Studies On Alternative Splicing Of Beclin1Gene And Generation Of Beclin1Conditional Knockout Mice

Part1Studies on alternative splicing of Beclin1geneObjective: To explore alternative splicing of Beclin1gene and its role in theregulation of autophagy in human B-acute lymphoblastic leukemia697cells.Methods:(1) Total RNA from697cells were extracted and reversely transcribed into cDNA.(2) The cDNA was used as the template for PCR amplication. The PCR primerswere located at5’UTR and3’UTR of Beclin1gene, respectively. PCR products weresequenced and aligned by DNAMAN Software.(3) Molecular mechanisms of Del-E11viriable transcript were analysed.(4) Lenti-GFP-Beclin1and Lenti-GFP-Del-E11expression vectors wereconstructed and transfected into Hela cells, and subcellular localization of fusionproteins were confirmed using confocal microscope.(5) Lenti-Beclin1and Lenti-Del-E11expression vectors were constructed andtransfected into697cells. Alternative splicing of Beclin1and its role in the regulation ofautophagy were tested by western blot and Co-IP assay.Results:(1) A transcript variant of Beclin1gene carrying a deletion of exon11, whichtermed as Del-E11, was found in human B-acute lymphoblastic leukemia697cells.Del-E11was shorter for95amino acid than its consensus sequence.(2) Lentiviral expression vectors of GFP-Beclin1and GFP-Del-E11fusion genewere successfully constructed. Vectors were packaged and virus were infected into Helacells, and finally obtained Hela-GFP-Beclin1and Hela-GFP-Del-E11cells. Fusionproteins were confirmed using western blot.(3) Lentiviral expression vectors of Beclin1and Del-E11gene were successfully constructed. Vectors were packaged and virus were infected into697cells, and finallyobtained697-Beclin1and697-Del-E11cells. Western blot results showed that Beclin1and Del-E11gene were normally transcribed and translated.(4) Subcellular localization of fusion proteins were confirmed using confocalmicroscope. Both the Beclin1and Del-E11fusion protein were found to be distributedin the cytoplasm of the cells.(5) Del-E11displayed a reduced activity in the induction of autophagic response.Conclusions:(1) Bioinformatics analysis suggested that alternative splicing of Beclin1pre-mRNA gives rise to a deletion of exon11and generates a C-terminal truncation ofBeclin1isoform, which termed as Del-E11.(2) Both the Beclin1and Del-E11fusion proteins were found to be distributed inthe cytosplasm of the cells, which indicated that there were no difference betweenBeclin1and Del-E11protein in distribution in cells.(3) Del-E11had lower binding affinity to Vps34, and failed to activate autophagicresponse. Part2Generation of Beclin1gene conditional knockout miceObjective: To generate Beclin1gene conditional knockout mice.Methods:(1) NeoRmurine embryonic fibroblasts as the feeder layer cells of e mbryonic stemcells were prepared.(2) Beclin1target vector was linearized by restriction enzyme AsiSI.(3) ES cells were cultured, electroporated, durg selected and genetyped for positiveES clones.(4) Vasectomized male mice and foster female mice were prepared.(5) The stage of estrous cycle in female mice were assessed. And superovulationwere induced according to estrous cycle.(6) Mice embryo were collected, microinjected and then transferred to the wombof the foster mice.(7) Chimeric male mice were screened and bred with C57BL/6J female mice. (8) N4generation of Beclin1f/+mice were genotyped and bred.(9) Vav-Beclin1+/-mice were genotyped and screened.Results:(1)18positive ES clones were genotype identificated from144ES cells.(2)3positive ES clones were selected, microinjected into145blastocysts, and thentransplanted to10foster mice. These blastocysts developed in the recipient mice tilldelivery and the offspring were chimeric mice. And finally we got24chimeras, inwhich14were male mice,10were female mice.(3)4high-degree chimeric male mice were selected and bred with C57BL/6J mice,and finally got19offspring mice, including12flox mice.(4)4positive Vav-Beclin1+/-mice were genotyped and screened.Conclusions: Beclin1gene conditional knockout mice model and Beclin1+/-hematopoietic system-specific mutant mice were successfully established.