Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The great advantage of the tetracycline-inducible system lies in its ability to address a large variety of biological questions in a time-dependent and tissue-specific manner. This study describes a transgenic mouse line, rTA(LAP)-1, which produces the reverse tetracycline transactivator under control of the liver activator protein (LAP) promoter. Two reporter lines with luciferase and LacZ reporter genes were used to demonstrate predominant expression in the kidney and liver when doxycycline was added to the drinking water. In the kidney, transgene expression was found primarily in cortical proximal tubules. No luciferase and beta-galactosidase activity was detected in mice without doxycycline in the drinking water, which attests to the tight control of this system. One of the advantages of the tet system lies in its reversibility, and indeed, a virtually complete remission of transgene activity in both the kidney and liver was observed when doxycycline was withdrawn. Also examined was transactivator activity during development by exposing the mothers producing the reverse transactivator to doxycycline before mating. Transgene activity was detected in newborn kidneys and liver, indicating that sufficient amounts of doxycycline had crossed the placental barrier. During nephron development, the LAP promoter appeared to be only active in the more mature proximal tubules. Finally, the rTA(LAP)-1 line was used to inducibly express the human PKD2 cDNA in proximal tubules of transgenic mice, but no cystic changes were detected, even after 6 mo of induction.
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PMID:Use of the tetracycline system for inducible protein synthesis in the kidney. 1287 58

Self-association of the transactivator HBx protein of hepatitis B virus was investigated using the yeast two-hybrid system. Expression vectors for the full-length HBx (X0) and its truncated mutants (X15 and X16) were constructed by separately ligating the DNA-binding (BD) and transactivation domains (AD) of Gal4. Co-transformants of the BD and AD constructs of HBx were selected using defined minimal medium and analyzed for the reconstitution of beta-galactosidase activity. No two-hybrid interaction was observed either between the full-length HBx molecules or its highly truncated mutant X16. However, a strong functional interaction between X0 and X15, X0 and X16, and X15 and X16 suggested that HBx could self-associate in a cellular environment through its carboxy-terminal region.
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PMID:Self-association of the hepatitis B virus X protein in the yeast two-hybrid system. 1509 70

To achieve conditional gene expression in the differentiated layers of the epidermis, we generated transgenic mice with the tetracycline-regulated transactivator proteins, tTA (tetracycline transactivator) and rtTA (reverse tetracycline transactivator), expressed from the human involucrin promoter. Interaction with tetracycline turns off or turns on the tTA and rtTA molecules, respectively, allowing for regulation of downstream target genes during development and postnatally. These transactivator lines were crossed with reporter mice driving LacZ expression from a tetracycline response element to analyze the specificity and levels of target gene expression. Quantitative beta-galactosidase experiments demonstrate a 30-fold induction, specific to epithelial tissues. Immunohistochemistry results illustrate that the beta-galactosidase staining follows that of endogenous involucrin expression. Induction initiates at embryonic day 14.5 with expression over the entire epidermal surface by E16.5. Together with other driver lines, expressing tetracycline transactivators in the mitotically active layers of the epidermis, these mice will allow investigators to specifically modulate expression of target genes to specific stages of epidermal differentiation.
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PMID:Tetracycline-regulated transactivators driven by the involucrin promoter to achieve epidermal conditional gene expression. 1524 31

Megakaryocyte (MK)-specific transgene expression has proved valuable in studying thrombotic and hemostatic processes. Constitutive expression of genes, however, could result in altered phenotypes due to compensatory mechanisms or lethality. To circumvent these limitations, we used the tetracycline/doxycycline (Tet)-off system to conditionally over-express genes in megakaryocytes and platelets in vivo. We generated 3 transactivator transgenic lines expressing the Tet transactivator element (tTA), under the control of the MK-specific platelet factor 4 promoter (PF4-tTA-VP16). Responder lines were simultaneously generated, each with a bidirectional minimal cytomegalovirus (CMV)-tTA responsive promoter driving prokaryotic beta-galactosidase gene, as a cellular reporter, and a gene of interest (in this case, the mitotic regulator Aurora-B). A transactivator founder line that strongly expressed PF4-driven tTA-viral protein 16 (VP16) was crossbred to a responder line. The homozygous double-transgenic mouse line exhibited doxycycline-dependent transgene overexpression in MKs and platelets. Using this line, platelets were conveniently indicated at sites of induced stress by beta-galactosidase staining. In addition, we confirmed our earlier report on effects of constitutive expression of Aurora-B, indicating a tight regulation at protein level and a modest effect on MK ploidy. Hence, we generated a new line, PF4-tTA-VP16, that is available for conditionally overexpressing genes of interest in the MK/platelet lineage in vivo.
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PMID:Conditional overexpression of transgenes in megakaryocytes and platelets in vivo. 1589 Jun 84

In view of recent serious adverse events and advances in gene therapy technologies, the use of regulatable expression systems is becoming recognized as indispensable adjuncts to successful clinical gene therapy. In the present work we optimized high-capacity adenoviral (HC-Ad) vectors encoding the novel tetracycline-dependent (TetOn)-regulatory elements for efficient and regulatable gene expression in the rat brain in vivo. We constructed two HC-Ad vectors encoding beta-galactosidase (beta-gal) driven by a TetOn system containing the rtTAS(s)M2 transactivator and the tTS(Kid) repressor under the control of the murine cytomegalovirus (mCMV) (HC-Ad-mTetON-beta-Gal) or the human CMV (hCMV) promoter (HC-Ad-hTetON-beta-Gal). Expression was tightly regulatable by doxycycline (Dox), reaching maximum expression in vivo at 6 days and returning to basal levels at 10 days following the addition or removal of Dox, respectively. Both vectors achieved higher transgene expression levels compared to the expression from vectors encoding the constitutive mCMV or hCMV promoter. HC-Ad-mTetON-beta-Gal yielded the highest transgene expression levels and expressed in both neurons and astrocytes. Antivector immune responses continue to limit the clinical use of vectors. We thus tested the inducibility and longevity of HC-Ad-mediated transgene expression in the brain of rats immunized against adenovirus by prior intradermal injections of RAds. Regulated transgene expression from HC-Ad-mTetON-beta-Gal remained active even in the presence of a significant systemic immune response. Therefore, these vectors display two coveted characteristics of clinically useful vectors, namely their regulation and effectiveness even in the presence of prior immunization against adenovirus.
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PMID:Regulatable gutless adenovirus vectors sustain inducible transgene expression in the brain in the presence of an immune response against adenoviruses. 1635 28

As serious side effects affected recent virus-mediated gene transfer studies, novel vectors with improved safety profiles are urgently needed. In the present study, replication-deficient retroviral vectors based on feline foamy virus (FFV) were constructed and analyzed. The novel FFV vectors are devoid of almost the complete env gene plus the internal promoter - accessory bel gene cassette including the gene for the viral transcriptional transactivator Bel1/Tas. In these Bel1/Tas-independent vectors, expression of the lacZ (beta-galactosidase) marker gene is directed by the heterologous, constitutively active human ubiquitin C promoter (ubi). Env-transcomplemented vectors have un-concentrated titers of more than 10(5) transducing units/ml. The vectors allow efficient transduction of a broad array of diverse target cells, which can be increased by repeated vector exposure. However, the number of lacZ marker gene expressing cells decreased slightly upon serial passages of the transduced cells. Vectors carrying a self-inactivating (SIN) deletion of the TATA box and most parts of the viral promoter were not rescued by wt FFV whereas those with the intact or a partially deleted promoter were readily reactivated. This finding indicates that the viral promoters are in fact non-functional, pointing to a highly advantageous safety profile of these new FFV-ubi-lacZ-SIN vectors.
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PMID:Construction and characterization of efficient, stable and safe replication-deficient foamy virus vectors. 1720 7

A key step towards understanding the development and function of the central nervous system is by characterizing the connections between neurons. Tetanus toxin C fragment (TTC) is transynaptically and retrogradely transported without the toxin's pathogenic effect, and therefore, recently it has been used as a genetic tracer combined with beta-galactosidase or green fluorescent protein. Here, we introduce a new fusion construct, APTTC, consisting of the truncated human placental alkaline phosphatase with TTC, and generating the transgenic mouse line, (tetracycline operator) tetO-APTTC, for inducible expression of APTTC regulated by tetO. We demonstrate that APTTC is transported retrogradely and transynaptically, and allows us to robustly visualize the inputs of the expressing neurons when transgenetically expressed in mice, exemplified in the striatal neuronal circuit. Therefore, tetO-APTTC transgenic mouse line can be widely used for visualization of neuronal connectivity when combined with mice carrying tetracycline-controlled transactivator (tTA) in any specific neurons.
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PMID:Inducible expression of retrograde transynaptic genetic tracer in mice. 1729 48

Through an established gene-targeting strategy, reverse tetracycline-dependent transactivator (rtTA) was targeted downstream of the murine progesterone receptor (PR) promoter. Mice were generated in which one (PR(+/rtTA)) or both (PR(rtTA/rtTA)) PR alleles harbor the rtTA insertion. The PR(+/rtTA) and PR(rtTA/rtTA) knockins exhibit phenotypes identical to the normal and the progesterone receptor knockout mouse, respectively. Crossed with the TZA reporter, which carries the TetO-LacZ responder transgene, the PR(+/rtTA)/TZA and PR(rtTA/rtTA)/TZA bigenics exhibit doxycycline-induced beta-galactosidase activity specifically in progesterone responsive target tissues such as the mammary gland, uterus, ovary, and pituitary gland. In the case of the PR(+/rtTA)/TZA mammary epithelium, dual immunofluorescence demonstrated that PR expression and doxycycline-induced beta-galactosidase activity colocalized; beta-galactosidase was not detected in the absence of doxycycline. Although both the PR(+/rtTA) and PR(rtTA/rtTA) knockins represent innovative animal models with which to further query progesterone's mechanism of action in vivo, the PR(rtTA/rtTA) mouse in particular promises to provide unique insight into the paracrine mechanism of action, which underpins progesterone's involvement in mammary morphogenesis with obvious implications for extending our understanding of this steroid's role in breast cancer progression.
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PMID:Targeting reverse tetracycline-dependent transactivator to murine mammary epithelial cells that express the progesterone receptor. 1794 Oct 46

We have generated a transgenic mouse that expresses Cre recombinase only in skeletal muscle and only following tetracycline treatment. This spatiotemporal specificity is achieved using two transgenes. The first transgene uses the human skeletal actin (HSA) promoter to drive expression of the reverse tetracycline-controlled transactivator (rtTA). The second transgene uses a tetracycline responsive promoter to drive the expression of Cre recombinase. We monitored transgene expression in these mice by crossing them with ROSA26 loxP-LacZ reporter mice, which express beta-galactosidase when activated by Cre. We find that the expression of this transgene is only detectable within skeletal muscle and that Cre expression in the absence of tetracycline is negligible. Cre is readily induced in this model with tetracycline analogs at a range of embryonic and postnatal ages and in a pattern consistent with other HSA transgenic mice. This mouse improves upon existing transgenic mice in which skeletal muscle Cre is expressed throughout development by allowing Cre expression to begin at later developmental stages. This temporal control of transgene expression has several applications, including overcoming embryonic or perinatal lethality due to transgene expression. This mouse is especially suited for studies of steroid hormone action, as it uses tetracycline, rather than tamoxifen, to activate Cre expression. In summary, we find that this transgenic induction system is suitable for studies of gene function in the context of hormonal regulation of skeletal muscle or interactions between muscle and motoneurons in mice.
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PMID:A tetracycline-inducible and skeletal muscle-specific Cre recombinase transgenic mouse. 1926 19


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