检测Cre重组酶活性的转基因报告猪的建立
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摘要
Cre/LoxP系统主要应用于制备人类疾病模型鼠,因此检测Cre重组酶活性的报告动物品系也全部是小鼠。由于猪与人类在解剖结构、生理学等更为接近,因此利用Cre/LoxP系统构建转基因疾病动物模型及其相应的转基因报告猪具有重要意义。为了实现Cre/LoxP转基因猪在人类疾病动物模型的应用,本实验构建了用于检测Cre重组酶活性的转基因报告猪。
首先构建条件性表达绿色荧光蛋白的真核表达载体,由CMV启动子调控条件性表达绿色荧光蛋白,两个同向的LoxP位点锚定一个筛选基因与终止子,当Cre重组酶不存在时,绿色荧光蛋白由于和启动子之间有终止子的存在,绿色荧光蛋白不表达;当Cre重组酶存在时,将LoxP间的序列删除,CMV启动子启动绿色荧光蛋白的表达。
将所构建的条件性表达绿色荧光蛋白的载体线性化后,用脂质体法转染到猪胚胎成纤维细胞,通过G418筛选,获得可以检测Cre重组酶活性的猪胚胎成纤维细胞,作为体细胞核移植的供体细胞。对所筛选的转基因猪胚胎成纤维细胞在基因水平、转录水平、表达水平进行了检测。并通过体细胞核移植技术,获得了11头健康的克隆猪。对所获得克隆猪进行PCR鉴定,并分离新生克隆猪尾部成纤维细胞,进行Cre重组酶的验证实验,鉴定结果表明,所获得的克隆猪均为转基因克隆猪。
The Cre-LoxP system has been developed as a powerful tool for manipulating DNA both in vitro and in vivo. Though standard gene knockouts have been highly informative, early embryonic lethality or complex phenotypes often obscure the roles of genes studied during later stages of development or in specific tissues. Conditional gene targeting provides a means to circumvent certain limitations of conventional gene targeting. the pig is thought to be the most suiTab non-human source of organs for xenotransplantation, and it is becoming more widely used as a model of human disease. Effective use of conditional Cre recombinase-LoxP gene modification requires Cre-expressing porcine strains with defined patterns of expression, for example by the use of a tissue-specific promoter. Such pigs would be a powerful tool for studying human gene expression. In order to monitor recombinant Cre expression, it is important to create reporter strains.
At first, we constructed a vector, in this vector, the EGFP gene is regulated by a CMV promoter, but its expression is inhibited by a LoxP-flanked intervening cassette containing the neo gene, followed by transcriptional termination sequences. The final vector was named pICE-STOP. To generate transgenic cells, porcine fetal fibroblasts were seeded in a 60mm dish at a density of 2×105 cells per dish on the day before transfection. Cells were cultured in DMEM supplemented with 15% fetal bovine serum and incubated at 39 oC. For the production of stably trasfected cell lines, the pICE-STOP plasmids were digested with NheI. On the following day, the pICE-STOP fragment was incorporated into Lipofectamine 2000 ? reagent, after cells were cultured in selection medium containing 300μg /ml of G418 antibiotic for an additional 7 days. The surviving cell colonies were picked and propagated and the cells were then frozen to be used as the donor cells.In order to examine if the gene have intergrated into genome, the genomic DNA of clones was used for PCR amplification and intergratd sites analyzed by high-efficiency thermal asymmetric interlaced PCR.
To examine excision-activated EGFP expression in these cells, the clones were individually assayed for expression of EGFP following infection with Ad-Cre at an MOI of 800 for 72 h. Images were collected on a laser scanning confocal microscope .At 72 h after Ad-Cre transfection, levels of EGFP mRNA were measured by RT–PCR using theprimers selected for detection of the EGFP sequence. FACS analysis was also used to characterize the expression of EGFP.
A positive cell clone was used to construct a transgenic pig by nuclear transfer (NT). To determine whether Cre can regulate the expression of EGFP, transgenic pig cells were isolated from tails that were infected with recombinant Cre adenovirus.Two days after rAd infection, EGFP gene expression was assessed with a laser scanning confocal microscope. Total RNA was purified and RT-PCR was performed.
We generate transgenic reporter pigs, which can be used to monitor Cre-mediated excision. The EGFP gene is expressed only after Cre-mediated excision of LoxP-flanked stop sequences. We expect that this reporter strain could facilitate the in vivo monitoring of Cre-mediated excision events in a variety of experimental contexts.
首先构建条件性表达绿色荧光蛋白的真核表达载体,由CMV启动子调控条件性表达绿色荧光蛋白,两个同向的LoxP位点锚定一个筛选基因与终止子,当Cre重组酶不存在时,绿色荧光蛋白由于和启动子之间有终止子的存在,绿色荧光蛋白不表达;当Cre重组酶存在时,将LoxP间的序列删除,CMV启动子启动绿色荧光蛋白的表达。
将所构建的条件性表达绿色荧光蛋白的载体线性化后,用脂质体法转染到猪胚胎成纤维细胞,通过G418筛选,获得可以检测Cre重组酶活性的猪胚胎成纤维细胞,作为体细胞核移植的供体细胞。对所筛选的转基因猪胚胎成纤维细胞在基因水平、转录水平、表达水平进行了检测。并通过体细胞核移植技术,获得了11头健康的克隆猪。对所获得克隆猪进行PCR鉴定,并分离新生克隆猪尾部成纤维细胞,进行Cre重组酶的验证实验,鉴定结果表明,所获得的克隆猪均为转基因克隆猪。
The Cre-LoxP system has been developed as a powerful tool for manipulating DNA both in vitro and in vivo. Though standard gene knockouts have been highly informative, early embryonic lethality or complex phenotypes often obscure the roles of genes studied during later stages of development or in specific tissues. Conditional gene targeting provides a means to circumvent certain limitations of conventional gene targeting. the pig is thought to be the most suiTab non-human source of organs for xenotransplantation, and it is becoming more widely used as a model of human disease. Effective use of conditional Cre recombinase-LoxP gene modification requires Cre-expressing porcine strains with defined patterns of expression, for example by the use of a tissue-specific promoter. Such pigs would be a powerful tool for studying human gene expression. In order to monitor recombinant Cre expression, it is important to create reporter strains.
At first, we constructed a vector, in this vector, the EGFP gene is regulated by a CMV promoter, but its expression is inhibited by a LoxP-flanked intervening cassette containing the neo gene, followed by transcriptional termination sequences. The final vector was named pICE-STOP. To generate transgenic cells, porcine fetal fibroblasts were seeded in a 60mm dish at a density of 2×105 cells per dish on the day before transfection. Cells were cultured in DMEM supplemented with 15% fetal bovine serum and incubated at 39 oC. For the production of stably trasfected cell lines, the pICE-STOP plasmids were digested with NheI. On the following day, the pICE-STOP fragment was incorporated into Lipofectamine 2000 ? reagent, after cells were cultured in selection medium containing 300μg /ml of G418 antibiotic for an additional 7 days. The surviving cell colonies were picked and propagated and the cells were then frozen to be used as the donor cells.In order to examine if the gene have intergrated into genome, the genomic DNA of clones was used for PCR amplification and intergratd sites analyzed by high-efficiency thermal asymmetric interlaced PCR.
To examine excision-activated EGFP expression in these cells, the clones were individually assayed for expression of EGFP following infection with Ad-Cre at an MOI of 800 for 72 h. Images were collected on a laser scanning confocal microscope .At 72 h after Ad-Cre transfection, levels of EGFP mRNA were measured by RT–PCR using theprimers selected for detection of the EGFP sequence. FACS analysis was also used to characterize the expression of EGFP.
A positive cell clone was used to construct a transgenic pig by nuclear transfer (NT). To determine whether Cre can regulate the expression of EGFP, transgenic pig cells were isolated from tails that were infected with recombinant Cre adenovirus.Two days after rAd infection, EGFP gene expression was assessed with a laser scanning confocal microscope. Total RNA was purified and RT-PCR was performed.
We generate transgenic reporter pigs, which can be used to monitor Cre-mediated excision. The EGFP gene is expressed only after Cre-mediated excision of LoxP-flanked stop sequences. We expect that this reporter strain could facilitate the in vivo monitoring of Cre-mediated excision events in a variety of experimental contexts.
引文
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