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PubMed1997

Upregulation of endogenous p53 and induction of in vivo apoptosis in B-lineage lymphomas of E(mu)-myc transgenic mice by deregulated c-myc transgene.

E(mu)-myc transgenic mice carry a constitutively overexpressed c-myc oncogene and develop B-lineage lymphomas. Previous studies have shown that c-myc overexpression can lead to in vitro apoptosis. Here, we investigated the in vivo effects of altered c-myc expression on cell proliferation versus death in spontaneously arising E(mu)-myc tumors. E(mu)-myc tumors display extensive in vivo apoptosis confined to small clusters of cells with greatly increased expression of both the c-myc transgene and the endogenous p53 gene as compared with that in normal, pretumor, or surrounding tumor tissue. This restricted overexpression of both the c-myc transgene and the endogenous p53 gene in small clusters of apoptotic tumor cells indicates that overexpression of these genes and apoptosis are not obligatory or uniform during tumor development and suggests that further somatic mutations or microenvironmental influences may be responsible for these properties. Nevertheless, the clear ability of tumor cells to undergo apoptosis in vivo may be exploitable for therapeutic purposes.

Animals↗

PubMed2006

Bean Yellow Dwarf Virus replicons for high-level transgene expression in transgenic plants and cell cultures.

A novel stable transgenic plant expression system was developed using elements of the replication machinery of Bean Yellow Dwarf Virus (BeYDV). The system contains two transgenes: 1) The BeYDV replicon vector with an expression cassette flanked by cis-acting DNA elements of BeYDV, and 2) The viral replication initiator protein (Rep) controlled by an alcohol-inducible promoter. When Rep expression was triggered by treatment with ethanol, it induced release of the BeYDV replicon from stably integrated T-DNA and episomal replication to high copy number. Replicon amplification resulted in substantially increased transgene mRNA levels (up to 80-fold) and translation products (up to 10-fold) after induction of Rep expression by ethanol treatment in tobacco NT1 cells and leaves of whole potato plants. Thus, the BeYDV stable transformant replicon system is a powerful tool for plant-based production of recombinant proteins.

Biotechnology↗

PubMed1995

A human (3.3 kb) haptoglobin-CAT transgene is modulated in lungs of transgenic mice by inflammation.

Four independent lines of transgenic mice were produced carrying integrated copies of a chimeric gene composed of 3.3 kb of the human haptoglobin 5' regulatory region fused to the CAT (chloramphenicol acetyl transferase) reporter gene. Although the endogenous mouse haptoglobin (Hp) and human haptoglobin (HP) genes express mainly in liver and lung, expression of the human 3.3-kb HP-CAT transgene was not detected until after induction of inflammation and then only in lungs. The results indicated that the transgene maintained the regulatory DNA elements required for lung specific responsiveness to inflammation in vivo but lacked the DNA sequence required for robust expression in liver. The DNA sequence(s) responsible for the normally high level of HP expression in liver either reside outside the 3.3-kb regulatory region of the HP chimeric gene or this region contains a suppressor sequence affecting tissue specific expression in the liver.

Animals↗

PubMed2002

Adenoviral-mediated, high-level, cell-specific transgene expression: a SYN1-WPRE cassette mediates increased transgene expression with no loss of neuron specificity.

Viral vectors are excellent tools for studying gene function in the brain, although a limitation has been the ability to effectively target transgene expression to specific neuronal populations. This generally cannot be overcome by the use of neuron-specific promoters, as most are too large to be used with current viral vectors and expression from these promoters is often relatively weak. We therefore developed a composite expression cassette, comprising 495 bp of the weak human SYN1 (synapsin-1) promoter and 800 bp of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Studies in hippocampal cultures, organotypic cultures, and in vivo showed that the 3' addition of the WPRE to the SYN1 element greatly increased enhanced green fluorescent protein expression levels with no loss of neuronal specificity. In vivo studies also showed that transgene expression was enhanced with no loss of neuronal specificity in dentate-gyrus neurons for at least 6 weeks following transfection. Therefore, unlike most powerful promoter systems, which mediate expression in neurons and glia, this SYN1-WPRE cassette can target powerful long-term transgene expression to central nervous system neurons when delivered at relatively low titers of adenovirus. Its use should therefore facilitate both gene therapy studies and investigations of neuronal gene function.

Adenoviridae↗

PubMed1992

Induced reciprocal translocation in transgenic mice near sites of transgene integration.

Transgenic mice (JCP0 #18), heterozygous for an insertion of approximately 50 copies of the rat peripheral myelin (P0) protein cDNA, displayed a pattern of reduced litter size that suggested a chromosome rearrangement. Chromosome banding studies of fetal cells disclosed the presence of an apparently balanced translocation between a Chromosome (Chr) 1 and 14 with breakpoints at bands 1H3 and 14C3. In situ hybridization of biotin-labeled P0 rat cDNA probe to chromosome spreads and detection of specific signal with fluorescein isothiocyanate-conjugated avidin revealed a strong signal on the 1(14) translocation chromosome at the site of the breakpoint. A weaker signal was present near the breakpoint on the 14(1) derivative chromosome. These results suggest an etiologic relationship between the insertion of the transgene and the origin of the translocation. To further elucidate possible mechanisms, we first mapped the endogenous P0 gene (gene symbol Mpp). As previously reported (You et al., Genomics 9: 751, 1991), we found that Mpp is located on Chr 1 in the region of the translocation breakpoint in JCP0 mice. Subsequently, we have carried out pulsed-field gel and standard Southern analyses with P0 gene probes, but found no evidence for a direct involvement of the endogenous P0 gene in the process that generated the balanced reciprocal translocation. Thus, we favor the hypothesis that, during repair of DNA strand breakage--possibly induced by the microinjection procedure--the transgene copies were ligated to broken ends of Chrs 1 and 14. According to convention, this translocation is designated T(1;14)1Po. Homozygotes are phenotypically normal and breed well; they will be useful for genetic and physical mapping of Chrs 1 and 14.

Animals↗

PubMed2025

Validation of caprine H11 and the Rosa26 platform for transgene integration via CRISPR-based system: investigations on stable transgene expression and genetic biosafety.

CRISPR/Cas9 technology is an efficient tool for site-specific livestock gene editing. However, to minimize potential disruption of host genome function, exogenous genes should be integrated into well-characterized genomic loci, such as H11 or Rosa26, which have been empirically validated for stable transgene expression. This study established a multi-dimensional assessment system to evaluate the biological applicability of the H11 locus and the widely used Rosa26 targeting platform as sites for targeted integration of exogenous genes in goats. Donor cells carrying the enhanced green fluorescent protein (EGFP) reporter gene at the H11 and Rosa26 loci were generated via CRISPR/Cas9-mediated homology-directed repair; this was followed by somatic cell nuclear transfer to produce transgenic cloned embryos and healthy offspring. Multi-dimensional analyses revealed the following. At the cellular level, there was stable and efficient EGFP expression at integration sites, with donor cells maintaining normal cell cycle progression, proliferation capacity, and apoptosis levels, and with no alterations in the transcriptional integrity of adjacent genes. At the embryonic level, there was sustained EGFP expression across pre-implantation embryonic stages, with developmental metrics statistically indistinguishable from wild-type embryos. Finally, at the individual level, cloned offspring exhibited growth phenotypes consistent with wild-type counterparts, and EGFP showed broad-spectrum expression in eight tissues. This study establishes the first CRISPR/Cas9-based crossscale (cellular-embryonic-individual) validation in goats, demonstrating that the H11 and Rosa26 loci support efficient and stable transgene integration in goats. These results provide a precise and predictable technical framework for livestock genetic improvement.

Animals↗

PubMed1991

Expression in brain sensory neurons of the transgene in transgenic mice carrying human tyrosine hydroxylase gene.

We have recently reported the production of transgenic (Tg) mice carrying the human tyrosine hydroxylase (TH) gene, and have described tissue-specific expression of the transgene in catecholaminergic (CAergic) neurons and adrenal glands. This paper describes the transgene expression in non-catecholaminergic (nCAergic) neurons in the brain of Tg mice by immunocytochemistry and in situ hybridization. In adult Tg mice, human TH was atypically expressed in the olfactory (typically, the anterior olfactory nucleus and pyriform cortex) and visual (typically, n. suprachiasmaticus and n. parabigeminalis) systems, in addition to typical CAergic neuron-rich nuclei in the brain. These results suggest the possibility that TH plays some novel roles in sensory systems.

Animals↗

PubMed1998

The in vivo mutagenicity and mutational spectrum at the lacI transgene recovered from the spleens of B6C3F1 lacI transgenic mice following a 4-week inhalation exposure to 1,3-butadiene.

1,3-Butadiene (BD) is carcinogenic and mutagenic in B6C3F1 mice. We determined the lacI mutant frequency and mutational spectrum in spleen following inhalation exposure to BD at levels that are known to induce tumors. B6C3F1 lacI transgenic mice were exposed to air or to 62.5, 625, or 1250 ppm BD for 4 weeks (6 h/day, 5 days/week) and euthanized 14 days after the last exposure. BD increased the lacI mutant frequency in spleen at all levels of BD examined. In BD-exposed mice, an increased frequency of G:C-->A:T transitions occurred at non-5'-CpG-3' sites. Exposure to BD in B6C3F1 lacI transgenic mice also increased the frequency of base substitution mutations that occurred at A:T base pairs when compared to air controls. The increased frequency of specific mutations at G:C base pairs in spleen was not observed in our previous studies in bone marrow and indicates tissue-specific differences in the BD-induced mutational spectrum. These data demonstrate that in vivo transgenic mouse mutagenicity assays can identify tissue-specific mutagenicity and mutational spectrum responses of genotoxic carcinogens at exposure levels that are known to induce tumors.

Administration, Inhalation↗

PubMed1997

Transgenic studies with a keratin promoter-driven growth hormone transgene: prospects for gene therapy.

Keratinocytes are potentially appealing vehicles for the delivery of secreted gene products because they can be transferred to human skin by the relatively simple procedure of grafting. Adult human keratinocytes can be efficiently propagated in culture with sufficient proliferative capacity to produce enough epidermis to cover the body surface of an average adult. However, the feasibility of delivering secreted proteins through skin grafting rests upon (i) the strength of the promoter in keratinocytes and (ii) the efficiency of protein transport through the basement membrane of the stratified epithelium and into the bloodstream. In this paper, we use transgenic technology to demonstrate that the activity of the human keratin 14 promoter remains high in adult skin and that keratinocyte-derived human growth hormone (hGH) can be produced, secreted, and transported to the bloodstream of mice with efficiency that is sufficient to exceed by an order of magnitude the circulating hGH concentration in growing children. Transgenic skin grafts from these adults continue to produce and secrete hGH stably, at approximately 1/10 physiological levels in the bloodstream of nontransgenic recipient mice. These studies underscore the utility of the keratin 14 promoter for expressing foreign transgenes in keratinocytes and demonstrate that keratinocytes can be used as effective vehicles for transporting factors to the bloodstream and for eliciting metabolic changes. These findings have important implications for considering the keratinocyte as a possible vehicle for gene therapy.

Adult↗

PubMed1997

Differential interaction of the human cholesteryl ester transfer protein with plasma high density lipoproteins (HDLs) from humans, control mice, and transgenic mice to human HDL apolipoproteins. Lack of lipid transfer inhibitory activity in transgenic mice expressing human apoA-I.

Plasma high density lipoproteins (HDLs) from humans, from transgenic mice to human apolipoprotein A-I (HuAITg mice), from transgenic mice to human apolipoprotein A-II (HuAIITg mice), from transgenic mice to human apolipoproteins A-I and A-II (HuAIAIITg mice), and from C57BL/6 control mice were isolated, and their ability to interact with the human cholesteryl ester transfer protein (CETP) was studied. Whereas cholesteryl ester transfer rates were gradually enhanced by the addition of moderate amounts of HDL from the different sources, striking differences appeared when HDL levels kept increasing beyond a maximal transfer value. Indeed, while a plateau value corresponding to maximal CETP activity was maintained when raising the concentration of HuAITg HDL and HuAIAIITg HDL, inhibitions could be observed with the highest levels of human, control mouse, and HuAIITg mouse HDL. The concentration-dependent inhibition of CETP activity could be reproduced by the addition of delipidated HDL apolipoproteins from control mice, but it was abolished by a 1-h preheating treatment at 56 degrees C. In contrast, no significant inhibition of CETP activity was observed with the delipidated protein moiety of HuAITg HDL, and cholesteryl ester transfer rates remained unchanged before and after a 1-h, 56 degrees C preheating step. Finally, the CETP-mediated transfer of radiolabeled cholesteryl esters from human low density lipoprotein to human HDL was significantly higher in the presence of lipoprotein-deficient plasma from HuAITg mice than in the presence of lipoprotein-deficient plasma from control mice. Interestingly, cholesteryl ester transfer rates measured with both control and HuAITg lipoprotein-deficient plasmas became remarkably similar following a 1-h, 56 degrees C preheating treatment. It is concluded that human, control mouse, and HuAIITg mouse HDL contain a heat-labile lipid transfer inhibitory activity that is absent from HDL of HuAITg and HuAIAIITg mice. Alterations in CETP-lipoprotein binding did not account for differential lipid transfer inhibitory activities.

Animals↗

PubMed2005

Analysis of rat insulin II promoter-ghrelin transgenic mice and rat glucagon promoter-ghrelin transgenic mice.

We developed and analyzed two types of transgenic mice: rat insulin II promoter-ghrelin transgenic (RIP-G Tg) and rat glucagon promoter-ghrelin transgenic mice (RGP-G Tg). The pancreatic tissue ghrelin concentration measured by C-terminal radioimmunoassay (RIA) and plasma desacyl ghrelin concentration of RIP-G Tg were about 1000 and 3.4 times higher than those of nontransgenic littermates, respectively. The pancreatic tissue n-octanoylated ghrelin concentration measured by N-terminal RIA and plasma n-octanoylated ghrelin concentration of RIP-G Tg were not distinguishable from those of nontransgenic littermates. RIP-G Tg showed suppression of glucose-stimulated insulin secretion. Arginine-stimulated insulin secretion, pancreatic insulin mRNA and peptide levels, beta cell mass, islet architecture, and GLUT2 and PDX-1 immunoreactivity in RIP-G Tg pancreas were not significantly different from those of nontransgenic littermates. Islet batch incubation study did not show suppression of insulin secretion of RIP-G Tg in vitro. The insulin tolerance test showed lower tendency of blood glucose levels in RIP-G Tg. Taking lower tendency of triglyceride level of RIP-G Tg into consideration, these results may indicate that the suppression of insulin secretion is likely due to the effect of desacyl ghrelin on insulin sensitivity. RGP-G Tg, in which the pancreatic tissue ghrelin concentration measured by C-RIA was about 50 times higher than that of nontransgenic littermates, showed no significant changes in insulin secretion, glucose metabolism, islet mass, and islet architecture. The present study raises the possibility that desacyl ghrelin may have influence on glucose metabolism.

Animals↗

PubMed2006

Immune regulation of transgene expression in the brain: B cells regulate an early phase of elimination of transgene expression from adenoviral vectors.

Cellular immune mechanisms that regulate viral gene expression within infected brain cells remain poorly understood. Previous work has shown that systemic immunization against adenovirus after vector delivery to the brain results in complete loss of brain cells infected by adenoviral vectors. Although T cells play an important role in this process, we demonstrate herein that B cells also significantly regulate transgene expression from the CNS. After the systemic immunization against adenovirus of animals injected via the brain with an adenoviral vector 30 days earlier, we uncovered substantial infiltration by CD19+ B cells of the area of the brain transduced by the virus. This suggests the involvement of B cells in the adaptive immune response-mediated loss of transduced cells from the brain. Confocal analysis of these brains demonstrated physical contacts between transduced brain cells and CD19+ cells. To test the hypothesis that B cells play a causal role in the loss of infected cells from the brain, we demonstrated that animals devoid of B cells were unable to eliminate transgene expression at early time points after immunization. This demonstrates that B cells play a necessary role in the loss of transgene expression at early, but not late, time points postimmunization. Thus, these data have important implications for our understanding of the role of B cells as immune effectors during the immune-mediated clearance of viral infections from the CNS, and also for understanding mechanisms operating in brain autoimmunity, as well as for the potential safety of clinical gene therapy for brain diseases.

Adenoviridae↗

PubMed2005

Long-term transgene expression and survival of transgene-expressing grafts following lentivirus transduction of bone marrow side population cells.

BACKGROUND: Successful transduction of hematopoietic stem cells is essential if gene therapy is to be used clinically to induce immunologic tolerance. METHODS: Hoechst 33342 staining was used to isolate a population of bone marrow cells enriched for stem cells, termed side population (SP) cells. Murine bone marrow SP cells were transduced with HLA-A2.1-expressing VSV-G-pseudotyped lentivirus or retrovirus vectors under identical conditions. RESULTS: After transduction without prestimulating cytokines, which minimizes cell cycling and helps maintain stem cell pluripotency, the HLA-A2.1 gene was found in the DNA of 56% of CFU-GM colonies derived from lentivirus-transduced SP cells, but in only 4% of colonies derived from retrovirus-transduced SP cells. Lentivirus and retrovirus transduction including cytokine prestimulation produced the same degree of integration as that following lentivirus-transduction of non-prestimulated cells. Transplantation of 5,000 lentivirus-transduced SP cells into lethally irradiated mice resulted in long-term expression of the HLA-A2.1 transgene in peripheral blood progeny of bone marrow SP cells and prolonged skin graft survival across this class I MHC barrier until the time of animal sacrifice. CONCLUSIONS: Recombinant lentivirus, but not retrovirus vectors, effectively transduced SP cells that were not prestimulated with cytokines and lentivirus-transduced SP cells successfully repopulated lethally irradiated C57BL/6 mice, animals where there is no selective advantage to repopulation with transduced cells. Transplantation of a relatively small number of transduced SP cells led to prolonged transgene mRNA expression and antigen-specific survival of grafts expressing the foreign MHC transgene.

Animals↗

PubMed2006

CD4-specific transgenic expression of human cyclin T1 markedly increases human immunodeficiency virus type 1 (HIV-1) production by CD4+ T lymphocytes and myeloid cells in mice transgenic for a provirus encoding a monocyte-tropic HIV-1 isolate.

Human immunodeficiency virus type 1 (HIV-1)-encoded Tat provides transcriptional activation critical for efficient HIV-1 replication by interacting with cyclin T1 and recruiting P-TEFb to efficiently elongate the nascent HIV transcript. Tat-mediated transcriptional activation in mice is precluded by species-specific structural differences that prevent Tat interaction with mouse cyclin T1 and severely compromise HIV-1 replication in mouse cells. We investigated whether transgenic mice expressing human cyclin T1 under the control of a murine CD4 promoter/enhancer cassette that directs gene expression to CD4(+) T lymphocytes and monocytes/macrophages (hu-cycT1 mice) would display Tat responsiveness in their CD4-expressing mouse cells and selectively increase HIV-1 production in this cellular population, which is infected primarily in HIV-1-positive individuals. To this end, we crossed hu-cycT1 mice with JR-CSF transgenic mice carrying the full-length HIV-1(JR-CSF) provirus under the control of the endogenous HIV-1 long terminal repeat and demonstrated that human cyclin T1 expression is sufficient to support Tat-mediated transactivation in primary mouse CD4 T lymphocytes and monocytes/macrophages and increases in vitro and in vivo HIV-1 production by these stimulated cells. Increased HIV-1 production by CD4(+) T lymphocytes was paralleled with their specific depletion in the peripheral blood of the JR-CSF/hu-cycT1 mice, which increased over time. In addition, increased HIV-1 transgene expression due to human cyclin T1 expression was associated with increased lipopolysaccharide-stimulated monocyte chemoattractant protein 1 production by JR-CSF mouse monocytes/macrophages in vitro. Therefore, the JR-CSF/hu-cycT1 mice should provide an improved mouse system for investigating the pathogenesis of various aspects of HIV-1-mediated disease and the efficacies of therapeutic interventions.

Animals↗

PubMed2000

Hemopoietic lineage commitment decisions: in vivo evidence from a transgenic mouse model harboring micro LCR-betapro-LacZ as a transgene.

A substantial body of published data suggests activation of lineage-specific genes in multipotential hemopoietic cells before their unilineage commitment. Because the behavior and plasticity of cells isolated in vitro away from microenvironmental constraints exercised in vivo may be altered, one wonders whether similar findings can be observed in a physiologic setting in vivo. We used a transgenic mouse model harboring human micro LCR together with beta promoter sequences as a transgene to examine activation of lineage-specific programs in vivo. By using LacZ as a reporter, we had the ability to detect, quantitate, and select live cells with different levels of LacZ activation. We found strong expression of LacZ by X-gal staining in 2 lineages-erythroid and megakaryocytic. Activation in the latter was a novel finding not previously observed when similar transgenes were used. We also found activation of muLCR-betapro at low levels in progenitor cells of granulocytic-macrophagic, erythroid, or megakaryocytic lineage detected by in vitro assays, suggesting activation before commitment to a specific lineage pathway. In particular, the expression of LacZ was graded among progenitors, so that in a proportion of them activation occurred only after commitment to erythroid or megakaryocytic lineage. In addition, we found quantitative reduction in LacZ expression between fetal liver and bone marrow-derived cells, the basis of which is unclear. Collectively our data provide in vivo evidence supporting the view that lineage-specific genes are expressed in a graded fashion in pluripotential cells before their irreversible unilineage commitment. (Blood. 2000;95:1274-1282)

Animals↗

PubMed1999

[Influence of chemotherapeutants and cytokines on growth and transgene expression of bone marrow cells from MT/p210(bcr-abl) transgenic mice].

The bone marrow cells of CML(chronic myelogenous leukemia) transgenic mice carrying metallothionein(MT) promoter/enhancer, bcr-abl(p210) cDNA and SV40 splicing/poly(A) signal sequences were cultured in liquid and soft agar with hydroxyurea (Hu), 5-fluorouracil(5-Fu), mouse stem cell factor (mSCF) and mouse interleukin-3(mIL-3) independently or combinatively. The cell or colony counting and transgene RT-PCR analysis showed that the proliferation, colony formation and transgene expression of the bone marrow cells were advanced in the combinative culture of mSCF with mIL-3, but they were significantly inhibited in the culture without any growth factors, or with mSCF, mIL-3 and Hu or 5-Fu. These findings suggest that the combinative utilization of chemotherapeutants and cytokines is a potentially effective strategy of the clinical treatment for CML.

Animals↗

PubMed1993

4-Hydroxynonenal content lower in brains of 25 month old transgenic mice carrying the human CuZn superoxide dismutase gene than in brains of their non-transgenic littermates.

Basic peroxidation in brains of normal 2 month old mice was compared to that in brains of 25 month old mice by measurement of 4-hydroxy-2-nonenal and protein carbonyl contents. No significant age-related differences in 4-hydroxy-2-nonenal were observed, showing that lipid peroxidation is not a determining factor in cerebral aging. However, protein carbonyls increased with age in brains, indicating accumulation of age-related cell constituent damage. The role of excess CuZn superoxide dismutase in basal lipid peroxidation in mouse brains was studied by comparison of eight 25 month old transgenic mice and eight non-transgenic littermates. Higher 4-hydroxy-2-nonenal concentrations were found in normal mouse brains. These data suggest an increased protective role of CuZn superoxide dismutase against lipid peroxidation in transgenic mouse brains.

Aging↗

PubMed1993

Progressive and regressive fate of lens tumors correlates with subtle differences in transgene expression in gamma F-crystallin-SV40 T antigen transgenic mice.

Regulatory elements of the mouse gamma F-crystallin gene were used to derive transgenic mice expressing SV40 large T antigen in terminally differentiating fiber cells of the ocular lens. The resulting gamma F-crystallin-T antigen mice developed either malignant or regressive lens tumors in a strain-dependent fashion. Developmental and RNA analyses revealed that in both 'tumor-progressing' and 'tumor-regressing' mouse strains expression of the transgene blocked morphological differentiation of lens fibers without appreciably affecting gamma-crystallin gene expression, a marker of terminal lens fiber cell differentiation. Strain-dependent differences in tumorigenic outcome could be correlated with both subtle differences in transgene expression and the ability of tumor cells to escape from the normal confines of the lens. The results implicate the importance of cellular environment to malignant tumor development and provide insight into those features of normal lens ontogeny that may render the lens refractory to the development of spontaneous tumors.

Animals↗