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PubMed1996

Variegated transgene expression in mouse mammary gland is determined by the transgene integration locus.

Mice carrying an ovine beta-lactoglobulin (BLG) transgene secrete BLG protein into their milk. To explore transgene expression stability, we studied expression levels in three BLG transgenic mouse lines. Unexpectedly, two lines exhibited variable levels of transgene expression. Copy number within lines appeared to be stable and there was no evidence of transgene rearrangement. In the most variable line, BLG production levels were stable within individual mice in two successive lactations. Backcrossing demonstrated that genetic background did not contribute significantly to variable expression. Tissue in situ hybridization revealed mosaicism of transgene expression within individual mammary glands from the two variable lines; in low expressors, discrete patches of cells expressing the transgene were observed. Transgene protein concentrations in milk reflected the proportion of epithelial cells expressing BLG mRNA. Furthermore, chromosomal in situ hybridization revealed that transgene arrays in both lines are situated close to the centromere. We propose that mosaicism of transgene expression is a consequence of the chromosomal location and/or the nature of the primary transgene integration event.

Animals↗

PubMed1995

Frequent deletions and sequence aberrations at the transgene junctions of transgenic mice carrying the papillomavirus regulatory and the SV40 TAg gene sequences.

Exogenous DNA microinjected into one-cell mouse zygotes either integrates into the host genome within a short time span, or is rapidly degraded. On integration, a transgene sequence is frequently reiterated. In this report, we describe the enzymatic amplification analysis of transgene junctions of 12 transgenic mice carrying different copy numbers of the same transgene with dissimilar ends. The transgene was composed of the regulatory sequence of the type 18 human papillomavirus linked to the TAg gene of the SV40 virus. Nucleotide sequences of 36 of these junctions were also determined. Deletions were found in 33 (91.7%) of the junctions analysed. At the crossover regions, 55.6% contained short overlapping sequences of one to six nucleotides. Insertions of 2-6 extraneous nucleotides were also found in 8.3% of the transgene junctions. Within a 10-nucleotide sequence on both sides of the transgene junctions, topoisomerase I (topo I) cleavage sites, runs of homogeneous purines or pyrimidiens, alternating purine-pyrimidine tracks and (A-T)-rich sequences were found frequently. Stringent control experiments were also performed to ascertain that the observations made were not artefacts resulting from the polymerase chain reaction. Our data therefore indicate that damage had occurred quite frequently and extensively in our transgene construct. Such transgene damage may also occur to various extents in mice carrying other transgenes. Primary structure of the nucleotide sequences of the injected DNA seems to influence the process of transgene reiteration and aberration.

Animals↗

PubMed1992

Transgenes expressing the Wnt-1 and int-2 proto-oncogenes cooperate during mammary carcinogenesis in doubly transgenic mice.

The Wnt-1 and int-2 proto-oncogenes are transcriptionally activated by mouse mammary tumor virus insertion mutations in virus-induced tumors and encode secretory glycoproteins. To determine whether these two genes can cooperate during carcinogenesis, we have crossed two previously characterized lines of transgenic mice to obtain bitransgenic animals carrying both Wnt-1 and int-2 transgenes under the control of the mouse mammary tumor virus long terminal repeat. Mammary carcinomas appear earlier and with higher frequency in the bitransgenic animals, especially the males, than in either parental line. Nearly all bitransgenic males develop mammary neoplasms within 8 months of birth, whereas only 15% of Wnt-1 transgenic males and none of the int-2 transgenic males have tumors. In virgin bitransgenic females, tumors occur approximately 2 months earlier than in their Wnt-1 transgenic siblings; int-2 transgenic females rarely exhibit tumors. Preneoplastic glands from the bitransgenic animals of either sex demonstrate pronounced epithelial hyperplasia similar to that seen in Wnt-1 transgenic virgin females and males, and both transgenes are expressed in the hyperplastic glands and mammary tumors. RNA from the int-2 transgene is more abundant in mammary glands from bitransgenic animals than from int-2 transgenic animals; the increase is associated with high levels of RNA specific for keratin genes 14 and 18, suggesting that Wnt-1-induced epithelial hyperplasia is responsible for the observed increase in expression of the int-2 transgene.

Animals↗

PubMed1997

Nephropathy in human immunodeficiency virus-1 transgenic mice is due to renal transgene expression.

HIV-associated nephropathy (HIVAN) is a progressive glomerular and tubular disease that is increasingly common in AIDS patients and one of the leading causes of end stage renal disease in African Americans. A major unresolved issue in the pathogenesis of HIVAN is whether the kidney disease is due to renal cell infection or a "bystander" phenomenon mediated by systemically dysregulated cytokines. To address this issue, we have used two different experimental approaches and an HIV-1 transgenic mouse line that develops a progressive renal disease histologically similar to HIVAN in humans. In the murine model, kidney tissue expresses the transgene and in heterozygous adults, renal disease develops shortly thereafter. We demonstrate by terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labeling assay that similar to the disease in humans, apoptosis of renal tubular epithelial cells is a component of the molecular pathogenesis. To determine whether apoptosis is due to transgene expression or environmental factors, we treated fetal kidney explants (normal and transgenic) with UV light to induce transgene expression. Apoptosis occurred in transgenic but not normal littermates after stimulation of transgene expression. To confirm a direct effect of HIV expression on the production of HIVAN, we transplanted kidneys between normal and transgenic mice. HIVAN developed in transgenic kidneys transplanted into nontransgenic littermates. Normal kidneys remained disease free when transplanted into transgenic littermates. Thus, the renal disease in the murine model is intrinsic to the kidney. Using two different experimental approaches, we demonstrate a direct effect of transgene expression on the development of HIVAN in the mouse. These studies suggest that in humans, a direct effect of HIV-1 expression is likely the essential cause of HIVAN, rather than an indirect effect of cytokine dysregulation.

AIDS-Associated Nephropathy↗

PubMed2000

Determination of transgene repeat formation and promoter methylation in transgenic plants.

The integration of transgenes into a plant host genome following Agrobacterium tumefaciens-mediated or direct transformation may occur as a single copy or in the form of tandem repeats. The latter has been associated with promoter methylation and silencing of transgenes. Thus, the early screening of such transgenic plants is desirable for ruling out future repeat-dependent transgene instability. We developed a simple PCR-based method in which primer pairs were specifically designed so that amplifications could only be obtained if the transgene was present in the form of multiple inserts in a transgenic line. The method was established using 35S-rolC transgenic aspen lines showing morphologically visible transgenic silencing. Later, it was possible to screen independent transgenic lines showing no visible marker gene expression. Furthermore, a method was developed in which positive PCR amplification was indicative of promoter methylation. The results were consistent and reproducible across different independent transgenic lines. The methods were quick, reliable, consistent and reproducible, and can be useful for routine screening of transgene silencing in lines derived from many different systems.

DNA Methylation↗

PubMed1992

Transgenic mice carrying intact HIV provirus: biological effects and organization of a transgene.

Twelve transgenic founder animals retaining intact copies of the infectious molecular clone of human immunodeficiency virus (HIV)-1 were obtained. All the founders appeared healthy during a 9- to 12-month observation period. However, transgenic offspring of one of the founders (female #13), died within the 1st month of life while manifesting several symptoms characteristic of human AIDS. To discover why only one transgenic lineage was affected and why the founder animal in the affected lineage remained healthy while all of her transgenic offspring were diseased, we compared the organization of the transgene in the transgenic lineages. Restriction enzyme analysis showed that the founder no. 13 was a mosaic carrying in each transgenic cell four tandemly arranged copies of the infectious molecular clone. All the units of the tandem repeat appeared to be correctly preserved with the exception of the 3'-most copy, which terminated near the start of human sequences that flank the 3' long terminal repeat (LTR). The unaffected founders and their transgenic litter usually carried a high number of copies of the provirus. The 5' terminus of the transgene in the unaffected animals appeared to be deleted or rearranged. None of the 12 transgenic founders carried a single copy of integrated provirus. We conclude that infectious molecular clone of HIV-1 can be expressed in transgenic mice, and that the mode of proviral integration similar to that seen during the retroviral infectious cycle (i.e., a single-copy provirus) may be incompatible with the postnatal survival.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2004

Characterization of a CD46 transgenic pig and protection of transgenic kidneys against hyperacute rejection in non-immunosuppressed baboons.

Human membrane cofactor protein (CD46) controls complement activation and when expressed sufficiently as a transgene protects xenografts against complement-mediated rejection, as shown here using non-immunosuppressed baboons and heterotopic CD46 transgenic pig kidney xenografts. This report is of a carefully engineered transgene that enables high-level CD46 expression. A novel CD46 minigene was validated by transfection and production of a transgenic pig line. Pig lymphocytes were tested for resistance to antibody and complement-mediated lysis, transgenic tissues were characterized for CD46 expression, and kidneys were transplanted to baboons without immunosuppression. Absorption of anti-Galalpha(1,3)Gal epitope (anti-GAL) serum antibodies was measured. Transgenic pigs expressed high levels of CD46 in all tissues, especially vascular endothelium, with stable expression through three generations that was readily monitored by flow cytometry of transgenic peripheral blood mononuclear cells (PBMC). Transgenic PBMC pre-sensitized with antibody were highly resistant to human complement-mediated lysis which readily lysed normal pig PBMC. Normal pig kidneys transplanted without cold ischemia into non-immunosuppressed adult baboons survived a median of 3.5 h (n = 7) whereas transgenic grafts (n = 9), harvested at approximately 24-h intervals, were either macroscopically normal (at 29, 48 and 68 h) or showed limited macroscopic damage (median > 50 h). Microscopic assessment of transplanted transgenic kidneys showed only focal tubular infarcts with viable renal tissue elsewhere, no endothelial swelling or polymorph adherence and infiltration by lymphocytes beginning at 3 days. Coagulopathy was not a feature of the histology in four kidneys not rejected and assessed at 48 h or later after transplantation. Baboon anti-GAL serum antibody titers were high before transplantation and, in one extensively analyzed recipient, reduced approximately 8-fold within 5.5 h. The data demonstrate that a single CD46 transgene controls hyperacute kidney graft rejection in untreated baboons despite the presence of antibody and complement deposition. The expression levels, tissue distribution and in vitro functional tests indicate highly efficient CD46 function, controlling both classical and alternative pathway complement activation, which suggests it might be the complement regulator of choice to protect xenografts.

Acute Disease↗

PubMed2006

[The use of transgenic animals in biomedical research in Germany. Part 2: Ethical evaluation of the use of transgenic animals in biomedical research and perspectives for the changeover in research to research animal-free methods].

As a rule, transgenic animals are being used in in vivo experiments to examine gene functions, their regulation or the contribution of genetic alterations to the development of diseases. Many transgenic animals already are affected in their wellbeing due to the genetic modification alone regardless of the procedures performed with them. Moreover, it is to be questioned whether the experimental use of transgenic animals led to results that were of such outstanding scientific relevance that they legitimated the suffering of the animals. In order to point to possible approaches to avoiding the use of transgenic animals in the areas of research identified, subsequent investigations aimed at collecting information on non-animal test methods that might be applied in pursuing the aforesaid questions. In particular, these were non-animal test methods that make use of genetic techniques. Amongst these are in vitro cell culture methods with genetically modified cells, such as the so called Transfected Cell Array, as well as in vitro test methods, in which specifically targeted genes can be turned on or off selectively for example by the so-called RNA interference technique or by antisense oligonucleotide genes. Since such technologies can also be applied to cell cultures with human cells, investigations with these methods enable direct information on the function of human genes. Even though a one to one replacement of animal experiments with transgenic animals by non-animal test methods is considered unlikely, from the point of view of animal welfare the broad spectrum of already available non animal test methods with which to study the function of genes and genetically caused pathophysiological reactions proves that waiving of animal tests with transgenic animals is possible without impeding biomedical research. Even if it cannot be totally excluded that some very specific questions linked to the respective animal experiment might not be pursued for the time being, nevertheless research that would be restricted to modern and ethically acceptable in vitro test methods would certainly conceive its very own questions to pursue and solve the problems currently faced by biomedical research. It is against this background that it is to be welcomed that the German Federal Government currently actively promotes the further development of genetechnological non-animal test methods. In order to ensure that these funding measures will make an effective contribution to reducing animal experiments, as spelled out by the government itself, the conversion of genetechnological research, just like biomedical research as a whole, to non-animal testing methods should be supported by concrete political actions. From the point of view of the German Animal Welfare Federation the following issues are to be requested: (1)In order to enable a fast and comprehensive advancement of promising genetechnological non-animal test methods, it should be ensured that public funding is provided with an adequate budget and over a sufficiently long period of time. (2)The legislator should initiate broad discussions on the question if society would be willing to dispense with certain pieces of knowledge if they would necessarily have to be gained at the expense of a certain degree of animal suffering. As the case may be, in the German Animal Welfare Act it should be laid down that certain procedures should not be considered acceptable as such. (3)As long as animal experiments with transgenic animals continue to be performed, concrete legal measures should be laid down in the German Animal Welfare Act to ensure that the distress of the animals (taking into account all factors relevant for transgenic animals) and the expected benefit of the research project are determined objectively so that the outcome of the ethical evaluation process becomes comprehensible. (4)The legislator should provide the authorities responsible for the licensing of research projects with concrete instructions in order to ensure that all aspects relevant for the welfare of the animals are fully taken into account when evaluating the ethical acceptability and scientific indispensability of projects and that special attention is given to research projects with transgenic animals. (5)The German Decree on the Reporting of Laboratory Animals should be amended to ensure that all individual transgenic animals are included in the official statistical reports regardless of whether they end up being used in scientific procedures or not. From the point of view of animal welfare it is possible to redesign biomedical research to do without transgenic animals without impeding necessary scientific progress. The survey in hand sought to make a contribution to providing a scientifically sound background for initiating these discussions.

Animal Testing Alternatives↗

PubMed1996

Chemically induced lung and forestomach neoplasias in transgenic mice carry mutant forms of the human c-Ha-ras transgene.

Susceptibility to lung carcinogens and genetic changes in neoplastic lesions were investigated in transgenic mice carrying a human hybrid c-Ha-ras gene, encoding a prototype p21 gene product. Nine-week-old male and female transgenic mice and non-transgenic littermates were injected i.p. with 6-nitrochrysene (6NC) three times biweekly or administered urethane in their drinking water for 3 weeks. Control mice were given dimethylsulfoxide (DMSO), the solvent for 6NC, alone. The incidences of lung adenocarcinomas were four out of seven female (57%) transgenic mice treated with 6NC and three out of three males (100%) and three out of three females (100%) receiving urethane. No adenocarcinomas were observed in control animals or non-transgenic mice. Adenomas developed in all treated groups, but the incidence and multiplicity were higher in transgenic animals than in their non-transgenic counterparts. In the 6NC-treated group, forestomach papillomas and squamous cell carcinomas were also observed in both male (25 and 50%) and female (56 and 33%) transgenic mice. PCR-SSCP and DNA sequence analysis of these induced lesions revealed point mutations at codon 61 of transgenic human c-Has-ras, from CAG (Gln) to CTG (Leu) or CAG (Gln) to AAG (Lyn) in lung hyperplasias (two out of three), an adenoma (one out of two), adenocarcinomas (five out of seven) and forestomach squamous cell carcinomas (four out of five). Mutations were not observed in forestomach papillomas. No changes in mouse Ha-ras or Ki-ras were found in any lesions. Furthermore, p21 overexpression was not evident in lung or forestomach tumors on immunohistochemical analysis. These findings indicate a high sensitivity to lung carcinogens in transgenic mice carrying the human c-Ha-ras gene and that this might be effected by mutational activation.

Animals↗

PubMed1995

Different 5'-flanking regions of the inhibin-alpha gene target transgenes to the gonad and adrenal in an age-dependent manner in transgenic mice.

The inhibin-alpha gene is expressed in a tissue-specific manner, and its protein product dimerizes with one of two beta-subunits to form bioactive heterodimers. To characterize the cis-acting elements involved in directing gonad- and adrenal-specific expression of inhibin-alpha, transgenic mice were generated that carried 2.5 or 6 kilobases (kb) of the 5'-flanking region of the mouse inhibin-alpha gene driving the human bcl-2 complementary DNA. Using an antibody specific for human Bcl-2, Western blotting and immunocytochemical analyses showed that both enhancer/promoter fragments direct transgene expression to the ovary, testis, and adrenal gland. The 6-kb fragment targeted the ovarian transgene expression in interstitial cells and young corpora lutea as well as granulosa and thecal cells of secondary, antral, and preovulatory follicles. In ovaries of animals with the 2.5-kb fragment, transgene expression was also detected in interstitial cells and young corpora lutea, but only in granulosa and thecal cells from antral and preovulatory follicles. The ovarian transgene expression in animals carrying the 6-kb inhibin-alpha promoter/bcl-2 construct was stimulated by gonadotropin treatment, with greater than 10-fold increases observed 2 days after PMSG stimulation. In the testes of both types of transgenic animals, immunoreactive Bcl-2 was predominantly detected in Sertoli cells of seminiferous tubules. Sporadic expression was also observed in some interstitial cells. In the adrenal gland, reporter protein was detected in the zona fasciculata of both types of transgenic animals during adult life; however, transgene expression was detected in zona fasciculata of young (21-day-old) animals with the 6-kb, but not the 2.5-kb, promoter construct. Thus, the 2.5-kb inhibin-alpha 5'-proximal DNA sequence directs transgene expression in mature ovarian follicles and testicular Sertoli cells. In contrast, enhancer elements in the 6-kb fragment are required for expression in preantral follicles and in the adrenal of immature animals. The inhibin-alpha promoter/enhancer used here represents unique DNA sequences for ovarian-specific transgene expression and is useful for future analysis of gonadal and adrenal cell functions.

Adrenal Glands↗

PubMed1999

Overexpression of spermidine/spermine N1-acetyltransferase under the control of mouse metallothionein I promoter in transgenic mice: evidence for a striking post-transcriptional regulation of transgene expression by a polyamine analogue.

We recently generated a transgenic mouse line overexpressing spermidine/spermine N1-acetyltransferase (SSAT) gene under its own promoter. The tissue polyamine pools of these animals were profoundly affected and the mice were hairless from early age. We have now generated another transgenic-mouse line overexpressing the SSAT gene under the control of a heavy-metal-inducible mouse metallothionein I (MT) promoter. Even in the absence of heavy metals, changes in the tissue polyamine pools indicated that a marked activation of polyamine catabolism had occurred in the transgenic animals. As with the SSAT transgenic mice generated previously, the mice of the new line (MT-SSAT) suffered permanent hair loss, but this occurred considerably later than in the previous SSAT transgenic animals. Liver was the most affected tissue in the MT-SSAT transgenic animals, revealed by putrescine overaccumulation, significant decrease in spermidine concentration and >90% reduction in the spermine pool. Even though hepatic SSAT mRNA accumulated to massive levels in non-induced transgenic animals, SSAT activity was only moderately elevated. Administration of ZnSO4 further elevated the level of hepatic SSAT message and induced enzyme activity, but not more than 2- to 3-fold. Treatment of the transgenic animals with the polyamine analogue N1,N11-diethylnorspermine (DENSPM) resulted in an immense induction, more than 40000-fold, of enzyme activity in the liver of transgenic animals, and minor changes in the SSAT mRNA level. Liver spermidine and spermine pools were virtually depleted within 1-2 days in response to the treatment with the analogue. The treatment also resulted in a marked mortality (up to 60%) among the transgenic animals which showed ultrastructural changes in the liver, most notably mitochondrial swelling, one of the earliest signs of cell injury. These results indicated that, even without its own promoter, SSAT is powerfully induced by the polyamine analogue through a mechanism that appears to involve a direct translational and/or heterogenous nuclear RNA processing control. It is likewise significant that overexpression of SSAT renders the animals extremely sensitive to polyamine analogues.

Acetyltransferases↗

PubMed2003

Additive transgene expression and genetic introgression in multiple green-fluorescent protein transgenic crop x weed hybrid generations.

The level of transgene expression in crop x weed hybrids and the degree to which crop-specific genes are integrated into hybrid populations are important factors in assessing the potential ecological and agricultural risks of gene flow associated with genetic engineering. The average transgene zygosity and genetic structure of transgenic hybrid populations change with the progression of generations, and the green fluorescent protein (GFP) transgene is an ideal marker to quantify transgene expression in advancing populations. The homozygous T(1) single-locus insert GFP/ Bacillus thuringiensis (Bt) transgenic canola ( Brassica napus, cv Westar) with two copies of the transgene fluoresced twice as much as hemizygous individuals with only one copy of the transgene. These data indicate that the expression of the GFP gene was additive, and fluorescence could be used to determine zygosity status. Several hybrid generations (BC(1)F(1), BC(2)F(1)) were produced by backcrossing various GFP/Bt transgenic canola ( B. napus, cv Westar) and birdseed rape ( Brassica rapa) hybrid generations onto B. rapa. Intercrossed generations (BC(2)F(2) Bulk) were generated by crossing BC(2)F(1) individuals in the presence of a pollinating insect ( Musca domestica L.). The ploidy of plants in the BC(2)F(2) Bulk hybrid generation was identical to the weedy parental species, B. rapa. AFLP analysis was used to quantify the degree of B. napus introgression into multiple backcross hybrid generations with B. rapa. The F(1) hybrid generations contained 95-97% of the B. napus-specific AFLP markers, and each successive backcross generation demonstrated a reduction of markers resulting in the 15-29% presence in the BC(2)F(2) Bulk population. Average fluorescence of each successive hybrid generation was analyzed, and homozygous canola lines and hybrid populations that contained individuals homozygous for GFP (BC(2)F(2) Bulk) demonstrated significantly higher fluorescence than hemizygous hybrid generations (F(1), BC(1)F(1) and BC(2)F(1)). These data demonstrate that the formation of homozygous individuals within hybrid populations increases the average level of transgene expression as generations progress. This phenomenon must be considered in the development of risk-management strategies.

Crops, Agricultural↗

PubMed2001

Progressive, age-related behavioral impairments in transgenic mice carrying both mutant amyloid precursor protein and presenilin-1 transgenes.

This study provides a comprehensive behavioral characterization during aging of transgenic mice bearing both presenilin-1 (PS1) and amyloid precursor protein (APP(670,671)) mutations. Doubly transgenic mice and non-transgenic controls were evaluated at ages wherein beta-amyloid (Abeta) neuropathology in APP+PS1 mice is low (5-7 months) or very extensive (15-17 months). Progressive cognitive impairment was observed in transgenic mice for both water maze acquisition and radial arm water maze working memory. However, transgenicity did not affect Y-maze alternations, circular platform performance, standard water maze retention, or visible platform recognition at either age, nor did transgenicity affect anxiety levels in elevated plus-maze testing. In sensorimotor tasks, transgenic mice showed a progressive increase in open field activity, a progressive impairment in string agility, and an early-onset impairment in balance beam. None of these sensorimotor changes appeared to be contributory to any cognitive impairments observed, however. Non-transgenic mice showed no progressive behavioral change in any measure evaluated. Given the age-related cognitive impairments presently observed in APP+PS1 transgenic mice and their progressive Abeta deposition/neuroinflammation, Abeta neuropathology could be involved in these progressive cognitive impairments. As such, the APP+PS1 transgenic mouse offers unique opportunities to develop therapeutics to treat or prevent Alzheimer's Disease through modulation of Abeta deposition/neuroinflammation.

Aging↗

PubMed2003

Analysis of cell-specificity and variegation of transgene expression driven by salmon prolactin promoter in stable lines of transgenic rainbow trout.

In order to identify the specificity and functionality of salmon prolactin (sPRL) promoter, transgenic rainbow trout carrying a construct comprising the 2.4 kb fragment of the 5' flanking region of Atlantic Chinook sPRL gene fused either to the reporter genes cat (sPRL-cat) or lacZ (sPRL-lacZ) were produced. sPRL-cat in transgenic F0 fish expressed strongly CAT only in the pituitary gland. Transgenic in F1-F4 lines harbouring sPRL-lacZ expressed beta-galactosidase (beta-gal) only in the follicular PRL-producing cells of the adenohypophysis. We observed heterocellular, mosaic distribution of beta-gal within PRL cell population and enormous variation of lacZ expression level between the littermates in the same transgenic line. Regardless of the transgene copy number, age or sex of transgenic fish, beta-gal expression was lactotroph-specific but variegated in all the nine F2 hemizygous lines analysed. One line harbouring a multicopy integration was followed up to F4 generation: the transgene was transmitted without modifications. Analysis of genomic DNA from pituitaries showed that lacZ sequences were highly methylated. LacZ expression was low and its transcripts, analysed by in situ hybridisation, showed a mosaic distribution within the pituitary gland. These data suggest that variegated expression of lacZ can occur at the transcription level owing to the silencing effect of lacZ gene. After proving the tissue-specific expression of reporter genes driven by the sPRL promoter, we tried to obtain the genetic ablation of PRL-producing cells,by transferring the same construct comprising diphtheria toxin DT-A gene (tox). However, the high mortality rate of sPRL-tox transformed embryos has embedded this study and no transgenic fish expressing tox were produced. The appropriateness of using transgenic strategies to analyse gene function in Salmonids is discussed, especially the implications of the multicopy integration patterns and of the variegated transgene expression.

Animals↗

PubMed1993

Frequent deletion of the transgene in T cell receptor beta chain transgenic mice.

TCR V beta 8.1 transgenic mice were generated using a genomic TCR V beta gene construct under the control of its promoter and enhancer. Among three lines of transgenic mice, one line expressed the transgenic TCR on only approximately 70% of peripheral T cells, while the other two lines expressed it on almost all mature T cells. T cells which lacked expression of the transgenic TCR beta chain expressed endogenous TCR beta chains. The molecular basis underlying the lack of transgene expression in T cells of this line of transgenic mice was investigated. The transgenic TCR- cells were isolated by two methods. First, Thy-1+ V beta 8.1/8.2- cells were purified from peripheral T cells using cell sorting. Second, transgenic TCR- T cell clones were established. In both cases, Southern blotting indicated that V beta 8.1- T cells had deleted the transgenic TCR gene. Thus, deletion of the transgenic TCR can occur in a high proportion of T cells, which allows rearrangement and expression of endogenous TCR beta chains.

Animals↗

PubMed1993

Induction of interleukin 2-responsiveness in thymocytes of the transgenic mice carrying lck-transgene.

The role of lck gene in T cell proliferation and differentiation was investigated with transgenic mice carrying human lck cDNA whose expression was regulated by the promoter of mouse H-2Kb and the enhancer element of mouse IgH. RNase protection assay revealed that the lck transgene was expressed in the thymus and spleen, whereas immunoblot analysis demonstrated that amounts of p56lck in freshly isolated lymphoid organs were almost equal between transgenic mice and negative littermates. Cell-surface marker analyses of the thymocytes and peripheral lymphocytes revealed no remarkable difference between both groups. Notable finding is that the thymocytes from transgenic mice showed a significant proliferative response to the stimulation with IL-2, but not the thymocytes from negative littermates. Further analysis revealed that CD4+8- single positive thymocytes proliferated in response to IL-2. While surface expression levels of IL-2R alpha and IL-2R beta of these CD4+8- thymocytes from transgenic and control mice were almost equal before stimulation with IL-2, the expression of IL-2R beta was induced only in transgenic thymocytes after stimulation with IL-2. Immunoblot analysis demonstrated that the expression of p56lck of transgenic thymocytes was not down-regulated at 4 hr after stimulation with IL-2, whereas p56lck of control ones were not detectable any more at 4 hr after stimulation with IL-2. Moreover, in vitro kinase assay substantiated such unchanged expression of p56lck in the thymocytes from transgenic mice: the kinase activities of p56lck did not decrease in thymocytes from transgenic mice after stimulation with IL-2, while kinase activities of control ones were significantly down-regulated by stimulation of IL-2. These results suggested that a significant proliferative response found in the thymocytes from lck-transgenic mice after the stimulation with IL-2 was caused by a constitutive expression of p56lck in these thymocytes even after the stimulation. Our findings, therefore, support a possibility that p56lck may play a role in the IL-2R-mediated signaling system in CD4+8- thymocytes.

Animals↗

PubMed2005

Conditional transgenic system for mouse aurora a kinase: degradation by the ubiquitin proteasome pathway controls the level of the transgenic protein.

Aurora A is a mitotic kinase that localizes to centrosomes. Expression of this protein is normally limited to the mitotic stage (G(2)-M) of the cell cycle, whereas human cancer cells frequently exhibit overexpression of Aurora A protein regardless of the cell cycle stage. In the present study, Aurora A transgenic mouse lines were generated with a new conditional expression system (cytomegalovirus immediate early enhancer-chicken beta-actin hybrid promoter-Z-enhanced green fluorescent protein) in order to analyze the function of this protein. Although transcripts for Aurora A were elevated in multiple organs of the transgenic mice, the corresponding protein was not detected in extracts analyzed by immunoblotting. The treatment of transgenic-derived embryonic fibroblasts (MEF) with proteasome inhibitors markedly increased the protein level of transgenic Aurora A, indicating that the transgenic Aurora A protein is readily degraded in normal mouse tissues. Under the exponential growth conditions of MEF cells, transgenic Aurora A was detected within the mitotic stage of the cell cycle and localized to centrosomes. In contrast, the marker of the transgenic promoter (enhanced green fluorescent protein) was continuously expressed throughout the cell cycle, indicating the constitutive transcription of transgenic mRNA. These results indicate that transgenic Aurora A is protected from degradation within G(2)-M but is immediately degraded after translation in the G(1)-S stage of the cell cycle. The findings obtained with this transgenic model and derived cells support that the transition from protection to degradation by the ubiquitin proteasome system at the end of mitosis is an important step in controlling the level of Aurora A protein during the cell cycle.

Animals↗

PubMed2002

Chromosome assignment of Cd36 transgenes in two rat SHR lines by FISH and linkage mapping of transgenic insert in the SHR-TG19 line.

The chromosome position of the Cd36 insert was determined by FISH in two rat transgenic lines (SHR/Ola-TgN(EF1aCd36)10Ipcv (SHR-TG10) and SHR/Ola-TgN(EF1aCd36)19Ipcv (SHR-TG19). The Cd36 transgene construct labelled with digoxigenin-11-dNTP was used as a probe in the FISH analysis. In accord with the previous finding that the SHR-TG10 harbours 6-8 copies of the transgene, the signals from both metaphase and interphase nuclei of SHR-TG10 preparations were rather strong and the probe hybridized to both copies of chromosome 1 at band q55. The probe hybridization to SHR-TG19 metaphase preparations also showed homozygosity of the transgene with localization of both copies to chromosome 11 at band q11. The signals were distinct but much weaker compared to the SHR-TG10, which again is in accord with the fact that the SHR-TG19 line harbours only a single copy of the transgene. In order to look for a possible impact of the insertion site neighbourhood upon the transgene phenotypic effect, we performed linkage mapping of the transgene in the SHR-TG19 line. By linkage mapping, the placement of the transgene to the proximal part of RNO11 was confirmed, the critical interval being 4 cM between D11Rat20 and D11Rat21, in good agreement with the RH map. Within the close neighbourhood of the inserted Cd36 transgene, there are several genes known to be expressed in kidney, and so the influence of some regulatory sequences enhancing kidney expression of the Cd36 transgene can be envisaged.

Animals↗