1. Conditional Knockout Of The First Exon Of Bcr/Abl Fusion Gene In Chronic Myeloid Leukemia 2. Application Of RNAi In Mammalian Animal Cells

Chronic myeloid leukemia (CML) is characterized by the reciprocal translocation of chromosome 9;22, which caused the bcr gene on chromosome 9 fused to the abl gene on chromosome 22. It has been reported that proteins encoded by Bcr/Abl fusion gene had constitutive kinase activity and was the main cause of CML. Further studies showed that specific domains coding by the first exon of bcr gene in bcr/abl (bE1) played a key role in the activation of proto-oncoprotein c-Abl. Therefore, it is necessary to explore the detailed effets of bE1 for better understanding of functions of bcr/abl oncoprotein and its roles in CML pathogenesis and progressions. In this study, a Cre-loxP system was applied to conditionally knockout bE1 gene in CML cell line K562 and changes of K652 in proliferation and carcinogenesis would be observed during gene targeting.Effectiveness of stuffs applied in the gene targeting systems was analyzed at first. 1) Chromosome kayrotyping analysis and fluorescent in situ hybridization (FISH)was performed to observe the chromosome variations and the distributions and copies of bcr/abl in K562 cells. Kayrotyping analysis showed that there were 67~69 chromosomes in K652 cells but Ph' chromosome did not exist among them. Results from FISH showed that there were more than three copies of bcr/abl or abl/bcr fusion gene in K562 cells , either bcr, abl or fusion genes were over-duplicated. It was suggested that K562 could be a useful tool in study of bcr/abl gene targeting. 2) To establish a controllable gene targeting system, vectors expressing rtTA protein and cre recombinase were constructed. Tetracycline-responsive elements (TRE) were also inserted into the cre-expressing vector so that expressions of cre would be under the control of doxycycline(Dox, derivative of tetracycline). As described previously, Dox is inducer of rtTA regulator. Binding of Dox to rtTA could effectively activate rtTA and activated rtTA then binds to TRE sequence, inducing downstream expressions. Levels of cre expressions induced by Dox were detected by western blot 48h after combined transfection of rtTA and cre expressing plasmids. Results showed that Dox induced the expression of cre in a dose-dependent manner, suggesting a controllable cre-expression system was successfully established. 3) To observe the carcinogenesis effects of K562 cells on NOD/SCID (non-obese diabetes/...