Upregulation of corin gene expression in hypertrophic cardiomyocytes and failing myocardium

High levels of plasma atrial natriuretic peptides (ANP) are associated with pathological conditions such as congestive heart failure (CHF). Recently, we have identified a cardiac serine protease, corin, that is the pro-ANP convertase. In this study, we examined the regulation of corin gene expression in cultured hypertrophic cardiomyocytes and in the left ventricular (LV) myocardium of a rat model of heart failure. Quantitative RT-PCR analysis showed that both corin and ANP mRNA levels were significantly increased in phenylephrine (PE)-stimulated rat neonatal cardiomyocytes in culture. The increase in corin mRNA correlated closely with the increase in cell size and ANP mRNA expression in the PE-treated cells ( r = 0.95, P < 0.01; r = 0.92, P < 0.01, respectively). The PE-treated cardiomyocytes had an increased activity in converting recombinant human pro-ANP to biologically active ANP, as determined by a pro-ANP processing assay and a cell-based cGMP assay. In a rat model of heart failure induced by ligation of the left coronary artery, corin mRNA expression in the noninfarcted LV myocardium was significantly higher than that of control heart tissues from sham-operated animals, when examined by Northern blot analysis and RT-PCR at 8 wk. These results indicate that the corin gene is upregulated in hypertrophic cardiomyocytes and failing myocardium. Increased corin expression may contribute to elevation of ANP in the setting of cardiac hypertrophy and heart failure.

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Human mononuclear cells express 12-LX: coordinated mRNA regulation with 5-LX and FLAP genes

Blood ◽

10.1182/blood.v87.1.331.bloodjournal871331 ◽

1996 ◽

Vol 87 (1) ◽

pp. 331-340

Author(s):

WE Kaminski ◽

E Jendraschak ◽

K Baumann ◽

R Kiefl ◽

S Fischer ◽

...

Keyword(s):

Gene Expression ◽

Mrna Expression ◽

Time Course ◽

Cell Activation ◽

Mononuclear Cells ◽

Constitutive Expression ◽

Ionophore A23187 ◽

Mrna Levels ◽

Rt Pcr ◽

Human Mononuclear Cells

Lipoxygenases (LXs) catalyze formation of leukotrienes and hydroxy-eicosatetraenoic acids (HETEs), proinflammatory, and spasmogenic autacoids that are critical for host defense systems. We studied the expression and regulation of LX genes (12-LX, 5-LX, and 15-LX) and the 5-lipoxygenase activating protein (FLAP) in human mononuclear cells (MNC) and granulocytes using a quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. We show that 12-LX mRNA is constitutively expressed in resting platelet-free MNC. 12-LX gene expression was upregulated by activation with lipopolysaccharide (LPS). The formation of 12-HETE was inducible with ionophore in MNC, as assessed by high-performance liquid chromatography (HPLC) and gas chromatography, and increased after LPS pretreatment. In addition to 12- LX, resting MNC expressed the genes for 5-LX and FLAP constitutively. Quantitative time course analyses of 12-LX, 5-LX, and FLAP gene expression suggested coregulation of 12-LX and FLAP mRNAs, and reciprocal regulation of 5-LX and FLAP mRNAs. During cell stimulation with LPS 5-LX mRNA levels remained unchanged, whereas FLAP gene expression increased. No 15-LX mRNA expression or 15-HETE formation was detectable in unstimulated and activated MNC. In contrast to MNC, quantitative RT-PCR mRNA analysis showed intermittent intraindividual expression of the 5-LX and FLAP genes in resting granulocytes. mRNAs for 12-LX and 15-LX were not expressed. On stimulation of granulocytes ex vivo, mRNA expression of 5-LX and FLAP was upregulated. Stimulation by LPS differed from that by ionophore A23187. Neither LPS nor ionophore induced gene expression of 12-LX or 15-LX in granulocytes. Our data indicate that resting human MNC and granulocytes express LX and FLAP genes in a cell-specific manner. Cell activation induces coordinated upregulation of 12-LX and FLAP genes in MNC, and 5-LX and FLAP genes in granulocytes, respectively. The constitutive expression of 12-LX mRNA, its upregulation on cell activation, and the formation of 12-HETE clearly indicate the presence of a functional 12-LX in human MNC.

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P3486Experimental model for heart failure in rats: apelin-13/apj and bnp-45 gene expression analysis

European Heart Journal ◽

10.1093/eurheartj/ehz745.0354 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

H R Helmi ◽

A P Sunjaya ◽

D Limanan ◽

A R Prijanti ◽

S W A Jusman ◽

...

Keyword(s):

Gene Expression ◽

Heart Failure ◽

Cardiac Hypertrophy ◽

Mrna Expression ◽

Rat Model ◽

Control Group ◽

P Value ◽

Sprague Dawley ◽

Hypoxia Exposure ◽

Systemic Hypoxia

Abstract Background Apelin, an adipokine peptide and its receptor has recently emerged as a key signaling pathway in maintaining cardiac performance at chronic pressure loads. Apelin has been linked to ventricular dysfunction and therefore maybe of pathophysiologic relevance as a candidate biomarker in HF patients. Purpose This study aims to investigate Apelin-13 gene expression and level, and Apelin receptor (APJ) level in a rat model of heart failure induced by chronic systemic hypoxia and their correlation to BNP-45 gene expression and level, the current gold standard biomarker for heart failure, and to cardiac histopathologic changes. The effect of chronic systemic hypoxia on cardiac hypertrophy, remodeling and heart failure parameters is also of interest. Methods Twenty-eight male Sprague-Dawley rats (8–12 weeks of age) were placed in special hypoxic chambers divided into 7 groups – a control group provided with normoxia (atmospheric O2 levels) and 6 exposure groups exposed to hypoxia (8% O2) for 6 hours, 1, 3, 5, 7 and 14 days respectively prior to measurement. Changes in the expression of Apelin and BNP-45 were measured using quantitative real-time PCR, whereas changes in Apelin-13, APJ and BNP-45 levels were measured using ELISA. Histopathology staining using Hematoxylin and Eosin was performed on cardiac tissues post-termination. Results Compared to control, BNP-45 mRNA expression in the hypoxic heart was only significantly different in day 14, whereas, Apelin mRNA expression had showed significantly higher values starting from day 7 onward. This is in line with the evidence of cardiac hypertrophy based on histopathologic examination present from day 7 onwards. BNP-45 and Apelin-13 levels were significantly higher compared to control from day 5 onwards with a peak on day 7. Although significantly higher than control, Apelin-13 and BNP-45 level decreases in day 14 as compared to day 7. Mean APJ levels showed a similar profile with Apelin-13 and BNP-45 levels with a peak in day 7 (4.619 ng/mL). The cardiac Apelin-13 level shows strong significant correlation with BNP-45 levels (r 0.823, p-value 0.0001). There was also a strong significant correlation between APJ receptor levels with Apelin-13 (r 0.9029, p-value 0.001) and BNP-45 (r 0.9062, p-value 0.0009) levels. Apelin-13, APJ and BNP-45 levels also showed strong significant positive correlation to the duration of hypoxia exposure. Conclusion Chronic (≥5 days) and not acute systemic hypoxia in an experimental rat model leads to increase in Apelin-13, APJ and BNP-45 levels. Apelin-13 and BNP-45 were found to significantly increase from 5 days onwards. Apelin mRNA expression was found to show significant increase earlier compared to BNP-45 mRNA expression. Hence, Apelin may serve as a new candidate biomarker for detection of HF due to oxidative stress compared to BNP-45. Exposure to chronic systemic hypoxia can serve as an easily replicable rat model for heart failure. Acknowledgement/Funding Department of Biochemistry and Molecular Biology, Faculty of Medicine, Tarumanagara University, Jakarta, Indonesia

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Human mononuclear cells express 12-LX: coordinated mRNA regulation with 5-LX and FLAP genes

Blood ◽

10.1182/blood.v87.1.331.331 ◽

1996 ◽

Vol 87 (1) ◽

pp. 331-340 ◽

Cited By ~ 12

Author(s):

WE Kaminski ◽

E Jendraschak ◽

K Baumann ◽

R Kiefl ◽

S Fischer ◽

...

Keyword(s):

Gene Expression ◽

Mrna Expression ◽

Time Course ◽

Cell Activation ◽

Mononuclear Cells ◽

Constitutive Expression ◽

Ionophore A23187 ◽

Mrna Levels ◽

Rt Pcr ◽

Human Mononuclear Cells

Abstract Lipoxygenases (LXs) catalyze formation of leukotrienes and hydroxy-eicosatetraenoic acids (HETEs), proinflammatory, and spasmogenic autacoids that are critical for host defense systems. We studied the expression and regulation of LX genes (12-LX, 5-LX, and 15-LX) and the 5-lipoxygenase activating protein (FLAP) in human mononuclear cells (MNC) and granulocytes using a quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. We show that 12-LX mRNA is constitutively expressed in resting platelet-free MNC. 12-LX gene expression was upregulated by activation with lipopolysaccharide (LPS). The formation of 12-HETE was inducible with ionophore in MNC, as assessed by high-performance liquid chromatography (HPLC) and gas chromatography, and increased after LPS pretreatment. In addition to 12- LX, resting MNC expressed the genes for 5-LX and FLAP constitutively. Quantitative time course analyses of 12-LX, 5-LX, and FLAP gene expression suggested coregulation of 12-LX and FLAP mRNAs, and reciprocal regulation of 5-LX and FLAP mRNAs. During cell stimulation with LPS 5-LX mRNA levels remained unchanged, whereas FLAP gene expression increased. No 15-LX mRNA expression or 15-HETE formation was detectable in unstimulated and activated MNC. In contrast to MNC, quantitative RT-PCR mRNA analysis showed intermittent intraindividual expression of the 5-LX and FLAP genes in resting granulocytes. mRNAs for 12-LX and 15-LX were not expressed. On stimulation of granulocytes ex vivo, mRNA expression of 5-LX and FLAP was upregulated. Stimulation by LPS differed from that by ionophore A23187. Neither LPS nor ionophore induced gene expression of 12-LX or 15-LX in granulocytes. Our data indicate that resting human MNC and granulocytes express LX and FLAP genes in a cell-specific manner. Cell activation induces coordinated upregulation of 12-LX and FLAP genes in MNC, and 5-LX and FLAP genes in granulocytes, respectively. The constitutive expression of 12-LX mRNA, its upregulation on cell activation, and the formation of 12-HETE clearly indicate the presence of a functional 12-LX in human MNC.

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Expression and homologous regulation of GH secretagogue receptor mRNA in rat adrenal gland

Acta Endocrinologica ◽

10.1530/eje.0.1470677 ◽

2002 ◽

pp. 677-688 ◽

Cited By ~ 23

Author(s):

ML Barreiro ◽

L Pinilla ◽

E Aguilar ◽

M Tena-Sempere

Keyword(s):

Biological Effects ◽

Biologically Active ◽

Pcr Analysis ◽

Mrna Levels ◽

R Gene ◽

Rt Pcr ◽

Short Term ◽

Opposite Pattern ◽

Corticosterone Secretion

OBJECTIVE: GH secretagogues (GHSs) elicit a variety of biological effects in several endocrine and non-endocrine target tIssues, including activation of the hypothalamic-pituitary-adrenal axis. The latter is mainly carried out through a central hypothalamic action; yet the possibility of additional effects directly at the adrenal level cannot be ruled out. The aims of this study were to evaluate the expression and homologous regulation of the GHS-receptor (GHS-R) gene in rat adrenal and to assess the effects of synthetic (GH releasing peptide-6 - GHRP-6) and natural (ghrelin) ligands of GHS-R upon basal and ACTH-stimulated corticosterone secretion in vitro. DESIGN AND METHODS: Analysis of adrenal expression of target mRNAs (GHS-R, GHS-R1a, ghrelin, and several steroidogenic factors) was conducted by means of primer-specific, semi-quantitative RT-PCR. Evaluation of corticosterone secretion by incubated adrenal tIssue was carried out by specific RIA. RESULTS: RT-PCR analysis demonstrated expression of the GHS-R gene, but not of the gene encoding the cognate ligand ghrelin, in rat adrenal. Moreover, expression of the mRNA coding for the type 1a GHS-R (GHS-R1a), i.e. the biologically active receptor form, was demonstrated. The adrenal expression of the GHS-R message appeared under the regulation of homologous signals in vitro, as short-term incubation of adrenal samples in serum-free medium induced a significant increase in GHS-R mRNA levels that was inhibited by exposure to different doses of GHRP-6 (10(-9)-10(-5) mol/l) or ghrelin (10(-7) mol/l). Notably, an opposite pattern of homologous regulation of GHS-R gene expression was observed at the pituitary. Finally, short-term stimulation with increasing concentrations of GHRP-6 (10(-9)-10(-5) mol/l) or ghrelin (10(-7) mol/l) failed to alter basal and ACTH-stimulated corticosterone secretion in vitro, neither did it modify ACTH-stimulated mRNA expression levels of several upstream elements in the steroidogenic route: the steroidogenic acute regulatory (StAR) protein, and the enzymes P450 cholesterol side-chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). CONCLUSIONS: Our study provides novel evidence for the expression and homologous regulation of the GHS-R gene in rat adrenal. However, our results cast doubts on the possibility of direct adrenal actions of ligands of the GHS-R in the regulation of corticosterone secretion in the rat.

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Effect of organic and inorganic dietary selenium supplementation on gene expression in oviduct tissues and Selenoproteins gene expression in Lohman Brown-classic laying hens

BMC Veterinary Research ◽

10.1186/s12917-021-02964-0 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

A. I. Muhammad ◽

A. M. Dalia ◽

T. C. Loh ◽

H. Akit ◽

A. A. Samsudin

Keyword(s):

Gene Expression ◽

Mrna Expression ◽

Laying Hens ◽

Egg Quality ◽

Selenium Supplementation ◽

Pcr Analysis ◽

Shell Gland ◽

Mrna Levels ◽

Active Growth ◽

Dietary Selenium

Abstract Background The oviduct of a hen provides a conducive environment for egg formation, which needs a large amount of mineral elements from the blood via trans-epithelial permeability. Eggshell is the calcified layer on the outside of an egg that provides protection and is critical for egg quality. However, little is known about the genes or proteins involved in eggshell formation, and their relationship to dietary microminerals. We hypothesized that dietary selenium supplementation in chickens will influence genes involved in eggshell biomineralization, and improve laying hen antioxidant capacity. The objective of this research was to investigate how organic and inorganic dietary selenium supplementation affected mRNA expression of shell gland genes involved in eggshell biomineralization, and selenoproteins gene expression in Lohman Brown-Classic laying hens. Results Shell gland (Uterus) and liver tissue samples were collected from hens during the active growth phase of calcification (15–20 h post-ovulation) for RT-PCR analysis. In the oviduct (shell gland and magnum) and liver of laying hens, the relative expression of functional eggshell and hepatic selenoproteins genes was investigated. Results of qPCR confirmed the higher (p < 0.05) mRNA expression of OC-17 and OC-116 in shell gland of organic Se hen compared to inorganic and basal diet treatments. Similarly, dietary Se treatments affected the mRNA expression of OCX-32 and OCX-36 in the shell gland of laying hens. In the magnum, mRNA expression of OC-17 was significantly (p < 0.05) higher in hens fed-bacterial organic, while OC-116 mRNA expression was down-regulated in dietary Se supplemented groups compared to non-Se supplemented hens. Moreover, when compared to sodium selenite, only ADS18 bacterial Se showed significantly (p < 0.05) higher mRNA levels in GPX1, GPX4, DIO1, DIO2 and SELW1, while Se-yeast showed significantly (p < 0.05) higher mRNA levels in TXNRD1 than the non-Se group. Conclusions Dietary Se supplementation especially that from a bacterial organic source, improved shell gland and hepatic selenoproteins gene expression in laying hens, indicating that it could be used as a viable alternative source of Se in laying hens. The findings could suggest that organic Se upregulation of shell gland genes and hepatic selenoproteins in laying hens is efficient.

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Abstract P007: Cardiomyocyte Damage-Released Extracellular S100A1 Protein Promotes Regeneration of Infarcted Myocardium by Modulating Cardiac Fibroblast Function

Circulation Research ◽

10.1161/res.109.suppl_1.ap007 ◽

2011 ◽

Vol 109 (suppl_1) ◽

Author(s):

David Rohde ◽

Gang Qiu ◽

Nicole Herzog ◽

Hugo A Katus ◽

Angelika Bierhaus ◽

...

Keyword(s):

Gene Expression ◽

Time Course ◽

Molecular Mechanisms ◽

Gene Expression Pattern ◽

Left Ventricular ◽

Apical Region ◽

Mrna Levels ◽

Rt Pcr ◽

Human Cardiomyocytes

Background: Similar to heart muscle-specific creatinkinase (CK-MB), S100A1 protein is released from damaged human cardiomyocytes in response to myocardial infarction (MI). Since S100A1-knock out (SKO) mice display rapid post-MI onset of adverse myocardial remodeling and accelerated transition to heart failure, we assessed the hypothesis that ischemia-related release of S100A1 protein modulates myocardial regeneration. Methods and Results: After LAD ligation in C57/B6 mice, S100A1 serum levels peaked at 10 µg/ml 8 hours post-MI, precisely mirroring the time course previously observed in MI patients. RT-PCR analyses in post-MI whole heart samples revealed significantly lower I-CAM (−50%) and IL-10 (−75%) mRNA abundance as well as heightened Collagen-1 (+40%) and VEGF (+80%) expression in SKO vs. WT mice (p<0.05, n=6 in each group). Interestingly, injection of an S100A1-neutralizing antibody prior to MI in WT mice mimicked the abnormalities observed in post-ischemic SKO animals. To further elucidate extracellular S100A1 biological activity, cardiomyocytes, cardiac fibroblasts (CF), endothelial and smooth muscle cells were exposed to S100A1 in vitro . A rapid internalization of S100A1 was exclusively found in CF, resulting in a phosphorylation of ERK1/2, JNK, and p38 with subsequent activation of NF-kappaB as assessed by Western Blot (WB) and EMSA. RT-PCR and WB analyses revealed significant alterations in CF gene expression in response to S100A1, including an increase in I-CAM (3,5-fold) and IL-10 (20-fold) mRNA levels and diminished Col-1 (−80%) expression. Similar effects were observed after direct injection of S100A1 protein into the left ventricular apical region of WT mice in vivo (S100A1- vs. PBS-injection, n=6). In SKO mice, intraperitoneal application of S100A1 prior to MI largely normalized the adverse gene expression pattern towards WT animals. Conclusions: Our study provides first evidence for cardiomyocyte damage-released S100A1 to act as an endogenous mediator of post-MI inflammation and tissue repair. Considering today's unability to manipulate these molecular mechanisms, extracellular S100A1 might represent a promising target for future therapies of MI.

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Abstract 835: Left Ventricular Expression of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 Serves as a Biomarker of Heart Failure in the Salt-Sensitive Dahl Rat Model of Hypertension

Circulation ◽

10.1161/circ.118.suppl_18.s_621-c ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Tomohide Takaya ◽

Tatsuya Morimoto ◽

Yoichi Sunagawa ◽

Hiromichi Wada ◽

Teruhisa Kawamura ◽

...

Keyword(s):

Heart Failure ◽

Rat Model ◽

Systolic Function ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Fold Increase ◽

Left Ventricular ◽

Low Density ◽

Mrna Levels ◽

Oxidized Low Density Lipoprotein

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) was originally identified as an endothelial receptor for oxidized LDL, and now is recognized as a multi-ligand receptor. LOX-1 expression in cardiomyocytes can be induced by oxidative stress and various hormonal stimuli. Activation of the LOX-1 pathway in cardiomyocytes induces apoptosis in vitro and deteriorates ischemia-reperfusion injury in vivo . However the role of LOX-1 in chronic heart failure is unknown. We examined the left ventricular (LV) expression of LOX-1 in a salt-sensitive Dahl (DS) rat model of hypertension. Compared with control salt-resistant Dahl (DR) rats, a high-salt diet, started at the age of 6 weeks, induced marked hypertension and apparent concentric LV hypertrophy on echocardiography in DS rats. The LV hypertrophy was moderate at the age of 11 weeks and marked at 18 weeks. The LV systolic function was preserved at 11 weeks, and decreased at 18 weeks. Quantitative real-time PCR revealed a 4.7-fold increase in LV levels of LOX-1 mRNA in DS rats compared with DR rats at 11 weeks, and a 32-fold increase at 18 weeks. The LV LOX-1 mRNA levels were significantly correlated with the LV end-systolic dimension (R=0.640, p=0.0002), and LV posterior wall thickness (R=0.555, p=0.0022), as well as the LV/body weight ratio (R=0.647, p=0.0002), and systolic blood pressure (R=0.751, p<0.0001). LOX-1 levels were also connected with an increase in LV mRNA levels of heme oxygenase-1 (R=0.692, p<0.0001), a marker of oxidative stress. Importantly, LOX-1 levels were strongly associated with a decrease in the LV ejection fraction (R=0.774, p<0.0001) and an increase in mRNA levels of BNP (R=0.814, p<0.0001), a representative marker of LV wall stress and heart failure. Immunohistochemistry demonstrated LOX-1 expression in cardiomyocytes as well as vessel walls of both DS and DR rat hearts. However, expression in cardiomyocytes was greater in DS than in DR. These findings demonstrate that LV cardiomyocyte expression of LOX-1 is markedly increased in proportion to the extent of heart failure and possibly involved in the deterioration of systolic function in the DS rat model of hypertension.

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Generation of Thyrotropin-Releasing Hormone Receptor 1-Deficient Mice as an Animal Model of Central Hypothyroidism

Molecular Endocrinology ◽

10.1210/me.2004-0017 ◽

2004 ◽

Vol 18 (6) ◽

pp. 1450-1460 ◽

Cited By ~ 49

Author(s):

Roland Rabeler ◽

Jens Mittag ◽

Lars Geffers ◽

Ulrich Rüther ◽

Michael Leitges ◽

...

Keyword(s):

Animal Model ◽

In Situ Hybridization ◽

Mrna Expression ◽

Pcr Analysis ◽

Target Cells ◽

Mrna Levels ◽

Rt Pcr ◽

Central Hypothyroidism ◽

R1 Gene

Abstract To provide an animal model of central hypothyroidism, mice deficient in the TRH-receptor 1 (TRH-R1) gene were generated by homologous recombination. The pituitaries of TRH-R1−/− mice are devoid of any TRH-binding capacity, demonstrating that TRH-R1 is the only receptor localized on TRH target cells of the pituitary. With the exception of some retardation in growth rate, TRH-R1−/− mice appear normal, but compared with control animals they exhibit a considerable decrease in serum T3, T4, and prolactin (PRL) levels but not in serum TSH levels. In situ hybridization histochemistry and real-time RT-PCR analysis revealed that in adult TRH-R1−/− animals TSHβ-mRNA expression is not impaired whereas PRL mRNA and GH mRNA levels are considerably reduced compared with control mice. The numbers of thyrotropes, somatotropes, and lactotropes, however, are not affected by the deletion of the TRH-R1 gene. The mutant mice are fertile, and the dams nourish their pups well, indicating that TRH is not a decisive factor for suckling-induced PRL release. In situ hybridization and quantitative RT-PCR analysis, furthermore, revealed that, as in control animals, pituitary PRL-mRNA expression in TRH-R1−/− is considerably increased during lactation, albeit strongly reduced as compared with lactating control animals.

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IDENTIFIKASI IKAN CUPANG (Betta imbelis) TRANSGENIK FOUNDER MEMBAWA GEN PENYANDI HORMON PERTUMBUHAN; Identification of Transgenic Founder Betta Fish (Betta imbelis) Carry Growth Hormone Gene.

Jurnal Riset Akuakultur ◽

10.15578/jra.11.3.2016.197-205 ◽

2017 ◽

Vol 11 (3) ◽

pp. 197

Author(s):

Eni Kusrini ◽

Alimuddin Alimuddin ◽

Mohammad Zairin ◽

Dinar Tri Sulistyowati

Keyword(s):

Gene Expression ◽

Growth Hormone ◽

Mrna Expression ◽

Transgenic Fish ◽

Pcr Analysis ◽

Growth Hormone Gene ◽

Rt Pcr ◽

Total Rna ◽

Gh Gene ◽

Dna Template

Penelitian dilakukan untuk mengidentifikasi keberhasilan introduksi gen penyandi hormon pertumbuhan (Growth Hormone, GH) pada induk F-0 ikan Betta imbellis. Ikan transgenik F-0 dibuat dengan menggunakan metode transfeksi. Identifikasi dilakukan menggunakan metode RT-PCR. RNA total diekstraksi dari embrio pooled sample hasil persilangan induk transgenik dan non-transgenik. Berdasarkan analisis ekspresi gen pada embrio juga menunjukkan adanya aktivitas ekspresi gen GH pada semua perlakuan dibandingkan dengan kontrol (embrio hasil persilangan non-transgenik x non-transgenik). Jumlah individu induk F-0 yang membawa gen GH eksogen berdasarkan analisis PCR dengan DNA template dari sirip ekor adalah sebanyak 16%. Individu positif membawa gen GH eksogen tersebut dibesarkan lebih lanjut untuk memproduksi Betta imbellis transgenik F-1. Kandidat ikan transgenik jantan F-0 dikawinkan dengan ikan non-transgenik betina, sedangkan transgenik F-0 betina dikawinkan dengan non-transgenik jantan. Sebanyak 30-50 butir embrio hasil pemijahan F-0 digabung, kemudian DNA genom diekstrak. Sebagian embrio digunakan untuk ekstraksi RNA total untuk analisis ekspresi mRNA GH eksogen. Hasil analisis PCR menunjukkan bahwa semua sampel embrio dari induk transgenik F-0 dapat terdeteksi gen GH eksogen, sedangkan untuk kontrol (non-transgenik) tidak terdeteksi. Ekspresi mRNA juga terdeteksi pada embrio F-1. Dengan demikian, metode transfeksi embrio Betta imbellis efektif digunakan untuk menghasilkan ikan transgenik, dan sangat berpotensi menghasilkan individu F-1 Betta imbellis dengan pertumbuhan lebih cepat.The study was conducted to identify the successful introduction of the growth hormone gene (Growth Hormone, GH) on the F-0 Betta imbellis broodstock. The F-0 transgenic fish was made through transfection methods. Identification was done using RT-PCR method. Total RNA was extracted from pooled embryos sample. Based on the analysis of gene expression in embryos also showed activity GH gene expression in all treatments compared to the control (non-transgenic x non-transgenic). The number of individuals F-0 which carried exogenous GH gene by PCR analysis of the DNA template of the tail fin was as much as 16%. Positive individuals carried the exogenous GH gene raised further to produce transgenic F-1 B. imbellis. Candidate of transgenic F-0 males fish were mated with non-transgenic female fish, whereas the transgenic F-0 females were mated with non-transgenic males. The 30-50 embryos obtained were combined, then their genomic DNA were extracted. Some of the embryos was used for the extraction of total RNA for analysis of mRNA expression of GH exogenous. The PCR analysis showed that all samples of embryos from the transgenic F-0 broodstock could be detected, whereas for the control (non-transgenic) was not detected. mRNA expression was also detected in embryos of F-1. The average weight of the F-0 broodstocks were 1.55 g and a total length was 12.97 cm. Thus, the transfection methods through betta embryos peaceful effectively generated transgenic fish, and potentially produced fast growth of individuals F-1 Betta imbellis.

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Molecular predictors of treatment response in colon and gastric cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.4_suppl.619 ◽

2012 ◽

Vol 30 (4_suppl) ◽

pp. 619-619

Author(s):

Paul Timothy Fanta ◽

Eric Roeland ◽

John P. Shen ◽

Kelly Anne Shimabukuro ◽

Michael Hwang ◽

...

Keyword(s):

Gene Expression ◽

Gastric Cancer ◽

Mrna Expression ◽

Response To Therapy ◽

Mrna Levels ◽

Metastatic Colon Cancer ◽

Rt Pcr ◽

Expression Levels ◽

Her 2 ◽

Median Expression

619 Background: With the noted exception of KRAS mutational status, currently there exists limited data regarding the incorporation of tumor-derived biomarkers in the clinical management of gastrointestinal malignancies. High ERCC1 levels have been associated with inferior results in platinum-treated patients with non-small cell lung cancer, esophageal cancers, and head and neck cancer. Lenz et al. concluded ERCC-1 gene expression levels may allow the selection of patients who may benefit from FOLFOX chemotherapy in metastatic colon cancer. Low intra-tumoral ERCC1 mRNA expression predicted improved PFS and OS in patients with esophageal adenocarcinoma who were treated with tri-modality therapy in the SWOG 0356 correlative study. Methods: To determine the prevalence and patterns of expression of select tumor biomarkers including ERCC1, TS, HER-2, KRAS, BRAF, and EGFR gene expression was measured in metastatic gastric and colorectal cancers using formalin fixed paraffin embedded tumor samples from 120 metastatic colorectal and 20 metastatic gastric cancer were dissected using laser-captured micro-dissection and analyzed for ERCC-1, TS, EGFR, RRM1, and VEGFR2a mRNA expression using a quantitative RT-PCR methodology. Gene expression values (relative mRNA levels) were recorded as ratios between the target gene and internal reference gene (beta-actin). A retrospective review of the patient’s response to therapy was planned. Results: In colorectal patients, the incidence of KRAS mutations was 50%, specifically Gly12Ser 4%, Gly12Val 11%, Gly12Asp 20%, Gly12 Cys 7% Gly12Ala 2% and Gly13Asp 8%. BRAF expression analysis displayed 91% wild type with 9% V600E mutations. Median expression values for ERCC1, TS, EGFR, RRM1, and VEGFR2A expression levels using RT-PCR were 1.23, 2.28, 1.90, 1.05, and 1.61 respectively in the colorectal subset. In gastric cancer, ERCC1, TS, and Her-2 median expression levels using RT-PCR were 1.54, 3.56, and 0.08 respectively. Correlation with clinical outcome is pending and will be reported later.

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