PCR Analysis Of Gene Expression Research Articles

TWO DISCRETE WAVES OF T CELL ACTIVATION FOLLOWING TRANSPLANTATION OF A SKIN ALLOGRAFT.

P1010 Aims: To visualize the response of alloreactive CD8+ T cells during the rejection of a skin allograft. Methods: 1x105 BM3 H2Kb-reactive TCR-transgenic CD8+ T cells (Tg-TCR+ T cells) were CFSE labeled and adoptively transferred into syngeneic CBARAG−/− mice one day before transplantation of a H2Kb+ skin allograft. Mice that received T cells but no skin graft or a skin graft in the absence of T cells served as controls. The activation and proliferation of the alloreactive T cells was followed in the axillary lymph nodes (draining (dLN) and contra-lateral (cLN)), mesenteric lymph nodes (MLN) and spleen 5, 10 and 15 days after transplantation. Skin allografts were harvested at the same timepoints for real-time PCR gene expression analysis and immunohistochemistry. Results: CBARAG−/− mice that had received Tg-TCR+ T cells acutely rejected B10 skin allografts (MST=23 days; n=6) whereas allografts transplanted onto unreconstituted CBARAG−/− recipients were accepted indefinitely (MST>100 days; n=4). 5 days after transplantation, Tg-TCR+ CD8+ T cells were found to have proliferated and expanded specifically in the dLN (the level of CFSE demonstrated on average 6 rounds of division). In contrast, 10 days after transplantation the CFSE+ T cells had the same level of fluorescent of CFSE in the dLN as those in the cLN, MLN and spleen, indicating that there was no priming of naive T cells at this time. However, by day 15 a further wave of T cell activation had taken place resulting in a dramatic expansion of T cells in the dLN (10 fold) and once again the level of CFSE on CFSE+ T cells was substantially reduced (30%+/−17% compared to CFSE+ T cells in the other lymphoid organs or untransplanted mice; n=3). CD3 mRNA expression and staining for CD8+ cells by immunohistochemistry confirmed that the alloreactive CD8+ T cells had infiltrated the skin allograft following the initial wave of T cell activation and were further increased following the second wave of priming. The infiltration of CD8+ T cells correlated with a profound increase in the mRNA expression of a number of proinflammatory molecules such as IFNγ (499 fold), Perforin (366 fold), RANTES/CCL5 (26 fold) and Lptn/XCL1 (102 fold) in rejecting skin grafts compared with expression levels in skin grafts transplanted onto unreconstituted CBARAG−/− mice (2-3 mice / group). Conclusions: Upon transplantation of an allogeneic skin graft we found that T cell priming was initiated around day 5 which was followed by a second wave of priming that occured between 10 and 15 days after transplantation. To our knowledge this is the first report demonstrating the existence of two waves of CD8+ T cell priming following skin transplantation.

Robust levels of long-term multilineage reconstitution in the absence of stem cell self-replication in W/Wv mice transplanted with purified stem cells.

Isolation of primitive blood stem cells by different methods results in cell populations with distinct biological activities. This study was aimed at resolving differences in the frequency of multilineage reconstituting cells (MRC) and their precursors (pMRC) in cell populations isolated by positive selection for Sca-1 compared to those isolated by negative selection for 15-1.1. Separation of wheat germ agglutinin-positive mouse bone marrow cells into 15-1.1neg or Sca-1+ subsets was performed by flow cytometry. The isolated cells were transplanted into W/Wv or normal irradiated recipient mice and reconstitution was evaluated over time. Sca-1+ cells were less frequent and contained more MRC than 15-1.1neg cells, while pMRC were found mainly among 15-1.1neg cells. MRC activity was exclusively contained within the Sca-1+ subpopulation of 15-1.1neg cells, but marrow from 7 robustly engrafted W/Wv mice did not contain donor-derived MRC, indicating that 15-1.1negSca-1+ cells contain low numbers of pMRC. Functional differences between 15-1.1neg and Sca-1+ cells were further confirmed by reverse transcriptase (RT)-PCR gene expression analysis. Early hematopoiesis-specific transcription factors (Scl, Gata-2, and Gata-1) were amplified from cDNA prepared from Sca-1+ but not 15-1.1neg cells. This study indicates that cell populations isolated as Sca-1+ are functionally distinct from those isolated as 15-1.1neg in that few pMRC are included among Sca-1+ cells and that MRC and pMRC are two distinct and separable cell populations.

Isolation, cDNA cloning and gene expression of an antibacterial protein from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros.

An antibacterial protein, designated rhinocerosin, was purified to homogeneity from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros immunized with Escherichia coli. Based on the amino acid sequence of the N-terminal region, a degenerate primer was synthesized and reverse-transcriptase PCR was performed to clone rhinocerosin cDNA. As a result, a 279-bp fragment was obtained. The complete nucleotide sequence was determined by sequencing the extended rhinocerosin cDNA clone by 5' rapid amplification of cDNA ends. The deduced amino acid sequence of the mature portion of rhinocerosin was composed of 72 amino acids without cystein residues and was shown to be rich in glycine (11.1%) and proline (11.1%) residues. Comparison of the deduced amino acid sequence of rhinocerosin with those of other antibacterial proteins indicated that it has 77.8% and 44.6% identity with holotricin 2 and coleoptrecin, respectively. Rhinocerosin had strong antibacterial activity against E. coli, Streptococcus pyogenes, Staphylococcus aureus but not against Pseudomonas aeruginosa. Results of reverse-transcriptase PCR analysis of gene expression in different tissues indicated that the rhinocerosin gene is strongly expressed in the fat body and the Malpighian tubule, and weakly expressed in hemocytes and midgut. In addition, gene expression was inducible by bacteria in the fat body, the Malpighian tubule and hemocyte but constitutive expression was observed in the midgut.

Rapid generation of homologous internal standards and evaluation of data for quantitation of messenger RNA by competitive polymerase chain reaction

Sensitive and quantitative measurement of messenger RNA (mRNA) is important for accurate assessment of gene expression. Conventional methods of mRNA measurement frequently lack the sensitivity required to detect mRNA expressed at low level, such as mRNA encoding receptors and intracellar signaling molecules. Thus, the extremely sensitive RT-PCR has become the method of choice for examination of gene expression. However, quantitation of mRNA by PCR is difficult because small variations in amplification efficiencies among sample tubes can lead to substantial differences in product yield, thereby rendering direct comparisons between samples invalid. Development of protocols for quantitative RT-PCR has relied on internal standards to monitor the efficiency of the RT-PCR in different reaction tubes. Technically, the two most serious limitations to routine successful application of competitive quantitative PCR is ready access to competitive internal standards and efficient methods for accurate quantitative analysis of the data. In the present manuscript, application and validation of a simple approach to generate homologous internal competitive standards and to quantitate data for rapid, accurate determination of the expression level of genes by quantitative PCR is described. Generation of the competitive standard from a previously amplified PCR product by the methods described requires only one additional primer pair, and an additional two-step reaction; it can be completed in 1–2 days. Analyzing the results of the competitive PCR reaction via phosphoimager analysis provides a simple, rapid method for accurate quantitation of results. Data presented here clearly illustrate that the methods described have been successfully applied, and that they should have wide application for competitive quantitative PCR analysis of gene expression.