Construction Of Drosophila Blood Development Candidate Gene CG8419 Transgenic Flies And Its Founctional Research

The CG8419 gene (a vertebrate homolog gene for TRIM45) was cloned and identified in our lab. It is a candidate gene which regulates development of hematopoietic stem cell. Bioinformatic analysis show that this gene has ring type zinc finger, B box type zinc finger, helix curly helix structure domain (ternary complex) and IG-FLMN protein structure domain. My room previous studies suggest that CG8419 gene may be associated with the development of hematopoietic cells.Extracted the RNA of wild type fruit flies, then reverse transcription it into cDNA. we design the primer clone the CG8419 gene of fruit flies, and we link the proliferation of genes and pMD18-T cloning vector together, establish a pMD18T-CG8419 plasmid and cut the process from the plasmid by use the enzyme of EcoRI and XhoI, translate the CG8419 gene into the plasmid pUAST expression vector and form the pUAST-CG8149 restructuring plasmid, mixing the pUAST-CG849 restructuring plasmid and p Δ 2-3 transposase plasmid, microscopic injection it into the 0 h-0.5 h fertilization embryos of white eye flies, and established 8 transgenic strains from the offspring through the mini-white in mark (orange eye) screening.In order to detect the insert point of CG8419 genes in transgenic strains, we cross eight transgenic strains with sco/cyo and double balance of sco/cyo; TM6/TM3 hybrid, then we find the genetically modified sco/cyo homozygous form, the results show that the CG8419 gene build in the third chromosome.In order to detect each genetically modified strain CG8419 in gene expression, the homozygous transgenic strains and Actin5c-GAL4 strains were hybrid, collect progeny 0 h-12 h embryos, protein extraction and Western Blot test, the result showed that in the no.2 in the strains CG8419 protein expression have increased significantly.In order to research whether the CG8419 gene’s over-expression cause the increase of blood cells or not, we cross 8 transgenic strains pure fit and W*; He-GFP hybrid strains, then check the distribution of larvae blood cells in fluorescence microscope, the result shows that the no.2 strain in their number increased obviously.Preparation mrj polyclonal antibody and detection CG8419 transgenic strains mrj in expression, found in CG8419 transgenic strains mrj expression in quantity rises.All in all, this paper successfully constructed 8 CG8419 gene genetically modified fly stocks, confirm the genetically modified chromosome loci, the CG8419 genes which build in the genome of transgenic strains can excessive expression, the over-expression of CG8419 gene can lead the hemocyte’s proliferation, it shows that CG8419 genes relate the development of drosophila blood cells.