phenotypic characterization of 23 whey acidic protein (WAP)-mutp53 transgenic mouse lines. The mutp53-expressing lines showed a mosaic expression pattern for the transgenes, leading to a heterogeneous yet mouse line-specific expression pattern for mutp53 upon induction. Only few lines were obtained, in which the majority of the induced mammary epithelial cells expressed the mutp53 transgene, most of the transgenic lines did not express mutp53, or expressed the transgene in less than 2% of the induced mammary epithelial cells. Hormone requirements for mutp53 transgene expression from the WAP-promoter differed in high and low expressing lines, being low in high expressing lines, and even lower in multiparous mutp53 mice, where persistent expression of the transgene occurred. Repeated induction of mutp53 expression through repeated parturition resulted in the formation of expanding mutp53-expressing foci within the mammary alveolar epithelium. The data suggest that epigenetic mechanisms play a role in modulating the expression of the mutp53 transgene. To support this idea, we crossed a nonexpressing WAP-mutp53 line with a strongly SV40 T-antigen-expressing WAP-T mouse line. In the bitransgenic mice, T-antigen-induced chromatin remodeling led to re-expression of epigenetically silenced mutp53 transgene(s). In these mice, mutp53 expression was much more variable compared to SV40 T-antigen expression, and seemed to depend on the coexpression of SV40 T-antigen. Mutp53 expression in this system thus resembles the situation in many human tumors, where one can observe a heterogeneous expression of mutp53, despite a homogeneous distribution of the p53 mutation in the tumor cells.

Animals↗
PubMed2003

Effects of transgene Oncostatin M on the development of retinal neuron in transgenic mice.

PURPOSE: Oncostatin M (OSM) is a cytokine released by macrophages and lymphocytes that can function as a growth regulator. A current study shows that leukemia inhibitory factor (LIF), a homologue of OSM, can prevent photoreceptor cell death when expressed in the lens of transgenic mice. We determined the efforts of lens-specific overexpression of OSM on the development of eye. METHODS: A truncated mouse OSM cDNA (-660 bp) was linked to the alpha A-crytallin promoter, and injected into single-cell embryos with microinjection. Then, transgenic mice were established. The mRNA expression of transgene OSM was detected by in situ hybridization. Immunohistochemistry was used to detect the expression of syntaxin, glial fibrillary acidic protein (GFAP), synaptophysin in the retinas of transgenic mice. RESULTS: At embryonic day (E 17.5), the expression of the syntaxin at the inner and mid portion of the retinas of transgenic mice was much higher than that of the retinas of non-transgenic mice. The expression of GFAP was detected in the retinas of transgenic mice, while no expression in non-transgenic normal FVB (FVB/N) mice was detected in this stage. At postnatal day one (P1), the expression of synaptophysin was detected in the retinas of transgenic mice, but there was no such expression in FVB/N mice. CONCLUSIONS: Lens-specific overexpression of OSM induces premature differentiation of amacrine cells, gial cells, and photoreceptors in vivo.

Animals↗
PubMed2006

Stable transmission and transcription of newfoundland ocean pout type III fish antifreeze protein (AFP) gene in transgenic mice and hypothermic storage of transgenic ovary and testis.

Here we describe the generation of transgenic mice carrying type III fish antifreeze protein (AFP) gene and evaluate whether AFP type III protects transgenic mouse ovaries and testes from hypothermic storage. AFPs exist in many different organisms. In fish, AFPs protect the host from freezing at temperatures below the colligative freezing point by adsorbing to the surface of nucleating ice crystals and inhibiting their growth. The transgenic expression of AFP holds great promise for conferring freeze-resistant plant and animal species. AFP also exhibits a potential for the cryopreservation of tissues and cells. In this study, we have generated 42 founder mice harboring the Newfoundland ocean pout (OP5A) type III AFP transgene and established one transgenic line (the line #6). This study demonstrated that AFP gene construct has been stably transmitted to the mouse progeny in the F3 generations in the line #6. Furthermore, the presence of AFP transcripts was confirmed by RT-PCR analysis on cDNAs from liver, kidney, ovarian, and testis tissues of the mouse from F3 generation in this line. These results indicate that ocean pout type III AFP gene could be integrated and transmitted to the next generation and stably transcribed in transgenic mice. In histological analysis of testis and ovarian tissues of nontransgenic control and AFP transgenic mice it has been shown that both tissues of AFP transgenic mice were protected from hypothermic storage (+4 degrees C). The AFP III transgenic mice obtained for the first time in this study would be useful for investigating the biological functions of AFP in mammalian systems and also its potential role in cryopreservation.

Animals↗
PubMed1996

Spatial and temporal regulation of a lacZ reporter transgene in a binary transgenic mouse system.

The transgenic mouse system is a powerful tool for the study of gene function. However, when the analysis involves genes that are critical for the normal developmental process, the usefulness of transgenic mouse systems is limited (for review see Hanahan, 1989; Westphal and Gruss, 1989; Byrne et al., 1991). This is due to potential transgene interference with development in case of ectopic or high level expression. As a result, establishing permanent transgenic mouse lines expressing these types of genes has proven difficult. To circumvent these difficulties, a binary transgenic mouse system has been established, termed the Multiplex System (Byrne and Ruddle, 1989). This is a two-tiered gene activation system in which expression of the gene of interest occurs only in offspring carrying transgenes encoding both components: transactivator and transresponder. Transactivator lines contain the gene encoding the VP16 protein of herpes simplex virus. Transresponder lines harbour the gene of interest linked to the IE promoter which includes recognition sequences for the VP16 transactivator. Previously, the inducibility of a chloramphenicol acetyltransferase reporter gene in newborn offspring that carried both a transactivator and transresponder transgene (Byrne and Ruddle, 1989) has been shown. Moreover, it has been demonstrated that expression of the VP16 protein was not detrimental to development and that transactivation appeared to be tissue specific. Here, the potential of the system for the expression of transgenes in early mouse embryogenesis was examined, using the Escherichia coli beta-galactosidase gene as a reporter in the transresponder mouse strain. To direct expression of VP16, the murine Hoxc-8 promoter, which is known to be active during early development, was used. Embryos from crosses of transactivators to transresponders were isolated at different stages of development and stained for beta-galactosidase activity. Transactivation, as demonstrated by strong beta-galactosidase staining, could be detected as early as eight days of development. At all stages examined, the pattern of lacZ transresponder gene expression accurately reflected the activity of the Hoxc-8 promoter controlling VP16 expression. It is demonstrated that the Multiplex System can be used to express transresponder transgenes in a spatially and temporally defined manner in multiple cell types early during mouse embryogenesis.

Animals↗
PubMed2000

Stable and unstable transgene integration sites in the human genome: extinction of the Green Fluorescent Protein transgene in K562 cells.

In gene transfer experiments including gene therapy studies, expression of the integrated transgenes in host cells often declines with time. The molecular basis of this phenomenon is not clearly understood. We have used the Green Fluorescent Protein (GFP) gene as both a selectable marker and a reporter to study long-term transgene integration and expression in K562 cells. Cells transfected with plasmids containing the GFP gene coupled to the HS2 or HS3 enhancer of the human beta-globin Locus Control Region (LCR) or the cytomegalovirus (CMV) enhancer were sorted by either fluorescence-activated-cell-sorting (FACS) alone or FACS combined with drug selection based on a co-integrated drug resistance gene. The two groups of selected cells were subsequently cultured for long periods up to 250 cell generations. Comparison of long-term GFP transgene integration and expression in these two groups of cells revealed that the K562 genome contains two types of transgene integration sites: i) abundant unstable sites that permit transcription but not long-term integration of the transgenes and thus eliminate the transgenes in 60-250 cell generations and ii) rare stable sites that permit both efficient transcription and long-term stable integration of the transgenes for at least 200 cell generations. Our results indicate that extinction of GFP expression with time is due at least in part to elimination of the gene from the host genome and not entirely to transcriptional silencing of the gene. However, long-term, stable expression of the transgene can be achieved in cells containing the transgene integrated into the rare, stable host sites.

Binding Sites↗
PubMed1998

Structures of transgene loci in transgenic Arabidopsis plants obtained by particle bombardment: junction regions can bind to nuclear matrices.

To clarify the molecular structure of the integration sites of transgenes, we used particle bombardment to examine the DNA sequences of transgene loci. Three transgenic Arabidopsis lines gave a single Southern hybridization band with a selectable gene as the probe. Junction regions flanked by the transgenes were cloned by the inverse polymerase chain reaction method, and the characteristics of the DNA sequences of the 10 junction regions were investigated. All but two of these were AT-rich sequences bearing motifs characteristic of a scaffold/matrix-attachment region (S/MAR). Calculations showed that seven of them should have a propensity for curvature. An assay of in-vitro binding to tobacco nuclear matrices showed that all the junction regions bound to nuclear matrices and that the two input DNAs did not bind. The 12 chromosome/transgene (CT) junctions in these three transgene loci were investigated. Cleavage sites for topoisomerase I were found at 10 of the 12, near the junction point. The other two junctions had sites within 6bp of the junction point. The sequence near one terminal of the transgene in the transgene loci was compared with that near the other terminal. Short, direct repeats consisting of 4-6bp were present within 10bp of the junction points in the sequence. We speculate that the transgene introduced by particle bombardment is delivered on AT-rich S/MAR that has a propensity for curvature, and then a nucleotide near the short, direct repeat on the transgene is joined near the cleavage sites on the genome for topoisomerase I.

Arabidopsis↗
PubMed1994

Generation of transgenic dairy cattle from transgene-analyzed and sexed embryos produced in vitro.

We have generated a transgenic calf from in vitro produced bovine embryos which had undergone transgene analysis and sexing prior to the embryo transfer. Bovine oocytes were isolated from slaughter-house-derived ovaries, matured and fertilized in vitro and subsequently microinjected with a dam-methylated gene construct consisting of genomic sequences encoding human erythropoietin and governed by bovine alpha S1-casein regulatory sequences. After 6 to 7 days in culture, the embryos were biopsied and while the embryo remained in culture, the biopsy was subjected to transgene analysis and sexing. The transgene analysis was accomplished with a combined treatment of the embryo lysates with DpnI restriction endonuclease and Bal31 exonuclease followed by polymerase chain reaction (PCR). The transgene analysis was based on the fact that DpnI only cleaves its recognition sequence if the adenine in the sequence is methylated. Pregnancy was induced by the transfer of three viable female embryos with a distinct transgene signal to a hormonally synchronized heifer recipient. Amniotic fluid analysis performed two months after the embryo transfer confirmed the presence of the transgene. The calf born was found to be transgenic by PCR analysis from blood, ear and fetal membranes. The presence of the transgene was also confirmed by Southern blotting.

Animals↗
PubMed2003

Targeted transgene integration into transgenic mouse fibroblasts carrying the full-length human AAVS1 locus mediated by HSV/AAV rep(+) hybrid amplicon vector.

Herpes simplex virus type 1/adeno-associated virus (HSV/AAV) rep(+) hybrid amplicon vectors containing AAV inverted terminal repeats (ITRs) and rep gene sequences can mediate site-specific integration into the human genome. In this study, we have generated and characterized the first transgenic mice that bear the full-length (8.2 kb) human AAVS1 locus. Immortalized mouse embryonic fibroblasts from this mouse line were transduced with the rep(+), rep(-) (containing only ITRs flanking the transgene) hybrid amplicon vectors, and the standard amplicon vector to determine stable integration frequency and the site of integration. Transduction of transgenic fibroblasts resulted in a 10-fold higher stable integration frequency with rep(+) hybrid amplicon vector than with rep(-) or standard amplicon vectors. Southern blot analysis of genomic DNA from transgenic cells stably transduced with the rep(+) hybrid amplicon vector revealed site-specific integration of transgenes at the AAVS1 locus in 50% of clones. Some site-specific and random integration events were limited to the ITR-flanked transgene cassette. In contrast, transduction of transgenic mouse cells with the rep(-) or standard amplicon vectors resulted in random integrations of the entire rep(-) hybrid amplicon or amplicon DNA that were incorporated into the host genome as a concatenate of various sizes. These results demonstrate for the first time that the genome of transgenic mice bearing the human AAVS1 locus serves as a platform for site-specific integration of AAV ITR-flanked transgene cassettes within the hybrid amplicon vector in the presence of Rep.

Animals↗
PubMed1989

Multiplex gene regulation: a two-tiered approach to transgene regulation in transgenic mice.

Transgenic mice have been used to study gene function and regulation by introducing inducible or tissue-specific transgenes. This approach is generally limited to studying gene function in adult mice since ectopic expression of many interesting genes is disease causing or may be lethal to the developing embryo. To extend the utility of the transgenic mouse system to the early stages of embryogenesis, we have developed a two-tiered method of gene regulation to control transgene expression. Our multiplex gene regulatory system (MGR) allows the establishment of transgenic lines that harbor inducible potentially lethal transgenes. These inducible transgenes are activated only when mated to a second transgenic animal. Induction in the MGR system provides a high degree of temporal and spatial control over transgene expression and should be suitable for engineering "gain of function mutations" for many developmental genes.

Animals↗
PubMed2006

An analytical model assessing the potential threat to natural habitats from insect resistance transgenes: continuous transgene input.

The potential effects of 'escape' of genetically modified material (transgenes) into natural communities is a major concern in their use. These effects may be limited in the first instance by limiting the proportion of transgene-carrying plants in the natural community. We previously presented an analytical model of the ecological processes governing the relative abundance and persistence of insect resistance (IR) transgenes in a natural community. In that paper, we illustrated the case in which the transgene is input into the community in a single season using data from oilseed rape (OSR) and its known herbivore, Plutella macropennis. We found that the transgene is unlikely to have a great impact on the natural community. Here, we extend the model for repeated input of crop pollen carrying the transgene. We show the model output, again using OSR, for continuous input of the transgene over 10 years, the projected commercial lifetime of a transgene without associated undesirable agronomic effects. Our results do not change our original conclusion that the IR transgene need not have a large impact on the natural community and our suggestions for assessing and mitigating any threat still stand.

Animals↗
PubMed1997

Activation of transgene expression by early region 4 is responsible for a high level of persistent transgene expression from adenovirus vectors in vivo.

The persistence of transgene expression has become a hallmark for adenovirus vector evaluation in vivo. Although not all therapeutic benefit in gene therapy is reliant on long-term transgene expression, it is assumed that the treatment of chronic diseases will require significant persistence of expression. To understand the mechanisms involved in transgene persistence, a number of adenovirus vectors were evaluated in vivo in different strains of mice. Interestingly, the rate of vector genome clearance was not altered by the complete deletion of early region 4 (E4) in our vectors. The GV11 (E1- E4-) vector genome cleared with a similar kinetic profile as the GV10 (E1-) vector genome in immunocompetent and immunocompromised mice. These results suggest that the majority of adenovirus vector genomes are eliminated from transduced tissue via a mechanism(s) independent of T-cell, B-cell, and NK cell immune mechanisms. While the levels of persistence of transgene expression in liver or lung transduced with GV10 and GV11 vectors expressing beta-galactosidase, cystic fibrosis transmembrane conductance regulator, or secretory alkaline phosphatase were similar in immunocompetent mice, a marked difference was observed in immunocompromised animals. Levels of transgene expression initially from both GV10 and GV11 vectors were the same. However, GV11 transgene expression correlated with loss of vector genome, while GV10 transgene expression persisted at a high level. Coadministration and readministration of GV10 vectors showed that E4 provided in trans could activate transgene expression from the GV11 vector genome. While transgene expression activity per genome from the GV10 vector is clearly activated, expression from a cytomegalovirus promoter expression cassette in a GV11 vector appeared to be further inactivated as a function of time. Understanding the molecular mechanisms underlying these expression effects will be important for developing persistent adenovirus vectors for chronic applications.

Adenoviridae↗
PubMed2005

Aberrant development of mammary glands, but precocious expression of beta-casein in transgenic females ubiquitously expressing whey acidic protein transgene.

It has been suggested that whey acidic protein (WAP) may function as a protease inhibitor. However, the actual function of WAP remains obscure. We investigated the histological development of the mammary glands of transgenic mice ubiquitously expressing WAP and CAG/WAP transgene. Ubiquitous expression of WAP induced aberrant development of the lobular alveoli of the mammary glands: mammary alveoli that were either aberrantly large or small in size increased in number in the developing mammary glands of these transgenic females during pregnancy and lactation. The expression of beta-casein was precociously induced in the mammary glands of the transgenic females during early pregnancy and accompanying this was a histological observation that abnormally developed lobular alveoli filled with milk proteins appeared in the mammary glands of transgenic females during early pregnancy. However, during lactation, the development of mammary glands was impaired in transgenic females. To investigate the possible paracrine action of WAP associated with mammary gland aberration, we transplanted the mammary tissue of CAG/EGFP transgenic females into the fat pad of virgin CAG/WAP transgenic females and initiated pregnancy by mating. The development of mammary tissue transplanted to the recipient was histologically examined on day 3 of lactation. The results revealed that the development of grafted mammary tissues was impaired in a manner similar to that of the mammary glands of CAG/WAP transgenic females, indicating that the inhibitory effect of WAP acts via a paracrine mechanism. In vitro experiments using HC11 cells with forced expression of exogenous WAP demonstrated the inhibitory function of WAP on proliferation of mammary epithelial cells.

Animals↗