Both megakaryocytopoiesis and erythropoiesis are induced in mice infected with a retrovirus expressing an oncogenic erythropoietin receptor [see comments]

Abstract Increasing direct and indirect evidence suggests that erythropoietin (Epo) promotes both erythropoiesis and megakaryocytopoiesis. Here we report that, in mice infected with a recombinant spleen focus-forming retrovirus (SFFV) expressing an oncogenic erythropoietin receptor (EpoR), there was an increase in platelet count preceding the ensuing erythrocytosis. Concurrently, there was a substantial increase in splenic megakaryocytes. Culture of the bone marrow and spleen cells from infected mice showed enhanced numbers of multipotent megakaryocytic progenitors. DNA polymerase chain reaction analysis of individual megakaryocyte-containing colonies showed recombinant SFFV (SFFVcEpoR) proviral integration. Immunofluorescence of spleen sections showed overexpression of EpoR protein in the megakaryocytes. Mice infected with a strain of SFFV also developed splenic megakaryocytosis without activating overexpression of the EpoR in megakaryocytes. This in vivo system shows that a relationship between erythropoiesis and thrombopoiesis can exist at the level of the Epo-EpoR signaling pathway. Also, SFFV-based vectors may be excellent vehicles for the introduction of genes into multipotent, hematopoietic progenitors, in vitro.

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Both megakaryocytopoiesis and erythropoiesis are induced in mice infected with a retrovirus expressing an oncogenic erythropoietin receptor [see comments]

Blood ◽

10.1182/blood.v82.8.2386.bloodjournal8282386 ◽

1993 ◽

Vol 82 (8) ◽

pp. 2386-2395 ◽

Cited By ~ 4

Author(s):

GD Longmore ◽

P Pharr ◽

D Neumann ◽

HF Lodish

Keyword(s):

Indirect Evidence ◽

Polymerase Chain Reaction Analysis ◽

Erythropoietin Receptor ◽

Spleen Cells ◽

Hematopoietic Progenitors ◽

Chain Reaction ◽

Proviral Integration ◽

Polymerase Chain

Increasing direct and indirect evidence suggests that erythropoietin (Epo) promotes both erythropoiesis and megakaryocytopoiesis. Here we report that, in mice infected with a recombinant spleen focus-forming retrovirus (SFFV) expressing an oncogenic erythropoietin receptor (EpoR), there was an increase in platelet count preceding the ensuing erythrocytosis. Concurrently, there was a substantial increase in splenic megakaryocytes. Culture of the bone marrow and spleen cells from infected mice showed enhanced numbers of multipotent megakaryocytic progenitors. DNA polymerase chain reaction analysis of individual megakaryocyte-containing colonies showed recombinant SFFV (SFFVcEpoR) proviral integration. Immunofluorescence of spleen sections showed overexpression of EpoR protein in the megakaryocytes. Mice infected with a strain of SFFV also developed splenic megakaryocytosis without activating overexpression of the EpoR in megakaryocytes. This in vivo system shows that a relationship between erythropoiesis and thrombopoiesis can exist at the level of the Epo-EpoR signaling pathway. Also, SFFV-based vectors may be excellent vehicles for the introduction of genes into multipotent, hematopoietic progenitors, in vitro.

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Specificity of reverse transcriptase polymerase chain reaction assays designed for the detection of circulating cancer cells is influenced by cytokines in vivo and in vitro

British Journal of Cancer ◽

10.1038/bjc.1998.653 ◽

1998 ◽

Vol 78 (9) ◽

pp. 1194-1198 ◽

Cited By ~ 108

Author(s):

R Jung ◽

W Krüger ◽

S Hosch ◽

M Holweg ◽

N Kröger ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcriptase ◽

Cancer Cells ◽

Chain Reaction ◽

Circulating Cancer Cells ◽

Polymerase Chain

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In vivo and in vitro regulation of erythropoietin mRNA: measurement by competitive polymerase chain reaction

Blood ◽

10.1182/blood.v81.3.617.617 ◽

1993 ◽

Vol 81 (3) ◽

pp. 617-623 ◽

Cited By ~ 140

Author(s):

J Fandrey ◽

HF Bunn

Keyword(s):

Polymerase Chain Reaction ◽

Hepatoma Cell ◽

Hepatoma Cell Line ◽

Mrna Levels ◽

Chain Reaction ◽

Human Hepatoma Cell ◽

Competitive Polymerase Chain Reaction ◽

Polymerase Chain

Abstract The regulation of erythropoietin (Epo) production was investigated by competitive polymerase chain reaction, a highly sensitive and accurate means of measuring Epo mRNA levels. Co-amplification of the test sample with added mutant Epo cDNA template corrects for variability in the efficiency of amplification. Epo mRNA levels were determined in tissues of normal rats and in animals with varying degrees of anemia. Reduction of the hematocrit level from 0.40 to 0.15–0.20 resulted in a 300-fold increase in kidney Epo mRNA, which comprised 80% of the total Epo mRNA versus 20% from the liver. In contrast, very low levels detected in lung and spleen were not significantly increased by anemia. The human hepatoma cell line, Hep3B, secretes high levels of Epo in response to hypoxia. This regulation is, to a large extent, transcriptional. When Hep3B cells were incubated in the presence of decreasing O2 tension from 160 to 7 mm Hg, there was a monotonic increase in Epo mRNA to 50 to 100 times the normoxic level. Hyperoxia did not suppress basal expression. When cells were incubated at a PO2 of 7 mm Hg, induction of Epo mRNA was first noted at 30 minutes and was maximal at 5 to 6 hours. After Epo mRNA was boosted by a 4-hour hypoxic incubation, cells were then exposed to normoxia, which shut off further transcription of the Epo gene. The decay of Epo mRNA levels closely followed first order kinetics with a half-life of 2 hours, an effective measurement of message stability.

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Analysis of TNF-receptor and ligand superfamily molecules in patients with lymphoproliferative disease of granular lymphocytes

Blood ◽

10.1182/blood.v96.2.647 ◽

2000 ◽

Vol 96 (2) ◽

pp. 647-654 ◽

Cited By ~ 15

Author(s):

Renato Zambello ◽

Livio Trentin ◽

Monica Facco ◽

Marta Siviero ◽

Silvia Galvan ◽

...

Keyword(s):

Cell Proliferation ◽

Annexin V ◽

Polymerase Chain Reaction Analysis ◽

Lymphoproliferative Disease ◽

Resting Cells ◽

Tnf Receptor ◽

Chain Reaction ◽

Polymerase Chain ◽

Granular Lymphocytes

Abstract In 21 patients with lymphoproliferative disease of granular lymphocytes (LDGL), we investigated the expression and the function of molecules belonging to TNF-receptor and TNF-ligand superfamilies (CD30/CD30L; CD40/CD40L; CD27/CD70; Fas [CD95]/FasL[CD95L]). Fourteen patients were characterized by a proliferation of granular lymphocytes (GLs) expressing the CD3+CD16+phenotype, whereas 7 cases showed the CD3−CD16+ CD56 ± phenotype. Our data show that both CD3+ and CD3-GLs are preferentially equipped with CD30, CD40, CD40L, CD70, and CD95 antigens; this pattern is usually associated with the lack of CD27 and CD30L antigens expression. CD95L was demonstrated in the cytoplasm in 14 of 21 cases by flow cytometry, but a definite signal was demonstrated in all cases studied using polymerase chain reaction analysis. On functional grounds, a stimulatory activity on rIL-2 mediated redirected-cytotoxicity against Fcγ+ P815 targets was demonstrated with anti-CD30, CD40, CD40L, CD70, CD95, and CD95L mAbs, although resting cells were unable to exhibit significant redirected-cell lysis. The addition of anti-CD30, CD30L, CD40, CD40L, CD95, and CD95L mAbs did not show any significant effect on cell proliferation at resting conditions or after rIL-2 stimulation, whereas anti-CD70 mAb mediated cell proliferation in 6 of 10 cases tested. This figure was not related to an increase in apoptotic cells, as investigated by Annexin-V expression. Our data indicate that both CD3+ and CD3− GLs are equipped with different costimulatory antigens, supporting the concept that these cells are in vivo activated and suggesting that these molecules might play a role in the cytotoxic mechanisms of GLs.

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Expression of MMP-8 and MMP-13 Genes in the Periodontal Ligament during Tooth Movement in Rats

Journal of Dental Research ◽

10.1177/154405910308200815 ◽

2003 ◽

Vol 82 (8) ◽

pp. 646-651 ◽

Cited By ~ 68

Author(s):

I. Takahashi ◽

M. Nishimura ◽

K. Onodera ◽

J.-W. Bae ◽

H. Mitani ◽

...

Keyword(s):

Gene Expression ◽

Periodontal Ligament ◽

Tooth Movement ◽

Orthodontic Tooth Movement ◽

Polymerase Chain Reaction Analysis ◽

Tension And Compression ◽

Polymerase Chain

Periodontal ligament tissue is remodeled on both the tension and compression sides of moving teeth during orthodontic tooth movement. The present study was designed to clarify the hypothesis that the expression of MMP-8 and MMP-13 mRNA is promoted during the remodeling of periodontal ligament tissue in orthodontic tooth movement. We used the in situ hybridization method and semi-quantitative reverse-transcription/polymerase chain-reaction analysis to elucidate the gene expression of MMP-8 and MMP-13 mRNA. Expression of MMP-8 and MMP-13 mRNA transiently increased on both the compression and tension sides during active tooth movement in vivo. The gene expression of MMP-8 and MMP-13 was induced by tension, while compression indirectly promoted the gene expression of MMP-8 and MMP-13 through soluble factors in vitro. Thus, we concluded that the expression of MMP-8 and MMP-13 is differentially regulated by tension and compression, and plays an important role in the remodeling of the periodontal ligament.

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Quantification of High-Capacity Helper-Dependent Adenoviral Vector Genomes In Vitro and In Vivo, Using Quantitative TaqMan Real-Time Polymerase Chain Reaction

Human Gene Therapy ◽

10.1089/hum.2006.17.ft-204 ◽

2006 ◽

Vol 0 (0) ◽

pp. 060801084750028

Author(s):

M. Puntel ◽

J.F. Curtin ◽

J.M. Zirger ◽

A.K.M. Muhammad ◽

W. Xiong ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Adenoviral Vector ◽

High Capacity ◽

Chain Reaction ◽

Polymerase Chain

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Amplifikasi In Vitro dan In Vivo Fragmen 0,4 KB D-Loop mtDNA Sampel Forensik

Jurnal Kimia Sains dan Aplikasi ◽

10.14710/jksa.9.2.25-29 ◽

2006 ◽

Vol 9 (2) ◽

pp. 25-29

Author(s):

Mukhammad Asy’ari ◽

A. Saifuddin Noer

Keyword(s):

Polymerase Chain Reaction ◽

Chain Reaction ◽

D Loop ◽

Polymerase Chain

Daerah DNA mitokondria (mtDNA) manusia yang mempunyai tingkat polimorfisme tertinggi adalah D-loop. Urutan nukleotida D-loop mtDNA dapat dijadikan alat untuk menentukan identitas genetik seseorang. Saat ini aplikasinya banyak digunakan untuk memecahkan kasus-kasus di bidang forensik. Isolasi fragmen 0,4 kilobasa (kb) D-loop mtDNA dapat dilakukan dengan mengekstrak mtDNA dari dalam sel, kemudian menggandakan (amplifikasi) mtDNA secara in vitro dengan metoda Polymerase Chain Reaction (PCR) dan secara in vivo dengan metoda Kloning. Pada penelitian ini telah berhasil diamplifikasi secara in vitro fragmen 0,4 kb mtDNA sampel forensik dengan metoda PCR menggunakan primer M1 (5’CACCATTAGCACCCAAAGCT-3’) dan M2 (5’GATTTCAC GGAGGATGGTG-3’) dan secara in vivo dengan metoda kloning menggunakan vektor pGEM-T dalam sel inang Eschericia coli JM 109. Identifikasi hasil PCR dilakukan dengan metoda elektroforesis gel agarosa 1% menggunakan pewarna fluorisen Etidium Bromida. Sedangkan sel rekombinan hasil kloning diseleksi menggunakan media selektif mengandung antibiotik ampisilin dan zat pewarna kultur rekombinan yaitu X-gal dan IPTG sebagai indusernya. Identifikasi plasmid rekombinan yang mengandung fragmen 0,4 kb D-loop mtDNA dilakukan menggunakan enzim restriksi Pst I. Hasil kloning diperoleh jumlah koloni putih (sel rekombinan) : biru (sel non rekombinan) adalah 2 : 1. Hasil analisis restriksi enzim PstI pada plasmid rekombinan diperoleh fragmen berukuran 3,4 kb, hal ini menunjukkan bahwa DNA insert berukuran 0,4 kb sesuai dengan fragmen mtDNA hasil PCR. Plasmid rekombinan selanjutnya digunakan pada penelitian berikutnya, yaitu penentuan urutan nukleotida dengan metode sekuensing sehingga dapat diketahui identitas genetika dari sampel forensik yang dianalisis.

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Effect of maturation on the expression of aquaporin 3 in mouse oocyte

Zygote ◽

10.1017/s0967199410000171 ◽

2010 ◽

Vol 19 (1) ◽

pp. 9-14 ◽

Cited By ~ 6

Author(s):

Jun Woo Jo ◽

Byung Chul Jee ◽

Chang Suk Suh ◽

Seok Hyun Kim ◽

Young Min Choi ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Maturation Process ◽

Chain Reaction ◽

Aquaporin 3 ◽

Immature Oocytes ◽

Antral Follicles ◽

Polymerase Chain ◽

Experimental Group

SummaryThis study aimed to investigate whether aquaporin 3 (Aqp3) mRNAs are expressed in immature oocytes and altered during in vitro maturation process. Five- to 6-week-old female ICR mice were primed by gonadotropin for 24 and 48 h. Immature oocytes obtained 48 h after priming were also matured in vitro for 17 to 18 h. In vivo matured oocytes were obtained after 48 h priming followed by hCG injection. Total RNAs were extracted from 80 to 150 oocytes in each experimental group, and the levels of Aqp3 mRNA were quantified by real-time reverse transcriptase polymerase chain reaction. The experiments were repeated twice using different oocytes. The Aqp3 mRNA was expressed in immature oocytes, as well as in in vitro and in vivo matured oocytes. The expression level was higher in immature oocytes obtained 48 h after priming (17.2 ± 8.6, mean ± SD) than those with no priming (5.7 ± 0.8) or obtained 24 h after priming (2.5 ± 0.8). The expression of Aqp3 mRNA decreased after in vitro maturation (1.2 ± 0.5), which was similar to in vivo matured oocytes (1.0 ± 0.0). Our work demonstrated that Aqp3 mRNA expression increased during the development of immature oocyte but decreased after completion of in vitro maturation. The results indicate that AQP3 is certainly needed for the acquisition of immature oocytes’ full growing potential within antral follicles.

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