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Results for “Reporter lines”

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At least 145 records · Page 8Linked to original sources

PubMed2000

Immunobiologic, cytogenetic and drug response features of a newly established cell line (SCRC-1) from renal small cell carcinoma.

PURPOSE: We describe the establishment and preliminary characterization of a cell line designated SCRC-1, which was derived from a primary renal small cell carcinoma. MATERIALS AND METHODS: Continuous cultures of a primary stage IVa renal small cell carcinoma and a xenograft in nude mice derived therefrom were characterized by immunohistology, electron microscopy, immunofluorescence/flow cytometry, cytogenetic analysis, and an in vitro drug resistance assay. RESULTS: SCRC-1 cells were reactive with antibodies to NSE, chromogranin-A, bombesin, Bcl-2, CD44s, CD44v6, CD44v7 to 8, vimentin and S100 protein (predominantly beta-subunit), and were unreactive with antibodies to EMA, CD54, EGFR(R1), URO-5, URO-7, URO-8 and URO-10. A similar immunoprofile was also found in both the primary tumor and the xenograft. Cytogenetic analysis revealed the following common clonal aberrations in all 50 metaphases analyzed: 45, XX, t (X;10;18) (p11;p11;q11), -der(18)t(X;10;18), indicating the clonal nature of this neoplasm. SCRC-1 cells showed low drug resistance to cyclophosphamide, doxorubicin, gemcitabine and fluorouracil, intermediate resistance to carmustine and mitomycin-C, and extreme resistance to cisplatin. CONCLUSION: We have documented the initial characterization of SCRC-1, which may be the first cell line reported to be derived from a primary small cell carcinoma of the kidney. This cell line can be used for further studies uncovering the biology and histogenesis of this rare cancer and delineating differences among small cell carcinomas of the kidney and other histological types.

Adult↗

PubMed2005

p53-dependent inhibition of progestin-induced VEGF expression in human breast cancer cells.

VEGF, a potent angiogenic growth factor, is up-regulated in many tumors including human breast tumors and stimulates growth of vascular networks that support tumor growth and metastasis. We previously reported that natural and synthetic progestins (P) increased VEGF mRNA and protein levels in progesterone receptor (PR) containing T47-D human breast cancer cells in a PR dependent manner, but not in PR positive ZR-75 and MCF-7, or in PR negative MDA-MB-231 cells. This indicated that factors beside PR are involved in progesterone-dependent VEGF regulation. We, therefore, tested additional tumor cell lines reported to contain PR for progestin-dependent VEGF induction. Out of nine PR-positive breast tumor cell lines, progestins induced VEGF in three cell lines that lack wild-type p53 (T47-D, BT-474, and HCC-1428) but not in cell lines that contained the wild-type p53 protein. The T47-D and BT-474 cells express mutant p53, while the p53 protein is absent HCC-1428 cells. The anti-progestin RU-486 blocked progestin-dependent induction of VEGF in T47-D and BT-474 cells but not in HCC-1428 cells. However, RU-486 partially blocked medroxyprogesterone acetate-dependent induction of VEGF in HCC-1428 cells. Estrogen receptor (ER) and PR agonists and antagonists also induce VEGF in HCC-1428 cells and this effect was partially blocked by anti-estrogen ICI-182, 780. Progestin-dependent VEGF induction was completely inhibited by PRIMA-1-activated p53 in all cell-types, but progestin-dependent transcription of a progesterone-regulated minimal promoter was only partially inhibited. PRIMA-1 induced activation of p53 in tumor cell lines was confirmed with a p53-responsive p21 reporter plasmid and by detecting increased levels of p21 proteins in cell lysates. PRIMA-1 induced p53 protein in the HCC-1428 cells while levels of mutant p53 protein in T47-D and BT-474 remained unaltered. Progestin-dependent induction of VEGF was also inhibited by stable transfection of wild-type p53 in T47-D cells. These results are consistent with the hypothesis that wild-type p53 blocks progestin-dependent induction of VEGF in breast cancer cells and this may be a novel anti-angiogenic mechanism for controlling the growth of progestin-dependent tumors.

Base Sequence↗

PubMed2005

Afr2 regulation occurs cell-autonomously in vitro but is not conferred on episomal DNA in transient assays.

Oncofetal antigens such as alpha-fetoprotein (AFP) are expressed in regenerating liver. The level of AFP gene expression during liver regeneration is regulated by the unlinked, autosomal gene, Alpha-fetoprotein regulator 2 (Afr2). C3H/HeJ (Afr2A/A) mice express 10-fold higher levels of AFP than C57BL/6J (Afr2B/B) mice. Here we show that primary hepatocytes isolated from C3H/HeJ and C57BL/6J mice exhibit differential expression of the endogenous AFP gene, which was attributed to the Afr2 gene locus and indicative of a cell-autonomous mechanism. We show that the Afr2-Response Element (ARE), between 1010 and 838 base pairs upstream of the AFP transcriptional start site, did not modulate reporter gene expression in transfection assays of Hep G2, Hep 3B, Hepa 1.6, and HeLa cell lines. Reporter gene expression in transiently transfected primary hepatocytes was also ARE-independent. Finally, gene expression from reporter constructs delivered by hydrodynamics-based transfection to the livers of C3H/HeJ and C57BL/6J mice after CCl4-induced liver regeneration was ARE-independent. In conclusion, ARE-dependent transcription was not found in transient assays performed in three different systems, two of which retained regulation of the endogenous AFP gene, suggesting that the ARE may not function as a simple transcription factor recognition site.

Animals↗

PubMed2026

The combination of morphogenic regulators BABY BOOM and GRF-GIF improves maize transformation efficiency and promotes leaf regeneration.

Transformation is an indispensable tool for plant genetics and functional genomics. Although stable transformation in maize is no longer a major obstacle, there remains a need for accessible and efficient methods for academic laboratories. Here, we present the GGB system, a rapid and efficient approach optimized for immature embryo transformation in B104 and other maize lines. This system combines two distinct morphogenetic regulators, the wheat GRF4-GIF1 chimera and the maize BABY BOOM (BBM) transcription factor (hence the name "GGB") with a modified QuickCorn protocol, enabling regeneration of transformed maize plantlets in c. 2 months with an efficiency 7-fold higher than when compared to either morphogenic factor used in isolation. Expression of both regulators did not significantly affect development, eliminating the need to excise them after regeneration. However, transmission of the transgenic GGB construct through pollen was significantly reduced, potentially aiding transgenic line containment. We show that the GGB system is adaptable for CRISPR-Cas9 editing and reporter line generation. Furthermore, stable GGB transformants exhibited high leaf regeneration capacity via somatic embryogenesis. RNA-seq time-course profiling of GGB leaf cultures identified additional factors that could promote regeneration and led to the discovery of asparagine and trehalose as additional media components that significantly enhanced leaf regeneration.

Zea mays↗

PubMed2005

Endothelial cell-specific expression of Cre recombinase in transgenic mice.

Endothelial cells participate in angiogenesis, vascular homeostasis, thrombosis, inflammation and vascular wall remodeling. To study the function of genes in endothelial cells using Cre-loxP system, we generated Tie2-Cre transgenic mice, in which expression of Cre recombinase is driven by Tie2 promoter. Total six founder mice carrying the Tie2-Cre transgene were identified by genomic PCR and Southern blot. The integration efficiency is 11%. In order to test the excision activity and tissue distribution of the Cre recombinase, the Tie2-Cre transgenic line was crossed with the mouse strain carrying the Smad4 conditional alleles (Smad4(Co/Co)) or the reporter line ROSA26. PCR of multiple tissue DNA from Tie2-Cre; Smad4(Co/+) mice revealed the Cre activity in all tissues containing endothelial cells. We detected pan-endothelial expression of the Cre transgene in Tie2-Cre; ROSA26 double transgenic embryos by lacZ staining. Therefore, this mouse line may serve as a valuable tool for endothelial cell lineage analyses and conditional gene ablation in endothelial cells.

Animals↗

PubMed2005

Reporting and classification of patient safety events in a cardiothoracic intensive care unit and cardiothoracic postoperative care unit.

OBJECTIVES: The objective was to evaluate a new mechanism for reporting and classifying patient safety events to increase reporting and identify patient safety priorities. METHODS: A voluntary patient safety event reporting system accessible by all health care workers was implemented in the Cardiothoracic Intensive Care and Post Anesthesia Care Units. Information collected included patient identifiers; date, time, and location of report and event; type and description of event; and severity score. Narrative descriptions of events were analyzed and coded to describe when in the care process the event occurred, what occurred, and a causal classification of why the event occurred. RESULTS: A total of 163 reports describing 157 events were received. These included 121 events reported from the intensive care unit (25.3 reported events per 1000 patient-days), a 3-fold increase compared with the preexisting on-line reporting system. A total of 113 reports (69%) came from nurses, 31 from physicians (19%), and 10 from other staff (6%). A majority of events (85, 54%) reached the patient but caused no harm. Multiple causes were identified for the majority of events. The most frequent causes were related to human factors (48%) and organizational factors (34%). CONCLUSIONS: Health care workers were willing to use the patient safety event reporting system, which yielded a broad range of patient safety data. Patient safety events are multifaceted and often have multiple causal factors. Application of a causal classification model for patient safety event coding in the intensive care and preoperative and postoperative care units is feasible and facilitates local communication of important event-related information.

Adult↗

PubMed2002

A novel doxycycline-inducible system for the transgenic analysis of mammary gland biology.

Normal developmental events such as puberty, pregnancy, and parity influence the susceptibility of the mammary gland to tumorigenesis in both humans and rodent model systems. Unfortunately, constitutive transgenic mouse models that rely on mammary-specific promoters to control transgene expression have limited utility for studying the effect of developmental events on breast cancer risk since the hormonal signals governing these events also markedly influence transgene expression levels. A novel transgenic mouse system is described that uses the MMTV-LTR to drive expression of the reverse tetracycline-dependent transactivator rtTA. Transgenic mice expressing rtTA in the mammary epithelium were crossed with reporter lines bearing tet operator-controlled transgenes. We tested the ability to spatially, temporally, and quantitatively control reporter gene expression after administration of doxycycline to bitransgenic mice. Transgene expression using this system can be rapidly induced and deinduced, is highly mammary specific, can be reproducibly titrated over a wide range of expression levels, and is essentially undetectable in the uninduced state. Homogeneous transgene expression throughout the mammary epithelium can be achieved. This system permits transgene expression to be restricted to any desired stage of postnatal mammary gland development. We have developed a mammary-specific, doxycycline-inducible transgenic mouse model for studying the effect of mammary gland development on transgene-mediated phenotypes. Unlike other mammary-specific, transgenic systems that have been described, this system combines spatially homogeneous transgene expression in the mammary epithelium during puberty, pregnancy, lactation, and involution with the use of an orally administered, inexpensive, and widely available inducing agent. This system offers new opportunities for the transgenic analysis of mammary gland biology in vivo.

Animals↗

PubMed1998

NF-AT-luciferase reporter T cell lines as tools to screen immunosuppressive drugs.

The development of safer analogues of immunosuppressants such as cyclosporin A and FK506 is an important goal for a number of clinical applications ranging from transplantation to the treatment of autoimmune diseases. Here we show the generation and the characterization of Jurkat T cell lines stably transfected with a reporter construct containing the firefly luciferase gene under the control of NF-AT. These lines specifically respond in a cyclosporin A-sensitive manner to T cell antigen receptor-derived signals. Due to the high levels of luciferase activity expression fewer than 1000 cells are required for detection of luciferase. In addition, a simplified luciferase assay allows to reduce both the manipulations and the time required for the assay, making these lines potentially useful models for the automated screening of cyclosporin A and FK506 analogues.

Cyclosporine↗

PubMed2004

Expression of Fas ligand is not a main mechanism used by tumors to counteract antitumor immunity.

The role of Fas ligand (FasL) in tumor immune privilege is controversial. In this study, 22 human tumor cell lines reported to be FasL+ were reevaluated by Western blot analysis, ELISA, and a functional assay. None of the cells lines expressed FasL. To assess whether human tumors express FasL in vivo, susceptibility to FasL-mediated killing was evaluated. About 75% of the 22 tumors tested were sensitive to FasL-mediated apoptosis, suggesting, therefore, that only about 25% could possibly express FasL. To investigate whether "FasL+" human tumor cells could suppress the proinflammatory effects of FasL in vivo, FasL transfectants were generated from two prototype "FasL+" tumor cell lines. The transfectants expressing FasL were rejected by SCID mice. In contrast, all the mice inoculated with parental tumor cells developed large tumors. These results suggested that human tumor cells that express FasL and resist both FasL-mediated apoptosis and inflammation are rare or nonexistant. We concluded that FasL expression is not a main mechanism that tumors use to counteract antitumor immunity.

Animals↗

PubMed2000

An "infobutton" for enabling patients to interpret on-line Pap smear reports.

We describe the development of a prototype application that would allow patients with little or no medical background to understand their Pap smear reports. This information button, or "infobutton", is attached to on-line text reports describing Pap smear results present in a medical record system intended for patient access (PatCIS). The infobutton application generates an explanation of terms present in the report and a list of questions related to the terms in the report, which link to publicly accessible resources on the Web.

Data Display↗

PubMed2004

A new allele of the mouse hairless gene interferes with Hox/LacZ transgene regulation in hair follicle primordia.

A new autosomal recessive mouse mutation, causing loss of hair in homozygous mice 2-3 weeks after birth, arose spontaneously in a colony at the National Institute for Medical Research (NIMR), Mill Hill, London in early 1998. Complementation analysis confirmed that this mutation was an allele of the hairless gene (hr). The gene symbol hr(rhbm) (hairless-rhino-bald Mill Hill) was assigned to reflect the source of the colony. Here we show the molecular defect in these mutants, which is a substantial deletion at the 3'-end of the hairless gene. Morphological and immunological analysis of the new hairless mutation was performed at early postnatal stages. In an effort to address the molecular and cellular mechanisms of the hairless phenotype, we analysed developmental stages before the establishment of alopecia. Using a HoxLacZ reporter line of transgenic mice, epidermal placode formation was followed in embryos. Homozygous mutant embryos (hr(rhbmh)/hr(rhbmh)), containing the LacZ reporter under the control of a Hoxb4 gene enhancer, display sharp loss of LacZ staining in epidermal cells invaginating to form the embryonic hair follicle placode. In the light of targeted mutagenesis data involving a Hox gene in the hair development, we discuss the potential implication of the hr(rhbmh) locus in cascades of Hox gene regulation during embryogenesis.

Alleles↗

PubMed2003

Tumor necrosis factor-alpha induces nuclear factor-kappaB-dependent TRPC1 expression in endothelial cells.

We investigated the role of tumor necrosis factor-alpha (TNF-alpha) in activating the store-operated Ca2+ channels in endothelial cells via the expression of transient receptor potential channel (TRPC) isoforms. We observed that TNF-alpha exposure of human umbilical vein endothelial cells resulted in TRPC1 mRNA and protein expression, whereas it had no effect on TRPC3, TRPC4, or TRPC5 expression. The TRPC1 expression was associated with increased Ca2+ influx after intracellular Ca2+ store depletion with either thrombin or thapsigargin. We cloned the 5'-regulatory region of the human TRPC1 (hTRPC1) gene which contained a TATA box and CCAAT sequence close to the transcription initiation site. We also identified four nuclear factor-kappaB (NF-kappaB)-binding sites in the 5'-regulatory region. To address the contribution of NF-kappaB in the mechanism of TRPC1 expression, we determined the effects of TNF-alpha on expression of the reporter luciferase after transfection of hTRPC1 promoter-luciferase (hTRPC1-Pro-Luc) construct in the human dermal microvascular endothelial cell line. Reporter activity increased >4-fold at 4 h after TNF-alpha challenge. TNF-alpha-induced increase in reporter activity was markedly reduced by co-expression of either kinase-defective IKKbeta kinase mutant or non-phosphorylatable IkappaB mutant. Treatment with NEMO-binding domain peptide, which prevents NF-kappaB activation by selectively inhibiting IKKgamma interaction with IKK complex, also blocked the TNF-alpha-induced TRPC1 expression. Thus, TNF-alpha induces TRPC1 expression through an NF-kappaB-dependent pathway in endothelial cells, which can trigger augmented Ca2+ entry following Ca2+ store depletion. The augmented Ca2+ entry secondary to TRPC1 expression may be an important mechanism of endothelial injury induced by TNF-alpha.

5' Untranslated Regions↗

PubMed2002

Efficient gene modulation in mouse epiblast using a Sox2Cre transgenic mouse strain.

We have generated a transgenic line that expresses the Cre gene product under the regulation of a 12.5 kb upstream regulatory sequence from the Sox2 gene. Using a R26R reporter line, we show that this transgenic line induces recombination in all epiblast cells by embryonic day (E) 6.5 but little or no activity in other extraembryonic cell types at this time. When crossed to a conditional allele of the Sonic hedgehog gene (Shh(c)), all Sox2Cre;Shh(n)/Shh(c) embryos displayed a phenotype indistinguishable from that of the Shh null mutant. Sox2Cre functioned more efficiently in epiblast-mediated recombination than the Mox2Cre (MORE) transgenic line, which has also been shown to drive Cre-mediated recombination exclusively in the embryonic component of the early mouse embryo. Although most MORE; shh(h)/shh(c) embryos have a shh hull phenotype, 33% displayed a milder skeletal phenotype, most likely result of incomplete recombination at egg cylinder stages. In agreement with these findings, Sox2Cre was active earlier and Sox2Cre-mediated recombination was more advanced than MORE-mediated recombination at early gastrulation stages. The Sox2Cre line is likely to be more effective in generating complete, epiblast-specific removal of gene activity, and the mosaic activity of the MORE line will be helpful in generating partial loss-of-function phenotypes in the embryo-proper.

Animals↗

PubMed1994

[Establishment of a subline of C6/36 adapted to growing in a serum-free medium and its usefulness for studies on the dengue virus].

Using the C6/36 clone of the Aedes albopictus cell line, a derived cell line was obtained capable of growing in a commercial serum-free medium facilitating this line to keep its morphologic characteristics and adhesion capacity. The growth speed rate reduction of the adapted cell line was compensated by its low maintenance cost. Its usefulness in the multiplication of dengue viruses 1 and 2 was tested; the reported line showed the same susceptibility for the 2 agents than the original cell line when the indirect fluorescence technique was applied.

Cell Division↗

PubMed2005

A Cre gene directed by a human TSPY promoter is specific for germ cells and neurons.

The testis-specific protein Y-encoded (TSPY) gene is a candidate for the gonadoblastoma locus on the Y chromosome and is expressed in normal testicular germ cells and gonadoblastoma cells of XY sex-reversed females. Although TSPY expression has been demonstrated in gonadoblastoma tissues, it is uncertain if such expression is involved in a causative or consequential event of the oncogenic process. We postulate that if TSPY is involved in gonadoblastoma development, its promoter should be functional in the female gonad before and/or at early stages of tumorigenesis. To test this hypothesis, we generated several lines of transgenic mice harboring a Cre-recombinase transgene directed by a 2.4-kb hTSPY promoter. These mice were crossed with the Z/EG reporter line that expresses EGFP only after a Cre-mediated recombination. Our results showed that hTSPY-Cre;Z/EG double transgenic mice expressed EGFP specifically in the germ cells of both male and female gonads. Further, neurons of the central and peripheral nervous systems also expressed EGFP as early as E12.5 embryonic stage. EGFP was particularly observed in the trigeminal nerve, trigeminal ganglion, dorsal root of the ganglia, and in postnatal and adult brains. These observations support the hypothesis that TSPY plays an active role in gonadoblastoma. The tissue-specific expression of the hTSPY-Cre transgene should also be useful in studies utilizing Cre-mediated gene activation/inactivation strategies in gamatogenesis and/or neurogenesis.

Animals↗

PubMed1983

Constitutive production of Interleukin-1 by the human continuous cell line, CM-S.

Supernatants from the human continuous cell line, CM-S, have been tested for Interleukin-1 (IL-1) activity and found to constitutively elaborate this factor. The CM-S-produced IL-1 activity has been partially purified and shown to be similar to IL-1 produced by human and mouse peripheral blood macrophages. To the best of our knowledge this is the first continuous human cell line reported that produces this monokine.

Cell Line↗

PubMed2006

Unique CCT repeats mediate transcription of the TWIST1 gene in mesenchymal cell lines.

TWIST1, a basic helix-loop-helix transcription factor, plays critical roles in embryo development, cancer metastasis and mesenchymal progenitor differentiation. Little is known about transcriptional regulation of TWIST1 expression. Here we identified DNA sequences responsible for TWIST1 expression in mesenchymal lineage cell lines. Reporter assays with TWIST1 promoter mutants defined the -102 to -74 sequences that are essential for TWIST1 expression in human and mouse mesenchymal cell lines. Tandem repeats of CCT, but not putative CREB and NF-kappaB sites in the sequences substantially supported activity of the TWIST1 promoter. Electrophoretic mobility shift assay demonstrated that the DNA sequences with the CCT repeats formed complexes with nuclear factors, containing, at least, Sp1 and Sp3. These results suggest critical implication of the CCT repeats in association with Sp1 and Sp3 factors in sustaining expression of the TWIST1 gene in mesenchymal cells.

Animals↗

PubMed2002

Efficient gene modulation in mouse epiblast using a Sox2Cre transgenic mouse strain.

We have generated a transgenic line that expresses the Cre gene product under the regulation of a 12.5 kb upstream regulatory sequence from the Sox2 gene. Using a R26R reporter line, we show that this transgenic line induces recombination in all epiblast cells by embryonic day (E) 6.5 but little or no activity in other extraembryonic cell types at this time. When crossed to a conditional allele of the Sonic hedgehog gene (Shhc), all Sox2Cre;Shhn/Shhc embryos displayed a phenotype indistinguishable from that of the Shh null mutant. Sox2Cre functioned more efficiently in epiblast-mediated recombination than the Mox2Cre (MORE) transgenic line, which has also been shown to drive Cre-mediated recombination exclusively in the embryonic component of the early mouse embryo. Although most MORE; shhh/shhc embryos have a shh hull phenotype, 33% displayed a milder skeletal phenotype, most likely result of incomplete recombination at egg cylinder stages. In agreement with these findings, Sox2Cre was active earlier and Sox2Cre-mediated recombination was more advanced than MORE-mediated recombination at early gastrulation stages. The Sox2Cre line is likely to be more effective in generating complete, epiblast-specific removal of gene activity, and the mosaic activity of the MORE line will be helpful in generating partial loss-of-function phenotypes in the embryo-proper.

Alleles↗