Since the first transgenic fish was produced in 1985, various transgenic fish had been created to meet different needs for human beings. In 2003, Guo et al. demonstrated that “All-fish” growth hormone (GH)-transgene promotes thymus development and thymocyte proliferation, and retards thymus degeneration. In order to find the molecular mechanism beneath the effect of GH on thymus development, a subtractive cDNA library of thymus in F3 generation of 4-month transgenic carp was constructed by suppression subtractive hybridization (SSH) techniques. After pre-screening and dot blot analysis, seven cDNA clones were eventually identified as differential expressed gene fragments, which were further confirmed with RT-PCR or virtual northern hybridization.
By using the technique of RACE, we have cloned complete cDNA sequences of differential expressed genes of CD8β、CD4、TCRγ、CD3γ/δ, which are all T cell marker genes. Tissue distribution of these genes in adult carp were analyzed by RT-PCR. Our results showed that they express predominantly in thymus, followed by intestine, gills.
The effects of GH on the generation of lymphoid cells during zebrafish development was studied by analyzing the expression of recombination activating gene 1 (Rag1) using whole mount in situ hybridization(WISH). Our results have shown that the expression of Rag1 in thymus at 72hpf can be enhanced by the GH mRNA (330pg) injection compared with the one of the control group, which indicating the stimulation of GH administration on the thymus development.
Detection with the CD8α and CD8β antisense RNA probes using WISH, the presence of CD8+ cells were found at intestine bulb and notochord in zebrafish at 4dpf and 5dpf stages. Furthermore, the corresponding signals could be largely suppressed by the treatment of 10μg/ml dexamethasone, an effective immune inhibitor, which suggesting the early CD8 positive cells developing in fish intestine and notochord area.
Plasmids that EGFP was driven by zebrafish CD8β5' genomic DNA fragment region from 500bp to 11kb were constructed. The strong enhanced GFP fluorescence could be observed at notochord of the CD8β5':EGFP transgenic zebrafish under fluorescence microscope.