人ATP7B转基因大鼠模型的建立及其在转基因大鼠体内的表达分析

Establishment of transgenic rat Expressing of Human ATP7B.

目的利用受精卵的雄性原核显微注射法,建立人ATP7B转基因大鼠模型。方法将构建7.1kb含有鸡β肌动蛋白启动子的人正常ATP7BcDNA,经显微注射法导入Wilson’s病动物模型LEC(Long-Evans Cinnamon)大鼠的受精卵,利用PCR法筛选、鉴定阳性转基因大鼠,以RT-PCR和Western bolt检测人ATP7B在转基因大鼠体内的表达,并从血液生化指标和临床症状上对16周龄内的阳性转基因大鼠进行分析。结果建立了3株独立的转基因大鼠株系,其中2株转基因大鼠的肝组织中可检测到完整的人ATP7B基因表达产物,血液生化和临床指征显示2株转基因大鼠具有明显的ATP7B相关功能恢复表型。结论人ATP7B在转基因大鼠的肝组织中获得完整和正确的表达,其弥补了转基因大鼠内源性Atp7b基因功能的缺陷。

Objective To establish the transgenic rat harboring the human ATP7B gene by using prenuclear microinjection.Methods The 7.1 kb transgene consisting of human ATP7B cDNA under the control of chiken β-actin promoter was introduced into the LEC rats which is an animal model of Wilson' s disease by microinjection.The positive transgenic rats were identified by PCR using the transgene specific primers.The expressions of human ATP7B transcript and protein in the transgenic rats were detected by RT-PCR and Western blot.The biochemical and clinic features relative to ATP7B phenotypes in transgenic rat were further investigated. Results Three ATP7B transgenic rat lines were established from the indedpendent founders.The expressions of human ATP7B mRNA and protein were detected in the liver of the two transgenic rat lines, which resulted in the restoration of ATP7B functions in both of the two lines.Consequently, AST and ALT were dramatic decreased, while the cerulo-plasmin ferroxidase activity and copper concentration were significantly increased in the plasma of transgenic rats.The transgenic rats did not showed clinic abnonnal till the age of 16 weeks as compared with the age matched non-transgenic rats. Conclusions The intact and correct product derived from human ATP7B gene compensated for the deficincy of the endogenous rattus protein and did function in transgenic rats.