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At least 577 records · Page 32Linked to original sources
PubMed2004

Colorado potato beetles compensate for tomato cathepsin D inhibitor expressed in transgenic potato.

We reported earlier the importance of digestive cathepsin D-like activity for initiating dietary protein hydrolysis in Colorado potato beetle, Leptinotarsa decemlineata Say [Brunelle et al. (1999) Arch. Insect Biochem. Physiol. 42:88-98]. We assessed here whether transgenic lines of potato (Solanum tuberosum L.) expressing a cathepsin D inhibitor (CDI) from tomato would show resistance to the beetle, or if the insect would compensate for the loss of cathepsin D activity after ingesting the recombinant inhibitor. Transgenic potato lines expressing tomato CDI were developed by Agrobacterium tumefaciens genetic transformation, and selected based on their relative amount of CDI. After confirming the absence of detectable visible effects of CDI on the plant's phenotype, diet assays with control and transgenic lines were carried out to assess the impact of the inhibitor on growth and development of the insect. Leaf consumption, relative growth rate, molting incidence, and digestive protease activity were monitored at 12-h intervals over 132 h for 3rd-instar larvae provided with transgenic potato foliage. Leaf consumption and relative growth rate were slightly reduced during the first 12 h for larvae fed CDI, but no significant differences were observed thereafter. In contrast, time for molting to the 4th larval stage was significantly longer for larvae fed modified plants, with developmental delays of approximately 10 h (0.5 day) compared to control larvae. Recombinant CDI also had an impact on the insect's digestive physiology, readily inducing overproduction of digestive proteases (rubiscases), followed by a gradual decrease of total and pepstatin-sensitive activity. Overall, these observations show the ability of Colorado potato beetle to compensate for the loss of cathepsin D activity by modulating its digestive protease complement in response to aspartate-type inhibitors in the diet. From a practical viewpoint, these data stress the importance of devising improved strategies for the effective inhibition of insect digestive proteinases in vivo, based on the use of hybrid inhibitors active against different protease classes.

Animal Feed↗
PubMed2005

A 7.1 kbp beta-myosin heavy chain promoter, efficient for green fluorescent protein expression, probably induces lethality when overexpressing a mutated transforming growth factor-beta type II receptor in transgenic mice.

The roles of transforming growth factor-beta (TGFbeta) in heart or skeletal muscle development and physiology are still the subject of controversies. Our aim was to block, in transgenic mice, the TGFbeta signalling pathway by a dominant negative mutant of the TGFbeta type II receptor fused to the enhanced green fluorescent protein (TbetaRII-KR-EGFP) under the control of a 7.1 kbp mouse beta-myosin heavy chain (betaMHC) promoter to investigate the roles of TGFbeta in the heart and slow skeletal muscles. First, we generated two transgenic lines overexpressing EGFP under the control of the 7.1 kbp betaMHC promoter. In embryos, EGFP was detectable as early as 7.5 days post coitum. In embryos, newborns and adults, EGFP was expressed mainly in the cardiac ventricles and in slow skeletal muscles. EGFP expression was intense in the bladder but weak in the intestines. In contrast to the endogenous betaMHC promoter, the activity of the 7.1 kbp betaMHC promoter in the transgene was not repressed after birth and remained high in adult transgenic mice. We obtained two founders with the transgene comprising the TbetaRII-KR-EGFP sequence under the control of the 7.1 kbp betaMHC promoter. These founders were generated at a very low frequency and expressed barely detectable levels of TbetaRII-KR-EGFP mRNA. Our failure to obtain transgenic lines overexpressing the dominant negative receptor suggests that the blocking of the TGFbeta signalling pathway in the heart and slow skeletal muscles could be embryonically lethal. To conclude, the 7.1 kbp betaMHC promoter directs high levels of transgene expression in the cardiac ventricles and in slow skeletal muscles of the mouse. Analysis of the consequences of the blocking of the TGFbeta signalling pathway in the heart will require the use of tissue specific means of conditional gene invalidation.

Animals↗
PubMed2002

Sertoli and granulosa cell-specific Cre recombinase activity in transgenic mice.

We have established transgenic mice expressing the Cre recombinase under the control of the anti-Müllerian hormone (AMH) gene promoter. Cre activity and specificity were evaluated by different means. In AMH-Cre mice, expression of the Cre recombinase mRNA was confined to the testis and ovary. AMH-Cre mice were crossed with reporter transgenic lines and the offspring exhibited Cre-mediated recombination only in the testis and the ovary. In male, histochemical analysis indicated that recombination occurred in every Sertoli cells. In female, Cre-mediated recombination was restricted to granulosa cells, but the protein was not evenly active in every cells. From these results, we conclude that potentially, this transgenic line possessing AMH promoter-driven expression of the Cre recombinase is a powerful tool to delete genes in Sertoli cells only, in order to study Sertoli cell gene function during mammalian spermatogenesis.

Animals↗
PubMed2000

GAP-43 N-terminal translocation signal targets beta-galactosidase to developing axons in a pan-neuronal transgenic mouse line.

Tracing neural connectivity is important in understanding the intricacy of the nervous system as this represents the functional unit throughout the system. Here, we provide evidence that beta-galactosidase (beta-gal) linked to the N-terminal axonal translocation signal of GAP-43 provides a reproducible and versatile reporter system for analyzing the developing nervous system in vivo. When expressed by the GAP-43 promoter in transgenic mice, the fusion protein is detected equally within the developing axons of the peripheral and the central nervous systems, directly reflecting the promoter activity.

Animals↗
PubMed2000

Transformation of Stomoxys calcitrans with a Hermes gene vector.

The ability of the Hermes transposable element to function as a germ line transformation vector was tested in the stable fly, Stomoxys calcitrans. Plasmid-based transposable element mobility assays indicated moderate mobility of Hermes in this species. Germline transformants were created using a Hermes element containing the enhanced green fluorescent protein (EGFP) under the regulatory control of the promoter from Actin5C gene of Drosophila melanogaster. Approximately 4% of the fifty-five adults that developed from the 1903 G(0) embryos injected with the vector produced transgenic progeny. In the four transgenic lines established, the EGFP expression pattern was distinctly nonuniform and levels of expression were low. Promoters other than the one from the Actin5C gene of D. melanogaster should be considered for widespread, constitutive expression. All transgenic lines contained multiple (2-4) integrated Hermes elements. Hermes integration events occurred through a canonical cut-and-paste mechanism.

Animals↗
PubMed1999

Hypersensitive site 2 specifies a unique function within the human beta-globin locus control region to stimulate globin gene transcription.

The human beta-globin locus control region (LCR) harbors both strong chromatin opening and enhancer activity when assayed in transgenic mice. To understand the contribution of individual DNase I hypersensitive sites (HS) to the function of the human beta-globin LCR, we have mutated the core elements within the context of a yeast artificial chromosome (YAC) carrying the entire locus and then analyzed the effect of these mutations on the formation of LCR HS elements and expression of the genes in transgenic mice. In the present study, we examined the consequences of two different HS2 mutations. We first generated seven YAC transgenic lines bearing a deletion of the 375-bp core enhancer of HS2. Single-copy HS2 deletion mutants exhibited severely depressed HS site formation and expression of all of the human beta-globin genes at every developmental stage, confirming that HS2 is a vital, integral component of the LCR. We also analyzed four transgenic lines in which the core element of HS2 was replaced by that of HS3 and found that while HS3 is able to restore the chromatin-opening activity of the LCR, it is not able to functionally replace HS2 in mediating high-level globin gene transcription. These results continue to support the hypothesis that HS2, HS3, and HS4 act as a single, integral unit to regulate human globin gene transcription as a holocomplex, but they can also be interpreted to say that formation of a DNase I hypersensitive holocomplex alone is not sufficient for mediating high-level globin gene transcription. We therefore propose that the core elements must productively interact with one another to generate a unique subdomain within the nucleoprotein holocomplex that interacts in a stage-specific manner with individual globin gene promoters.

Animals↗
PubMed2006

Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter.

In contrast to what we know on development of endocrine pancreas, the formation of exocrine pancreas remains poorly understood. To create an animal model that allows observation of exocrine cell differentiation, proliferation, and morphogenesis in living animals, we used the zebrafish elastaseA (elaA) regulatory sequence to develop transgenic zebrafish that display highly specific exocrine pancreas expression of GFP in both larvae and adult. By following GFP expression, we found that the pancreas in early development was a relatively compact organ and later extended posterior along the intestine. By transferring the elaA:gfp transgene into slow muscle omitted mutant that is deficient in receiving Hedgehog signals, we further showed that Hedgehog signaling is required for exocrine morphogenesis but not for cell differentiation. We also applied the morpholino knockdown and toxin-mediated cell ablation approaches to this transgenic line. We showed that the development of exocrine pancreas is Islet-1 dependent. Injection of the diphtheria toxin A (DTA) construct under the elastaseA promoter resulted in selective ablation of exocrine cells while the endocrine cells and other endodermal derivatives (liver and intestine) were not affected. Thus, our works demonstrated the new transgenic line provided a useful experimental tool in analyzing exocrine pancreas development.

Amino Acid Sequence↗
PubMed2006

Selective expression of an aP2/Fatty Acid Binding Protein 4-Cre transgene in non-adipogenic tissues during embryonic development.

Mouse strains expressing the site-specific Cre recombinase facilitate conditional ablation or activation of genomic sequences when one or several exons of a gene of interest are flanked by loxP sites. Recently, several strains targeting Cre expression to adipocytes have been developed using promoter sequences from the aP2 (Fatty Acid Binding Protein 4, FABP4) gene for adipose tissue-specific gene expression studies. aP2/FABP4 is predominantly expressed in adipose tissue, and while this promoter provides adipocyte-restricted expression postnatally, its expression throughout embryonic development had not been previously characterized. In this report, we demonstrate that the aP2-Cre transgene is expressed and consistently localized within the embryo from mid-gestation stage 9.5 dpc. By 15.5 dpc, beta-gal activity was detected primarily in the brown adipose tissue, trigeminal ganglia, dorsal root ganglia, cartilage primordia and vertebrae. Immunofluorescence staining for Cre recombinase and FABP4 protein showed a corresponding staining pattern similar to that of beta-gal, confirming that Cre recombinase was produced in the transgenic line at late stages of development, and overlapped with endogenous aP2/FABP4 production. Further, fat-specific oil red O staining of tissue sections validated the presence of lipids in the stained tissues indicating that adipocytes and/or adipocyte-like cells were indeed present in these tissues. This is the first report to our knowledge to describe and confirm aP2/FABP4 promoter expression in this transgenic line during development in the mouse embryo and indicates that aP2/FABP4 expression occurs not only in mature adipocytes, but has a wider embryonic expression pattern than previously appreciated.

Adipose Tissue↗
PubMed1989

Developmental regulation of hepatitis B surface antigen expression in two lines of hepatitis B virus transgenic mice.

Two lines of hepatitis B virus (HBV) transgenic mice, designated G7 and G26, show preferential expression of the 2.1-kilobase hepatitis B surface antigen (HBsAg) RNA transcript in liver and kidney tissues (R. D. Burk, J. A. DeLoia, M. K. ElAwady, and J. D. Gearhart, J. Virol 62:649-654, 1988). This transcript was first identified in transgenic mice at gestational day 14 and was detected at similar or increased levels through birth and early development. However, in contrast to 2.1-kilobase HBsAg mRNA levels, HBsAg protein levels in serum decreased shortly after birth. Thereafter, serum HBsAg increased 100-fold to adult levels, with a corresponding 5- to 10-fold increase in HBsAg mRNA levels. In addition, adult males have higher levels of HBsAg in serum than females. HBsAg in serum in males was reduced approximately 50% by surgical castration and was restored to near-normal levels by testosterone supplementation. Since both transgenic lines show similar patterns of gene expression, we suggest that HBsAg gene expression is determined by viral regulatory elements in response to host factors. Whether tissue specificity, developmental regulation, and sexual dimorphism of expression of the exogenous HBV sequences were determined by single or multiple HBV regulatory elements remains to be determined.

Age Factors↗
PubMed2003

Self-fertile apple resulting from S-RNase gene silencing.

Self-incompatibility (SI) restricts fertilisation and fruit setting in many tree fruit crops. In apple, we have produced transgenic trees harbouring extra copies of the endogenous S-gene controlling SI. Two independent transgenic genotypes were characterised in detail. Controlled self- and cross-pollination of the flowers of trees from both genotypes over a 3-year-period showed that the transgenic lines produced normal levels of fruit and seeds after selfing. In contrast, the controls produced much less fruit following self- compared to cross-pollination. Fruit set data correlated with the results of microscopic evaluation of pollen tube growth through the pistil, which revealed inhibition after selfing in the controls but not in the transgenic lines. The self-fertile phenotype was associated with the complete absence of pistil S-RNase proteins, which are the products of the targeted S-gene. These results confirm that self-fertility was due to inhibition of expression of the S-RNase gene in the pistil, resulting in un-arrested self-pollen tube growth, and fertilisation.

Alleles↗
PubMed2006

Does the desaturase-2 locus in Drosophila melanogaster cause adaptation and sexual isolation?

The desaturase-2 (desat2) locus of Drosophila melanogaster has two alleles whose frequencies vary geographically: one (the "Z" allele) is found primarily in east Africa and the Caribbean, and the other (the "M" allele) occurs in other parts of the world. It has been suggested that these alleles not only cause sexual isolation between races, but that their distribution reflects differential adaptation to climate: Z alleles are supposedly adapted to tropical conditions and M alleles to temperate ones. This has thus been viewed as a case of reproductive isolation evolving as a pleiotropic byproduct of adaptation. Here we reinvestigate this presumed climatic adaptation, using transgenic lines differing in the nature of their desat2 alleles. We were unable to replicate earlier results showing that carriers of M alleles are uniformly more cold resistant and less starvation resistant than carriers of Z alleles. It is thus doubtful whether the distribution of these alleles reflects natural selection involving climate. Mating studies of transgenic lines show some evidence for sexual isolation due to desat2. However, work on other, wild-type lines, as well as observations on the nature of sexual isolation, suggest that this conclusion--and thus the relationship between this locus and mating discrimination between races of D. melanogaster--may also be doubtful.

Adaptation, Physiological↗
PubMedDate not supplied

Functional consequences of oncogene-induced photoreceptor degeneration in transgenic mice.

This study evaluated retinal function in mice following the expression of oncogenes under the control of photoreceptor-specific promoters in transgenic mice. Electroretinograms (ERGs) were recorded under stimulus conditions chosen to elicit rod- or cone-mediated components. In one transgenic line (MOT1), the simian virus 40 large tumor antigen was expressed under the control of the mouse opsin promoter. MOT1 mice exhibited an age-related decline in the amplitude of the rod-mediated ERG a-wave. In comparison, cone-mediated responses recorded from MOT1 mice remained normal up to four months of age, the oldest age tested. In the second transgenic line (CMYC), the rat c-myc gene was expressed under control of the human interphotoreceptor-retinoid binding protein promoter. CMYC mice exhibited a rapid reduction of cone-mediated responses and a gradual loss of the rod ERG a-wave. Analysis of rod ERG a-waves obtained from young MOT1 and CMYC mice indicated that the rod ERG abnormalities reflect a reduction in the number of rods contributing to the response with the retention of normal response properties in rods that remain. These results support the possibility that aberrant expression of oncogenes may underlie some forms of human rod and cone-rod dystrophy.

Animals↗
PubMed2005

Distinct functions for Bmp signaling in lip and palate fusion in mice.

Previous work suggested that cleft lip with or without cleft palate (CL/P) is genetically distinct from isolated cleft secondary palate (CP). Mutations in the Bmp target gene Msx1 in families with both forms of orofacial clefting has implicated Bmp signaling in both pathways. To dissect the function of Bmp signaling in orofacial clefting, we conditionally inactivated the type 1 Bmp receptor Bmpr1a in the facial primordia, using the Nestin cre transgenic line. Nestin cre; Bmpr1a mutants had completely penetrant, bilateral CL/P with arrested tooth formation. The cleft secondary palate of Nestin cre; Bmpr1a mutant embryos was associated with diminished cell proliferation in maxillary process mesenchyme and defective anterior posterior patterning. By contrast, we observed elevated apoptosis in the fusing region of the Nestin cre; Bmpr1a mutant medial nasal process. Moreover, conditional inactivation of the Bmp4 gene using the Nestin cre transgenic line resulted in isolated cleft lip. Our data uncover a Bmp4-Bmpr1a genetic pathway that functions in lip fusion, and reveal that Bmp signaling has distinct roles in lip and palate fusion.

Animals↗
PubMed2005

Cardiac-specific and ligand-inducible target gene expression in transgenic mice.

Conditional transgene expression in the heart is a useful approach to explore the physiological basis of the cardiac phenotype. The present study describes the development of a binary transgenic system in which transgene expression in the mouse heart can be turned on/off by administration/withdrawal of an exogenous compound. We generated a transgenic line (alphaMHC-Glp 65) harboring a mifepristone (RU486)-controlled chimeric transcription factor (Glp 65) under the regulatory control of the cardiac-specific alpha-myosin heavy chain (alphaMHC) promoter. In the presence of RU486, Glp 65 expressed in the heart is able to bind to a target gene promoter containing four copies of the 17-mer GAL4 binding site, resulting in ligand-inducible transactivation of the target gene. We tested this system by crossing the transgenic mice, alphaMHC-Glp 65, with a transgenic line harboring human growth hormone (hGH) target gene. We observed that expression of hGH could be induced in adults as well as in the embryonic hearts of bigenic mice by RU486. The basal hGH expression was very low and the inducible level in the heart was estimated over 800-fold higher versus the basal level after 4 days of administration of RU486 at 500 microg/kg body weight per day at 2-5 months of age. The level of the transgene returned to the basal level within 7 days after withdrawal of RU486. This system can be used to control cardiac-specific expression of transgene in a time- and dose-dependent manner.

Animals↗
PubMed2000

Overexpression of Bcl-x(L) in beta-cells prevents cell death but impairs mitochondrial signal for insulin secretion.

To study effects of Bcl-x(L) in the pancreatic beta-cell, two transgenic lines were produced using different forms of the rat insulin promoter. Bcl-x(L) expression in beta-cells was increased 2- to 3-fold in founder (Fd) 1 and over 10-fold in Fd 2 compared with littermate controls. After exposure to thapsigargin (10 microM for 48 h), losses of cell viability in islets of Fd 1 and Fd 2 Bcl-x(L) transgenic mice were significantly lower than in islets of wild-type mice. Unexpectedly, severe glucose intolerance was observed in Fd 2 but not Fd 1 Bcl-x(L) mice. Pancreatic insulin content and islet morphology were not different from control in either transgenic line. However, Fd 2 Bcl-x(L) islets had impaired insulin secretory and intracellular free Ca(2+) ([Ca(2+)](i)) responses to glucose and KCl. Furthermore, insulin and [Ca(2+)](i) responses to pyruvate methyl ester (PME) were similarly reduced as glucose in Fd 2 Bcl-x(L) islets. Consistent with a mitochondrial defect, glucose oxidation, but not glycolysis, was significantly lower in Fd 2 Bcl-x(L) islets than in wild-type islets. Glucose-, PME-, and alpha-ketoisocaproate-induced hyperpolarization of mitochondrial membrane potential, NAD(P)H, and ATP production were also significantly reduced in Fd 2 Bcl-x(L) islets. Thus, although Bcl-x(L) promotes beta-cell survival, high levels of expression of Bcl-x(L) result in reduced glucose-induced insulin secretion and hyperglycemia due to a defect in mitochondrial nutrient metabolism and signaling for insulin secretion.

Animals↗
PubMedDate not supplied

Differential expression of alpha-fetoprotein genes on the inactive X chromosome in extraembryonic and somatic tissues of a transgenic mouse line.

During development of the female mouse embryo, one of the two X chromosomes is inactivated in a random manner in most cell lineages. However, in the extraembryonic trophectoderm and primary endoderm lineages there is preferential inactivation of the paternally derived X chromosome. The inactivated X chromosomes of the extraembryonic and somatic tissues appear equally inactive at the level of the expression of X-linked genes. However, there are differences in the timing of their replication and the extent of DNA modification as determined by gene transfer. The identification of transgenic animals carrying X-linked modified alpha-fetoprotein (AFP) genes allowed us to examine whether the inactivation process extends to an autosomal gene which is normally expressed at high levels in specific extraembryonic and somatic cells, and if so, whether the inactivation process is different in these two tissues. Our results demonstrate that the X-linked AFP genes were expressed on the inactive X chromosome in the visceral endoderm of the yolk sac but not in fetal liver. Thus, the transcriptional activity of the AFP minigene on the inactive X chromosome is dependent on the tissue in which it resides, and most probably reflects differences in the nature of the maintenance of the inactive state of the extraembryonic and embryonic X chromosomes.

Animals↗
PubMed1998

Engraftment of human myelodysplastic syndrome derived cell line in transgenic severe combined immunodeficient (TG-SCID) mice expressing human GM-CSF and IL-3.

A transgenic SCID (TG-SCID) mouse expressing the human cytokines interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) has been generated with the aim of making a model system allowing the in vivo proliferation of human hematopoietic cells. Using TG-SCID mice expressing high levels (30-35 ng/ml in the serum) of human GM-CSF and IL-3, we attempted to engraft a human myeloid leukemia cell line, F-36P, derived from a myelodysplastic syndrome (MDS) patient. When F-36P cells were transferred intravenously into sublethally irradiated TG-SCID mice, extensive proliferation of F-36P cells was found 4-6 wk later. Successful engraftment, however, required the preadministration of a monoclonal antibody to mouse interleukin-2 receptor (IL-2R) beta chain, neutralizing NK activity. Surprisingly, all the transplanted TG-SCID mice engrafted with F-36P cells developed hind leg paralysis approximately 6 wk after transfer. Histological analysis demonstrated extensive invasion and formation of osteolytic lesions by the F-36P cells in the vertebrae. These data indicate that transgenic SCID mice expressing human IL-3 and GM-CSF provide a useful system for the study of human leukemia. In addition, NK cells appear to play an important role in rejection of human cells.

Animals↗