Inducible expression of an hsp68-lacZ hybrid gene in transgenic mice

Development ◽  
1989 ◽  
Vol 105 (4) ◽  
pp. 707-714 ◽  
Author(s):  
R. Kothary ◽  
S. Clapoff ◽  
S. Darling ◽  
M.D. Perry ◽  
L.A. Moran ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Constitutive Expression ◽  
Heat Shock Gene ◽  
Preimplantation Embryos ◽  
Northern Analysis ◽  
Sequence Information ◽  
Hybrid Gene ◽  
Stage Of Development ◽  
Beta Galactosidase

Transgenic mice have been generated that express the E. coli beta-galactosidase gene under the control of the promoter from the mouse heat-shock gene, hsp68. Sequences from −664 to +113 relative to the start of transcription of the hsp68 gene were sufficient to direct stress-induced expression of the beta-galactosidase gene in adult tail tissue and various tissues of fetal stages of development. Expression was detected in situ by staining with the chromogenic substrate, X-gal. The hybrid gene was refractory to induction in preimplantation embryos until the blastocyst stage of development, as reported for the endogenous hsp68 gene. No constitutive expression was observed by in situ staining or Northern analysis at any stage of development, even in tissues that constitutively express the endogenous hsp68 gene. We conclude that the hsp68 promoter region included in the construct contains sufficient sequence information for heat and arsenite inducibility, but it does not contain sequences controlling tissue-specific expression during development. This tightly regulated inducible promoter may provide a useful tool for short-term inducible gene expression in transgenic mice.

The initiation of basal disc formation in Dictyostelium discoideum is an early event in culmination

Development ◽  
1996 ◽  
Vol 122 (3) ◽  
pp. 753-760 ◽  
Author(s):  
K. Jermyn ◽  
D. Traynor ◽  
J. Williams
Keyword(s):  
Early Event ◽  
Double Staining ◽  
Forward Movement ◽  
Basal Disc ◽  
Stage Of Development ◽  
Glucuronidase Reporter ◽  
Tip Formation ◽  
Beta Galactosidase ◽  
Disc Formation

We have analysed expression of the ecmA and ecmB genes of Dictyostelium by enzymatic double staining using beta-galactosidase and beta-glucuronidase reporter gene constructs. Cells expressing the ecmA gene first appear as scattered cells at the mound stage of development and we show that this is also true for cells expressing the ecmB gene. During tip formation the ecmA-expressing cells move to the apex of the mound, while the ecmB-expressing cells accumulate in the base. The ecmB-expressing cells constitute part of the basal disc if the culminant is formed in situ but are discarded if a migratory slug is formed. During slug migration they are replaced by a band of ecmB-expressing cells, situated in the front half of the prespore zone and tightly apposed to the substratum. When culmination is triggered these cells rapidly move to the back half of the prestalk zone, possibly acting as a point of attachment to the substratum. Ultimately, they are joined by cells at the back of the slug, the rearguard cells, to form the basal disc. Thus, contrary to previous belief, basal disc formation is initiated very early during culmination and occurs by the forward movement of cells located in the anterior of the prespore zone.

In situ detection of beta-galactosidase in lenses of transgenic mice with a gamma-crystallin/lacZ gene

Science ◽  
1987 ◽  
Vol 235 (4787) ◽  
pp. 456-458 ◽  
Author(s):  
D. Goring ◽  
J Rossant ◽  
S Clapoff ◽  
M. Breitman ◽  
L. Tsui
Keyword(s):  
Transgenic Mice ◽  
Lacz Gene ◽  
Gamma Crystallin ◽  
In Situ Detection ◽  
Beta Galactosidase

In Situ Localization of PPAR Gamma and Uncoupling Protein in Mouse Embryo Sections Using Digoxigenin-Labeled Riboprobes

1996 ◽  
Vol 54 ◽  
pp. 32-33
Author(s):  
C. Jennermann ◽  
S. A. Kliewer ◽  
D. C. Morris
Keyword(s):  
Alkaline Phosphatase ◽  
Room Temperature ◽  
Uncoupling Protein ◽  
Ppar Gamma ◽  
Nuclear Hormone Receptor ◽  
Full Length ◽  
Northern Analysis ◽  
Peroxisome Proliferator

Peroxisome proliferator-activated receptor gamma (PPARg) is a member of the nuclear hormone receptor superfamily and has been shown in vitro to regulate genes involved in lipid metabolism and adipocyte differentiation. By Northern analysis, we and other researchers have shown that expression of this receptor predominates in adipose tissue in adult mice, and appears first in whole-embryo mRNA at 13.5 days postconception. In situ hybridization was used to find out in which developing tissues PPARg is specifically expressed.Digoxigenin-labeled riboprobes were generated using the Genius™ 4 RNA Labeling Kit from Boehringer Mannheim. Full length PPAR gamma, obtained by PCR from mouse liver cDNA, was inserted into pBluescript SK and used as template for the transcription reaction. Probes of average size 200 base pairs were made by partial alkaline hydrolysis of the full length transcripts. The in situ hybridization assays were performed as described previously with some modifications. Frozen sections (10 μm thick) of day 18 mouse embryos were cut, fixed with 4% paraformaldehyde and acetylated with 0.25% acetic anhydride in 1.0M triethanolamine buffer. The sections were incubated for 2 hours at room temperature in pre-hybridization buffer, and were then hybridized with a probe concentration of 200μg per ml at 70° C, overnight in a humidified chamber. Following stringent washes in SSC buffers, the immunological detection steps were performed at room temperature. The alkaline phosphatase labeled, anti-digoxigenin antibody and detection buffers were purchased from Boehringer Mannheim. The sections were treated with a blocking buffer for one hour and incubated with antibody solution at a 1:5000 dilution for 2 hours, both at room temperature. Colored precipitate was formed by exposure to the alkaline phosphatase substrate nitrobluetetrazoliumchloride/ bromo-chloroindlylphosphate.

scholarly journals Constitutive expression of cyclo-oxygenase 2 transgene in hepatocytes protects against liver injury

2008 ◽  
Vol 416 (3) ◽  
pp. 337-346 ◽  
Author(s):  
Rafael Mayoral ◽  
Belen Mollá ◽  
Juana Maria Flores ◽  
Lisardo Boscá ◽  
Marta Casado ◽  
...  
Keyword(s):  
Liver Injury ◽  
Transgenic Mice ◽  
Genetic Model ◽  
Acute Liver Injury ◽  
Constitutive Expression ◽  
Transgenic Animals ◽  
Hepatocyte Proliferation ◽  
Nuclear Antigen ◽  
Cox 2 ◽  
Inflammatory Profile

The effect of COX (cyclo-oxygenase)-2-dependent PGs (prostaglandins) in acute liver injury has been investigated in transgenic mice that express human COX-2 in hepatocytes. We have used three well-established models of liver injury: in LPS (lipopolysaccharide) injury in D-GalN (D-galactosamine)-preconditioned mice; in the hepatitis induced by ConA (concanavalin A); and in the proliferation of hepatocytes in regenerating liver after PH (partial hepatectomy). The results from the present study demonstrate that PG synthesis in hepatocytes decreases the susceptibility to LPS/D-GalN or ConA-induced liver injury as deduced by significantly lower levels of the pro-inflammatory profile and plasmatic aminotransferases in transgenic mice, an effect suppressed by COX-2-selective inhibitors. These Tg (transgenic) animals express higher levels of anti-apoptotic proteins and exhibit activation of proteins implicated in cell survival, such as Akt and AMP kinase after injury. The resistance to LPS/D-GalN-induced liver apoptosis involves an impairment of procaspase 3 and 8 activation. Protection against ConA-induced injury implies a significant reduction in necrosis. Moreover, hepatocyte commitment to start replication is anticipated in Tg mice after PH, due to the expression of PCNA (proliferating cell nuclear antigen), cyclin D1 and E. These results show, in a genetic model, that tissue-specific COX-2-dependent PGs exert an efficient protection against acute liver injury by an antiapoptotic/antinecrotic effect and by accelerated early hepatocyte proliferation.

scholarly journals The c-myc proto-oncogene regulates cardiac development in transgenic mice.

1990 ◽  
Vol 10 (7) ◽  
pp. 3709-3716 ◽  
Author(s):  
T Jackson ◽  
M F Allard ◽  
C M Sreenan ◽  
L K Doss ◽  
S P Bishop ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Mrna Expression ◽  
Cardiac Myocyte ◽  
Cellular Proliferation ◽  
Cardiac Development ◽  
Constitutive Expression ◽  
Transgenic Animals ◽  
Hypertrophic Growth ◽  
Myocyte Proliferation

During the maturation of the cardiac myocyte, a transition occurs from hyperplastic to hypertrophic growth. The factors that control this transition in the developing heart are unknown. Proto-oncogenes such as c-myc have been implicated in the regulation of cellular proliferation and differentiation, and in the heart the switch from myocyte proliferation to terminal differentiation is synchronous with a decrease in c-myc mRNA abundance. To determine whether c-myc can influence myocyte proliferation or differentiation, we examined the in vivo effect of increasing c-myc expression during embryogenesis and of preventing the decrease in c-myc mRNA expression that normally occurs during cardiac development. The model system used was a strain of transgenic mice exhibiting constitutive expression of c-myc mRNA in cardiac myocytes throughout development. In these transgenic mice, increased c-myc mRNA expression was found to be associated with both atrial and ventricular enlargement. This increase in cardiac mass was secondary to myocyte hyperplasia, with the transgenic hearts containing more than twice as many myocytes as did nontransgenic hearts. The results suggest that in the transgenic animals there is additional hyperplastic growth during fetal development. However, this additional proliferative growth is not reflected in abnormal myocyte maturation, as assessed by the expression of the cardiac and skeletal isoforms of alpha-actin. The results of this study indicate that constitutive expression of c-myc mRNA in the heart during development results in enhanced hyperplastic growth and suggest a regulatory role for this proto-oncogene in cardiac myogenesis.

scholarly journals Rapid Detection, Identification, and Enumeration ofEscherichia coli Cells in Municipal Water by Chemiluminescent In Situ Hybridization

2001 ◽  
Vol 67 (1) ◽  
pp. 142-147 ◽  
Author(s):  
Henrik Stender ◽  
Adam J. Broomer ◽  
Kenneth Oliveira ◽  
Heather Perry-O'Keefe ◽  
Jens J. Hyldig-Nielsen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Sensitivity And Specificity ◽  
Bacterial Species ◽  
Rdna Sequence ◽  
Sequence Information ◽  
Sequence Alignments ◽  
E Coli ◽  
Municipal Water

ABSTRACT A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturableEscherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35°C, individual microcolonies of E. coliwere detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targetingP. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other thanE. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.

scholarly journals Overexpression of antizyme in the hearts of transgenic mice prevents the isoprenaline-induced increase in cardiac ornithine decarboxylase activity and polyamines, but does not prevent cardiac hypertrophy

2000 ◽  
Vol 350 (3) ◽  
pp. 645-653 ◽  
Author(s):  
Caroline A. MACKINTOSH ◽  
David J. FEITH ◽  
Lisa M. SHANTZ ◽  
Anthony E. PEGG
Keyword(s):  
Transgenic Mice ◽  
Ornithine Decarboxylase ◽  
Prolonged Exposure ◽  
Constitutive Expression ◽  
Decarboxylase Activity ◽  
Cardiac Growth ◽  
Polyamine Content ◽  
Transgenic Lines ◽  
Response To Stress ◽  
Polyamine Uptake

Two lines of transgenic mice were produced with constitutive expression of antizyme-1 in the heart, driven from the cardiac α-myosin heavy chain promoter. The use of engineered antizyme cDNA in which nucleotide 205 had been deleted eliminated the need for polyamine-mediated frameshifting, normally necessary for translation of antizyme mRNA, and thus ensured the constitutive expression of antizyme. Antizyme-1 is thought to be a major factor in regulating cellular polyamine content, acting both to inhibit ornithine decarboxylase (ODC) activity and to target it for degradation, as well as preventing polyamine uptake. The two transgenic lines had substantial, but different, levels of antizyme in the heart, as detected by Western blotting and by the ability of heart extracts to inhibit exogenous purified ODC. Despite the high levels of antizyme, endogenous ODC activity was not completely abolished, with 10– 39% remaining, depending on the transgenic line. Additionally, a relatively small decrease (30–32%) in cardiac spermidine content was observed, with levels of putrescine and spermine unaffected. Interestingly, although the two lines of transgenic mice had different antizyme expression levels, they had almost identical cardiac polyamine content. When treated with a single acute dose of isoprenaline (isoproterenol), cardiac ODC activity and putrescine content were substantially increased (by 14-fold and 4.7-fold respectively) in non-transgenic littermate mice, but these increases were completely prevented in the transgenic mice from both founder lines. Prolonged exposure to isoprenaline also caused increases in cardiac ODC activity and polyamine content, as well as an increase in cardiac growth, in non-transgenic mice. Although the increases in cardiac ODC activity and polyamine content were prevented in the transgenic mice from both founder lines, the increase in cardiac growth was unaffected. These transgenic mice thus provide a valuable model system in which to study the importance of polyamine levels in cardiac growth and electrophysiology in response to stress.

scholarly journals Development of an IL-15–autocrine CD8 T-cell leukemia in IL-15–transgenic mice requires the cis expression of IL-15Rα

Blood ◽  
2011 ◽  
Vol 117 (15) ◽  
pp. 4032-4040 ◽  
Author(s):  
Noriko Sato ◽  
Helen Sabzevari ◽  
Song Fu ◽  
Wei Ju ◽  
Michael N. Petrus ◽  
...  
Keyword(s):  
T Cells ◽  
Transgenic Mice ◽  
Cd8 T Cells ◽  
Constitutive Expression ◽  
Γδ T Cells ◽  
Selective Advantage ◽  
Intraepithelial Lymphocytes ◽  
Leukemic Transformation ◽  
Nk T Cells ◽  
Growth Promoting

AbstractIL-15 has growth-promoting effects on select lymphoid subsets, including natural killer (NK) cells, NK T cells, intraepithelial lymphocytes (IELs), CD8 T cells, and γδ-T cells. Constitutive expression of murine IL-15 in IL-15–transgenic mice was reported to cause T-NK leukemia. We investigated whether IL-15 expression is sufficient for leukemic transformation using a human IL-15–transgenic (IL-15Tg) mouse model. We noted that 100% of the mice observed over a 2-year period (n > 150) developed fatal expansions of CD8 T cells with NK markers, and determined that these cells expressed IL-15 receptor alpha (IL-15Rα). The expression of IL-15Rα on CD8 T cells appears to be required for uncontrolled aggressive lymphoproliferation, because none of the IL-15Rα−/−–IL-15Tg mice that we followed for more than 2 years developed the fatal disease despite controlled expansion of CD8 T cells. In addition, in contrast to IL-15Tg mice, in which leukemia-like CD8 T cells expressed IL-15Rα persistently, acutely activated normal CD8 T cells only transiently expressed IL-15Rα. Inhibition of DNA methylation enabled sustained IL-15Rα expression induced by activation. We present a scenario for IL-15Tg mice in which CD8 T cells that acquire constitutive persistent IL-15Rα expression are at a selective advantage and become founder cells, outgrow other lymphocytes, and lead to the establishment of a leukemia-like condition.

A 182 bp fragment of the mouse pro alpha 1(II) collagen gene is sufficient to direct chondrocyte expression in transgenic mice

1995 ◽  
Vol 108 (12) ◽  
pp. 3677-3684 ◽  
Author(s):  
G. Zhou ◽  
S. Garofalo ◽  
K. Mukhopadhyay ◽  
V. Lefebvre ◽  
C.N. Smith ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Reporter Gene ◽  
Type Ii Collagen ◽  
Mouse Embryos ◽  
Collagen Gene ◽  
Specific Expression ◽  
Intact Mouse ◽  
Endogenous Gene ◽  
Intron 1 ◽  
Beta Galactosidase

Type II collagen is a major chondrocyte-specific component of the cartilage extracellular matrix and it represents a typical differentiation marker of mature chondrocytes. In order to delineate cis-acting elements of the mouse pro alpha 1(II) collagen gene that control chondrocyte-specific expression in intact mouse embryos, we generated transgenic mice harboring chimeric constructions in which varying lengths of the promoter and intron 1 sequences were linked to a beta-galactosidase reporter gene. A construction containing a 3,000 bp promoter and a 3,020 bp intron 1 fragment directed high levels of beta-galactosidase expression specifically to chondrocytes. Expression of the transgene coincided with the temporal expression of the endogenous gene at all stages of embryonic development. Successive deletions of intron 1 delineated a 182 bp fragment which targeted beta-galactosidase expression to chondrocytes with the same specificity as the larger intron 1 fragment. Transgenic mice harboring a 309 bp Col2a1 promoter lacking intron 1 tester sequences showed no beta-galactosidase expression in chondrocytes. Reduction of the 182 bp fragment to a 73 bp subfragment surrounding a decamer sequence previously reported to be involved in chondrocyte specificity, resulted in loss of transgene expression in chondrocytes. When the Col2a1 promoter was replaced with a minimal beta-globin promoter, the 182 bp intron 1 sequence was still able to target expression of the transgene to chondrocytes. We conclude that a 182 bp intron 1 DNA segment of the mouse Col2a1 gene contains the necessary information to confer high-level, temporally correct, chondrocyte expression on a reporter gene in intact mouse embryos and that Col2a1 promoter sequences are dispensable for chondrocyte expression.

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