Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Comparative genomic hybridization of mouse normal bone marrow and leukemia cells induced by HoxA10 forced expression

ABSTRACT: The Rosa-rtTAnls/tetO-HoxA10 mouse model is a Tetracycline-On (tetO) system in which the expression level of HoxA10 can be tightly regulated and overexpressed under administration of the Doxycycline (Doxy) in the drinking bottle of mice or fed with Ciproxine (Cipro) as a negative control of HoxA10 expression.

In this experiment, we analyzed molecular karyotype derived from CGH arrays analysis of HoxA10 Acute Myeloid Leukemia (AML) cells induced in primary recipient mice (1ary) and in secondary recipient mice, with HoxA10 overexpression (DOXY) or after turning off the HoxA10 overexpression (CIPRO).

Concerning the biological design, we compared leukemia induced in primary recipient mice (1ary) to normal bone marrow (normal control). We compared also primary leukemia (1ary) to leukemia induced in secondary recipient mice induce by continuous expression of HoxA10 (Doxy). Finally we compared leukemia induced in secondary recipient mice induce by continuous expression of HoxA10 (Doxy) to leukemia induced in secondary recipient mice after turning off expression of HoxA10 (Cipro) to analyse relapse growth of leukemic cells after turning off HoxA10.

Concerning the protocol, the leukemic cells originated from two independent primary AML donor mice (cl1 and cl2) are injected in secondary recipient mice, which are monitored for reoccurrence of leukemia. AML are induced by Doxycycline (Doxy) or Ciproxine as negative control (Cipro), by administration of 0.2g/mL continuously in the drinking bottle until development of AML. In sick mice, femurs and tibias were removed and cleaned of soft tissue, bones were crushed, and the cell suspension was filtered over a 100-m pore size cell strainer filter (Falcon; Becton Dickinson). Total DNA from BM cells of Cipro and Doxy receiving recipient mice suffering from leukemia was extracted from bone marrow cells (Qiagen). DNA concentration and integrity were respectively determined by NanoDrop spectrophotometry (NanoDrop technologies) and a Bioanalyser (Agilent technology). Hybridization to Mouse CGH microarray 244A (Agilent) were performed by the Atlas Biolabs (Germany).

ORGANISM(S): Mus musculus

DISEASE(S): normal

SUBMITTER: Quere Ronan

PROVIDER: E-MEXP-2473 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The Rosa-rtTAnls/tetO-HoxA10 mouse model is a Tetracycline-On (tetO) system in which the expression level of HoxA10 can be tightly regulated and overexpressed under administration of the Doxycycline (Doxy) in the drinking bottle of mice or fed with Ciproxine (Cipro) as a negative control of HoxA10 expression. In this experiment, we analyzed expressed transcripts derived from microarray analysis of HoxA10 Acute Myeloid Leukemia (AML) cells induced in secondary recipient mice, with HoxA10 overexpression (Doxy) or after turning off the HoxA10 overexpression (Cipro). Concerning the biological design, we compared leukemia in secondary recipient mice induced by continuous expression of HoxA10 (Doxy) to leukemia induced after turning off expression of HoxA10 (Cipro) and analyse relapse growth of leukemic cells after turning off HoxA10. Concerning the protocol, the leukemic cells originated from two independent primary AML donor mice (cl3 and cl4) are injected in secondary recipient mice, which are monitored for reoccurrence of leukemia. AML are induced by Doxycycline (Doxy) or Ciproxine as negative control (Cipro), by administration of 0.2g/mL continuously in the drinking bottle until development of AML. In sick mice, femurs and tibias were removed and cleaned of soft tissue, bones were crushed, and the cell suspension was filtered over a 100-m pore size cell strainer filter (Falcon; Becton Dickinson). Total DNA from BM cells of Cipro and Doxy receiving recipient mice suffering from leukemia was extracted from bone marrow cells (Qiagen). DNA concentration and integrity were respectively determined by NanoDrop spectrophotometry (NanoDrop technologies) and a Bioanalyser (Agilent technology). Hybridization to Mouse CGH microarray 244A (Agilent) were performed by the Atlas Biolabs (Germany). Reverse transcription using Illumina TotalPrep RNA Amplification Kit (Ambion) and hybridization to Mouse WG-6 Expression v2 BeadChips (Illumina) were performed by the Swegene Center for Integrative Biology at Lund University (SCIBLU).

Project description:The Rosa-rtTAnls/tetO-HoxA10 mouse model is a Tetracycline-On (tetO) system in which the expression level of HoxA10 can be tightly regulated and overexpressed under administration of the Doxycycline (Doxy) in the drinking bottle of mice or fed with Ciproxine (Cipro) as a negative control of HoxA10 expression. In this experiment, we analyzed expressed transcripts derived from microarray analysis of HoxA10 Acute Myeloid Leukemia (AML) cells induced in secondary recipient mice, with HoxA10 overexpression (Doxy) or after turning off the HoxA10 overexpression (Cipro). Concerning the biological design, we compared leukemia in secondary recipient mice induced by continuous expression of HoxA10 (Doxy) to leukemia induced after turning off expression of HoxA10 (Cipro) and analyse relapse growth of leukemic cells after turning off HoxA10. Concerning the protocol, the leukemic cells originated from two independent primary AML donor mice (cl1 and cl2) are injected in secondary recipient mice, which are monitored for reoccurrence of leukemia. AML are induced by Doxycycline (Doxy) or Ciproxine as negative control (Cipro), by administration of 0.2g/mL continuously in the drinking bottle until development of AML. In sick mice, femurs and tibias were removed and cleaned of soft tissue, bones were crushed, and the cell suspension was filtered over a 100-m pore size cell strainer filter (Falcon; Becton Dickinson). Total DNA from BM cells of Cipro and Doxy receiving recipient mice suffering from leukemia was extracted from bone marrow cells (Qiagen). DNA concentration and integrity were respectively determined by NanoDrop spectrophotometry (NanoDrop technologies) and a Bioanalyser (Agilent technology). Hybridization to Mouse CGH microarray 244A (Agilent) were performed by the Atlas Biolabs (Germany). Reverse transcription using Illumina TotalPrep RNA Amplification Kit (Ambion) and hybridization to Mouse WG-6 Expression v1.1 BeadChips (Illumina) were performed by the Swegene Center for Integrative Biology at Lund University (SCIBLU).

Project description:The Rosa-rtTAnls/tetO-HoxA10 mouse model is a Tetracycline-On (tetO) system in which the expression level of HoxA10 can be tightly regulated and overexpressed under administration of the Doxycycline (DOXY) in the drinking bottle of mice or fed with Ciproxine (CIPRO) as a negative control of HoxA10 expression. In this experiment, we analyzed expressed genes derived from microarray analysis of HoxA10 Acute Myeloid Leukemia (AML) cells induced in primary recipient mice (1ary) and in secondary recipient mice, with HoxA10 overexpression (DOXY) or after turning off the HoxA10 overexpression (CIPRO). Concerning the biological design, we compared leukemia induced in primary recipient mice (1ary) to normal bone marrow (normal BM). We compared also primary leukemia (1ary) to leukemia induced in secondary recipient mice (2ary) induce by continuous expression of HoxA10 (DOXY) to leukemia induced in secondary recipient mice after turning off expression of HoxA10 (CIPRO) and analyse relapse growth of leukemic cells after turning off HoxA10. Concerning the protocol, the leukemic cells originated from two independent primary AML donor mice (cl1, cl2, cl3, cl4) are injected in secondary recipient mice, which are monitored for reoccurrence of leukemia. AML are induced by Doxycycline (Doxy) or Ciproxine as negative control (Cipro), by administration of 0.2g/mL continuously in the drinking bottle until development of AML. In sick mice, femurs and tibias were removed and cleaned of soft tissue, bones were crushed, and the cell suspension was filtered over a 100-m pore size cell strainer filter (Falcon; Becton Dickinson). Total RNA from BM cells of ciproxine and doxycycline receiving recipient mice suffering from leukemia was extracted from bone marrow cells (Qiagen). RNA concentration and integrity were respectively determined by NanoDrop spectrophotometry (NanoDrop technologies) and a Bioanalyser (Agilent technology). Reverse transcription using Illumina TotalPrep RNA Amplification Kit (Ambion) and hybridization to Mouse WG-6 Expression v1.1 BeadChips (Illumina) interrogating more than 45,000 transcripts were performed by the Swegene Center for Integrative Biology at Lund University (SCIBLU).

Project description:To design an effective antibody therapy to improve clinical outcomes in leukemia, the identification of novel cell surface antigens is needed. Herein, we demonstrate a role for transmembrane tumor necrosis factor-αin leukemia. To characterize tmTNF-α expression in acute leukemia, normal hematopoietic cells, and non-hematopoietic tissues, we used a monoclonal antibody, termed C1, which specifically recognizes the tmTNF-α domain. We found that tmTNF-α was preferentially expressed by acute leukemia and leukemia stem cells. More abundant expression correlated with poor risk stratification, extramedullary infiltration, and adverse clinical parameters. Moreover, knockdown of tmTNF-α+ expression rendered leukemia cells more sensitive to chemotherapy in vitro and delay regeneration of leukemia in NOD–SCID mice. Targeting tmTNF-α by C1 resulted in leukemia cell killing via antibody-dependent cell-mediated and complement -dependent cytotoxicity in vitro, and inhibited leukemia cell growth in vivo while simultaneously sparing normal hematopoietic cells. Notably, C1 administration impaired the regeneration of leukemia in secondary serial transplantationin to NOD-SCID mice. To further understand the different expression patterns in leukemia cells and potential mechanisms underlying the effects caused by decreased expression of tmTNF-α, total RNA was extracted with TRIzol from SKM-1 stably transfected with control or tmTNF-α shRNA, tmTNF-α+ or - cells sorted from a primary AML sample, and human CD45+ leukemia cells from micetreated with C1 or IgG1 after first transplantation of 7052, and were then subjected to the microarray analysis. We extracted total RNA from SKM-1 stably transfected with control or tmTNF-α shRNA, tmTNF-α+ or - cells sorted from a primary AML sample, and human CD45+ leukemia cells from mice treated with C1 or IgG1 after first transplantation of 7052 (leukemia cell line initially derived from patients with primary acute leukemia who remained stably transplantable in NOD–SCID mice). These were then subjected to the GeneChip probe arrays (Affymetrix GeneChip® Human Genome U133 Plus 2.0). Using SKM-1 stably transfected with control (NC), tmTNF-α+ cells sorted from the primary AML sample and CD45+ leukemia cells from mice treated with IgG1 as reference samples respectively, we compared the different expression patterns in leukemia cells caused by decreased expression of TNF-α (SKM-1 89-1/NC-1, 89-2/NC-2; 70-C1-2/70-IgG-1, 70-C1-3/70-IgG-2; AMLTNFα- 1/+ 1; AMLTNFα- 2/+ 2). Two replicates were included for each pair of samples.

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Comparative genomic hybridization of mouse normal bone marrow and leukemia cells induced by HoxA10 forced expression

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