Studies on macrophage-mediated compensatory growth and on embryonic growth: Development of a messenger-RNA phenotyping method

We have developed a technique called 'single-cell mRNA phenotyping' which has the characteristics of extreme sensitivity (as few as 10 copies of mRNA), rapid qualitative screening for multiple mRNA transcripts, and a resolution of less than 3-fold differences in RNA input. The technique consists of linked methods for purifying single cell RNA, reverse transcription primed by oligo dT or sequence specific primers, and sequence-specific primed polymerase chain reaction. We have used the technique to mRNA phenotype cells participating in compensatory and embryonic growth.;a. Macrophage growth factor expression in wound healing. Ablation of macrophages slows the regeneration of cells required for wound healing. The cell types which divide during wound healing in vivo are stimulated to divide by various growth factors in vitro. We tested the hypothesis that wound macrophages express these growth factors in vivo by isolating wound macrophages from sub-epidermal cylinders in mice and phenotyping their growth factor mRNA. We found that wound macrophages express TGF-;b. Other models of macrophage- and platelet-mediated compensatory growth. We have found that macrophages express growth factors which influence nerve regeneration that are expressed in regenerating nerve, and that stimulated macrophages liberate EGF (see Chapter 4 for detail). We have also found that megakaryocytes express TGF-;c. Preimplantation mouse embryo growth factor expression. We tested the hypothesis that mouse embryos might express endogenous growth factors by mRNA phenotyping. We found that preimplantation mouse embryos express TGF-...