条件性缺氧诱导因子-1αRNAi转基因小鼠模型的建立

Generation of Inducible HIF-1α RNAi Transgenic Mice

缺氧诱导因子1α(hypoxia inducible factor-1α,HIF-1α)是细胞在缺氧等条件下稳定表达的具有转录活性的蛋白,通过与多种靶基因调控区的缺氧反应元件(hypoxia response element,HRE)结合,调控靶基因表达,使机体对缺氧、缺血等病理生理过程产生适应性反应。为从整体动物水平研究HIF-1α的作用,需要建立HIF-1α相关遗传修饰小鼠。分别针对HIF-1αmRNA序列的两个靶位点合成两对互补的寡核苷酸链,构建可诱导的RNA干扰真核表达载体HIF-AB和HIF-CD。分别将CRE重组酶真核表达载体CRE-ER^(T2)与HIF-AB或HIF-CD转染入RAW264.7细胞,筛选得到稳定表达CRE-ER^(T2)与HIF-AB,或CRE-ER^(T2)与HIF-CD的稳定细胞系。在用4-HT诱导去除上述细胞系中HIF-AB或HIF-CD所含的Neo基因后,用CoCl_2诱导HIF-1α表达,采用半定量RT-PCR检测HIF-AB或HIF-CD对HIF-1α基因表达的影响。结果发现干扰载体(HIF-AB和HIF-CD)对HIF-1αmRNA序列的沉默效果分别为85%和72%。选择干扰效率较高的表达载体HIF-AB经显微注射获得HIF-1α基因敲低小鼠模型,经PCR以及测序验证获得2个转基因阳性小鼠(Founders,G_0代)。G_0代雄鼠与FVB/N雌鼠交配后获得2只F_1代(first filial generation)转基因阳性小鼠,经与EIIA-Cre转基因小鼠交配,得到EIIA-Cre;HIFRNAi^(flox/+)小鼠,RT-PCR结果显示,EIIA-Cre;HIFRNAi^(flox/+)小鼠肝、肺、肾等组织的HIF-1αmRNA水平明显降低,分别约为正常对照的44%、38.2%和23.5%。该小鼠模型的建立为进一步研究HIF-1α的功能及作用机制提供了新的手段。

Hypoxia inducible factor-1α （HIF-1α） is a transcription factor that responds to changes in oxygen concentration. In this study, we constructed two vectors, HIF-AB and HIF-CD, to transcribe functional short interfering RNA against different region of mouse HIF-1 tx. The oligonucleotide encoding small hairpin RNAs against mouse HIF-1 tx was inserted into the downstream of U6 promoter of pBSK/U6-NEO plasmid. Cre-expression vector CRE-ERr2 with either HIF-AB or HIF-CD was transfected into RAW264.7 cell line, after selection with G418 and hygromycin, to obtain cell lines with stabilized expression of CRE-ERT2 and HIF-AB, or CRE-ER^T2 and HIF-CD. The expression levels of HIF-1α of these stable cell lines were detected by semi-quantitative RT-PCR following treatment of COCl2, a HIF-1 a expression inducer. The HIF-1α mRNA expression was reduced by 85% and 72% by HIF-AB and HIF-CD respectively. HIF-AB vector was microinjected into pronucleus of zygotes to generate transgenic mice. We got two founder and two ftrst filial generation transgenic mice containing HIF-AB and these transgenic mice were crossed with EIIA-Cre transgenic mouse to get EIIA-Cre;HIFRNAiflx/＋ mice. The conditional knock down mice were viable and developed normally. The results of RT-PCR indicated that the HIF-1α mRNA expression of liver, lung and kidney were reduced significantly compared with the normal control.