Preliminary Study Of The Development Of Bcr Promotor-driven EGFP Expressing Transgenic Mice

The most important characteristic of Chronic Myeloid Leukemia is the existence of Ph chromosome, which is the result of t(9;22) reciprocal chromosomal translocation in a hematopoietic stem cell. The fusion gene results in expression of a chimeric protein that not only create a novel fusion protein but also places transcriptional control of abl expression under the bcr promoter. The bcr-abl protein is leukemgenic because the Abl-derived tyrosine kinase is constitutively activated, leading to cell malignant transformation by interfering with apoptosis and proliferation. People attempt to set up BCR-ABL transgenic mouse models with different promoters at all time, but so far no transgenic mouse which would develop disease like human chronic myeloid leukemia were obtained. Most mouse models which were developed in past resulted in acute pre-B cell lymphoblastic leukemia. It suggests that those selected promoter might not direct the P210bcr-abl special expression in haematopoietic stem cell and myeloid progenitors.As the BCR-ABL fusion gene's natural promoter, bcr promoter should be the preferred promoter for BCR-ABL transgenic mouse model. However, whether the promoter has specificity of tissues and organs has not been reported in domestic and foreign literature. In this research, we made bcr-EGFP transgenic mice by means of microinjection to study this promoter's specificity in vivo level, and the ultimate goal is to find a right promoter for BCR-ABL transgenic mouse model.1, Construction and verification of eukaryotic expression vector pbcr-EGFP: The 1.1 kb bcr promoter was amplified from total RNA of K562 cells(chronic myeloid leukemia cell lines) by RT-PCR and was cloned into the upstream of EGFP reporter gene in pEGFP-N1 vector replacing its CMV promoter. The recombinant plasmid pbcr-GEFP was transfected into K562 cells and NIH3T3 cells with liposome. After 24 hours the expression was observed with fluorescent. The results showed that we constructed this vector successful.2, The generation of bcr-EGFP transgenic mice:1486 eggs of C57BL/6 were microinjected with the 2.1 kb fragment digested from the expression vector pbcr-EGFP with AseⅠand AflⅡ. 683 eggs were transferred into 35 forster mothers, 30 of which were pregnant and 110 pups were obtained, among which 3 pups (1♀, 2♂) were transgenic mice. 4 pups of No.4(♀) were transgenic mice, the rate of integration was 44.4% (4/9); 5 pups of No. 81(♂) were transgenic mice, the rate of integration was 50.0% (5/10); No.36(♂) transgenic mouse has no pups currently.We made blood smears with tail blood from F0 transgenic mice. Observing this smears by fluorescence microscope, the results showed that there were no expression of EGFP. Further analysis of specific reasons are needed.