bcr启动子驱动增强绿色荧光蛋白表达转基因小鼠的制备和初步鉴定

Preliminary Study on Development of bcr Promotor Driven EGFP Expressing Transgenic Mice

目的制备bcr启动子驱动．增强绿色荧光蛋白（bcr—EGFP）转基因小鼠模型，在活体水平验证bcr启动子的启动特异性。方法以慢性髓性细胞白血病（cML）细胞株K562细胞基因组为模板，PCR扩增1．1kbbcr启动子，在扩增产物的上、下游引物加入了AseI、EcoRI酶切位点．AseI、EcoRI双酶切pEGFP—N1真核表达质粒载体，去除载体自身的CMv启动子，并将1．1kbbcr启动子定向克隆到EGFP绿色荧光蛋白报告基因上游，构建pbcr-EGFP真核表达载体，并将此重组质粒瞬时转染K562细胞和NIH3T3细胞，进行表达验证。显微注射法制备bcr-EGFP转基因小鼠，采用PCR方法进行初步整合检测。结果显微注射583枚受精卵，移植到30只受体鼠输卵管中，受体鼠妊娠26只，共生产首建鼠90只，经过PCR检测，甜（♀）、36挣（♂）和81#（♂）等3只F0代为转基因整合阳性鼠。结论成功制备了bcr—EGFP转基因小鼠，为进一步研究bcr启动子在CML转基因小鼠模型制备的安全性和可靠性提供线索和帮助。

Objective To establish the bcr promoter drived green fluorescerce protein（bcr-EGFP） transgenic mouse model and to analyze the specificity of bcr promoter for further in vivo study. Methods The 1.1 kb bcr promoter was obtained from the genome of K562 cells （chronic myeloid leukemia cell line）, at the same time, two restriction sites of Ase I, EcoR I were introduced into the up and down streams of this promoter. The CMV promoter of pEGFP-N1 plasmid was replaced by 1.1 kb bcr promoter to restructure pbcr-EGFP plasmid. Transfect the new recombinant plasmid into K562 cells and NIH3T3 cells by liposomes, after 24 hours, the expression was observed with fluorescentce. The results showed that this vector was constructed successfully. The bcr-EGFP recombinant fragment was obtained from pbcr-EGFP plasmid, and used to establish bcr-EGFP transgenic mouse by microinjection and the integration of target gene was detected by PCR. Results Total of 583 eggs of C57BL/6 were microinjected with the bcr-EGFP fragment. These eggs were transferred into 30 foster female mice, and 26 of which were pregnant and 90 pups were obtained, among which 3 pups （1♀,2♂ ） were transgenic mice. Conclusion The successful establishment of bcr-EGFP transgenic mouse model has paved the way for future study of the specificity of bcr promoter.