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 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).

Project description:Measurement of specific gene expression in clinical samples is a promising approach for monitoring the recipient immune status to the graft in organ transplantation. Identification of biomarker genes closely associated with tolerance or rejection is critical for this monitoring protocol. Unlike previous studies, our microarray analysis focused on donor antigen-reactive T cells, which were prepared by collecting CD69+ T cells from cocultures of recipient peripheral T cells and donor antigen-presenting cells. A comparison of different recipient groups enabled us to identify several tolerance- and rejection-correlated biomarker genes, including previously unknown genes. By measuring biomarker gene expression in the CD69+ T cell fraction using quantitative reverse-transcription polymerase chain reaction, we were able to precisely detect the immune status of recipients relative to their graft. Full-thickness donor (BDF1; C57BL/6 x DBA/2) tail skin was grafted onto recipient (C57BL/6) mice. Tolerance to the graft was induced by donor specific transfusion combined with administration of anti-CD40L Ab (MR-1). Total T lymphocytes were prepared from spleen and lymph nodes of tolerant recipient mice on day 100 after skin transplantation, untreated recipient mice on day 7, and ungrafted, healthy C57BL/6 mice and used as tolerant, rejecting and naïve T lymphocyte samples, respectively. In order to isolate alloantigen-reactive cells, T lymphocytes were cocultured with donor T cell-depleted splenocytes for 20-21 hours and then separated into two subpopulations based on CD69 expression using fluorescence cell sorter. Total RNA was extracted from these CD69+ and CD69- T cells and subjected to microarray using Illunima MouseWG-6 Expression BeadChip. All data analysis and visualization of differentially expressed genes was conducted using Illumina GenomeStudio v2009.2 (Gene Expression Module v1.5.4) and R statistical language v2.6.1. Candidate tolerance genes were defined as the common genes satisfying the following criteria; 1) 1.5 fold or higher expression in CD69+ T cells isolated from tolerant recipients than those from naïve mice, 2) 1.5 fold or higher expression in CD69+ T cells than CD69- T cells from tolerant mice, 3) 500 or higher average signal measured in CD69+ T cells from tolerant mice. The criteria for candidate rejection genes were 1) 1.5 fold or higher expression in CD69+ T cells isolated from rejecting recipients than those from tolerant mice, 2) 1.5 fold or higher expression in CD69+ T cells than CD69- T cells from rejecting mice, 3) 500 or higher average signal measured in CD69+ T cells from rejecting mice.

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:Measurement of specific gene expression in clinical samples is a promising approach for monitoring the recipient immune status to the graft in organ transplantation. Identification of biomarker genes closely associated with tolerance or rejection is critical for this monitoring protocol. Unlike previous studies, our microarray analysis focused on donor antigen-reactive T cells, which were prepared by collecting CD69+ T cells from cocultures of recipient peripheral T cells and donor antigen-presenting cells. A comparison of different recipient groups enabled us to identify several tolerance- and rejection-correlated biomarker genes, including previously unknown genes. By measuring biomarker gene expression in the CD69+ T cell fraction using quantitative reverse-transcription polymerase chain reaction, we were able to precisely detect the immune status of recipients relative to their graft. Full-thickness donor (BDF1; C57BL/6 x DBA/2) tail skin was grafted onto recipient (C57BL/6) mice. Tolerance to the graft was induced by donor specific transfusion combined with administration of anti-CD40L Ab (MR-1). Total T lymphocytes were prepared from spleen and lymph nodes of tolerant recipient mice on day 21 and 100 after skin transplantation, untreated recipient mice on day 7, and ungrafted, healthy C57BL/6 mice and used as early tolerant, tolerant, rejecting and naïve T lymphocyte samples, respectively. In order to isolate alloantigen-reactive cells, T lymphocytes were cocultured with donor BM-DCs for 20-21 hours and then separated into two subpopulations based on CD69 expression using fluorescence cell sorter. Total RNA was extracted from these CD69+ and CD69- T cells and subjected to microarray using Illunima MouseRef-8 Expression BeadChip. All data analysis and visualization of differentially expressed genes was conducted using Illumina BeadStudio v3.1.3 (Gene Expression Module v3.3.8), Array Assist 5.5.1 (Stratagene) and R 2.4.1(www.r-project.org). Candidate tolerance genes were defined as the common genes satisfying the following criteria; 1) 1.5 fold or higher expression in CD69+ T cells isolated from tolerant recipients than those from naïve mice, 2) 1.5 fold or higher expression in CD69+ T cells than CD69- T cells from tolerant mice, 3) 500 or higher average signal measured in CD69+ T cells from tolerant mice. The criteria for candidate rejection genes were 1) 1.5 fold or higher expression in CD69+ T cells isolated from rejecting recipients than those from tolerant mice, 2) 1.5 fold or higher expression in CD69+ T cells than CD69- T cells from rejecting mice, 3) 500 or higher average signal measured in CD69+ T cells from rejecting mice.

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).

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