慢病毒载体介导的转人ahsp基因的β654地中海贫血小鼠的制备

目的：经慢病毒载体介导,制备转人α血红蛋白稳定蛋白基因（ahsp）的β654地中海贫血小鼠。方法：用巢式PCR从人血DNA中获得人ahsp基因,构建含有人ahsp基因的慢病毒载体,制备假病毒,通过卵周隙显微注射手段将其导入β654地贫小鼠的受精卵,经移植至假孕母鼠输卵管,最终孕育出转人ahsp基因的β654地贫小鼠;分析小鼠体内外源ahsp基因的表达情况及其遗传稳定性。结果：共获得了8只人ahsp阳性小鼠,转基因阳性率为32%（8/25）,其中3只同时具有β654突变基因;人ahsp基因在小鼠体内的表达水平维持在小鼠自身ahsp表达量的1%左右,且可稳定遗传至子代。结论：制备了转人ahsp基因小鼠,并可遗传至子代,为在个体水平上研究α血红蛋白稳定蛋白与β地贫之间的关系提供了工具。     

人地中海贫血β654基因作为外源基因用于转基因的制备方法

目的探讨人地中海贫血β654基因为外源基因用于转基因小鼠制作时,外源基因的制备方法,为进一步制作转基因小鼠打下基础。方法采用反向点杂交法确诊人地中海贫血β654纯合子DNA,以PCR法扩增获得人地中海贫血β654基因,分离纯化后,通过连接酶反应将其克隆入含βLCR调控序列的基础载体pBGT51质粒中,经PCR扩增、酶切、反向点杂交及DNA测序鉴定重组质粒,用EcoRⅤ酶切获得转基因所用的外源基因。结果将人地中海贫血β654基因作为外源基因克隆入基础载体pBGT51质粒中构建了重组质粒βBGT51,用EcoRV酶切获得含人β654基因及βLCR调控序列的6.5kb的DNA片段,制备了可用于显微注射转基因的外源基因。结论采用该方法构建的含人地中海贫血β654基因的重组质粒,可获得用于显微注射转基因的外源基因。     

2型腺相关病毒载体介导正常人β珠蛋白基因在重型地贫流产胎儿造血细胞中的表达

目的:探讨2型重组腺相关病毒(recombinant adeno-associated virus 2,rAAV2)载体介导人β-珠蛋白基因转染地贫患者造血细胞治疗β-地中海贫血的可行性.方法:分离β 41-42/β 654杂合子型重型β-地贫流产胎儿造血细胞,经rAAV2-β-globin病毒转染(MOI=50)后移植入经X射线照射的BALB/c裸小鼠体内,分别于移植后14d、21d处死受体小鼠,RT-PCR及等位基因特异性PCR法检测人珠蛋白基因在受体小鼠体内的表达.结果:RT-PCR方法于3只受体小鼠骨髓样本中成功检测到人β-actin和人β-珠蛋白基因的表达,而21d处死的转染与对照小鼠外周血样本中未检测到人β-珠蛋白基因表达;等位基因特异性PCR方法在所有受体鼠体内同时检测到β 41-42和β 654突变基因,以及正常β-珠蛋白基因的表达,但rAAV2-β-globin转染组小鼠体内正常人β-珠蛋白基因表达水平明显高于对照组.结论:rAAV2可有效转染β 41-42/β 654杂合子型重型地中海贫血患者造血细胞,并通过exvivo途径介导β-珠蛋白转基因的体内表达,提高红系细胞内正常β-珠蛋白基因的表达水平.     

红系特异的GFP基因在转基因小鼠中的整合和表达

应用荧光定量PCR技术对由位点控制区LCR的HS2元件和 β 珠蛋白基因启动子指导的红系特异表达绿色荧光蛋白 (GFP)基因的转基因小鼠中外源基因拷贝数进行测定 ,使用荧光显微镜和流式细胞仪检测小鼠外周血中GFP的表达水平 ,并运用荧光原位杂交技术 (FISH)确定了其中两只转基因小鼠中外源基因的整合位点 ,结果表明 :在转基因小鼠中外源基因的拷贝数各不相同且相差较大 ,而且拷贝数与GFP基因的表达量之间未呈现出相关性 ;FISH分析确定出两只转基因小鼠的外源基因整合于不同的染色体上 ;杂交信号的强弱与拷贝数的多少相一致     

hOPG基因启动子驱动报告基因LacZ的转基因小鼠模型的建立

目的:建立带有人类骨保护素OPG基因启动子驱动报告基因LacZ的转基因小鼠模型,为OPG体内转录水平的表达调控研究和药物筛选创造条件。方法:将克隆到的人类OPG基因5′端上游6.0kb非翻译序列作为启动子,大肠杆菌编码β半乳糖苷酶的LacZ基因作为报告基因,构建表达载体pCINeoOPGLacZ。经显微操作注射到受精卵原核中,经PCR以及Southern印迹杂交鉴定转基因阳性小鼠;用RTPCR分析LacZ在组织中的表达;利用邻硝基苯βD半乳吡喃糖苷(ONPG)作为底物反应后比色分析组织中的β半乳糖苷酶活性。结果:构建完成的表达载体pCINeoOPGLacZ质粒经酶切和测序鉴定序列正确,线性化后显微注射。PCR以及Southern印迹杂交鉴定获得了10只转基因小鼠(Founders),经交配繁育,建立了5个转基因小鼠系,RTPCR分析表明其中一个系小鼠组织中表达LacZ基因,与内源OPG表达模式一致,组织中可以广泛检测到相应的β半乳糖苷酶活性。结论:成功建立了人类OPG基因启动子驱动报告基因LacZ的转基因小鼠,为体内研究OPG转录水平的表达及药物筛选提供了理想的动物模型。     

复制型HBV转基因小鼠遗传稳定性研究

目的：提高复制型HBV转基因小鼠的遗传稳定性。方法：应用回交传代及双杂交育种法,经荧光定量PCR、ELISA和化学发光法研究HBV基因在小鼠体内的复制与表达。结果：HBV转基因小鼠已稳定传至第23代,血清HBsAg达4122.31±2044.74IU/ml,93.93%的转基因小鼠血清HBV DNA达104-106copies/ml,表达复制水平较早期有显著提高并稳定传代;雌雄小鼠之间表达水平无显著性差异。结论：该转基因小鼠经过培育传代,已成为一个高表达且遗传稳定的复制型HBV小鼠模型。     

应用荧光原位杂交技术检测人β^E珠蛋白基因小鼠染色体上的整合状态

为将荧光原位杂交技术应用于基因定位研究中,探讨一种能有效地检测转基因动物染色体上外源基因整合状态的实验方法,对小鼠腹腔注射秋水仙素后,取转基因小鼠骨髓制备中期染色体,将传统的FISH方法加以改进,检测外源基因在转基因小鼠染色体上的整合状态。检测结果表明,外源人β^E珠蛋白基因已稳定地整合于小鼠染色体上,FISH能直观地反映外源基因在转基因动物染色体上的整合状态,该方法可对转基因动物及基因转移研究中的外源基因整合反进行染色体定位检测。     

MiR-34a通过抑制Pgc-1β和线粒体生成促进小鼠骨骼肌脂肪异位沉积

过氧化物酶体增殖物激活受体γ辅激活因子-1β（peroxisome proliferative activated receptor γ coactivator 1 β,Pgc-1β）与线粒体生成相关。已有研究证明,miR-34a在肝组织脂肪异位沉积中发挥重要作用,但是否与骨骼肌的脂肪异位沉积相关尚不清楚。本研究以C57Bl/6J小鼠为研究对象,通过尾静脉注射miR-34a模拟物,探讨miR-34a过表达对小鼠骨骼肌脂肪沉积的影响。组织切片进行油红O染色及甘油三酯含量测定揭示,miR-34a过表达的小鼠骨骼肌组织中脂滴积累及甘油三酯含量显著增加。实时荧光定量PCR（qRT-PCR）显示,与对照鼠比较,miR-34a处理的小鼠骨骼肌组织中的脂肪酸合成酶(Fas）表达显著上调,而脂肪酸氧化分解相关基因产物肉毒碱棕榈酰基转移酶1α（Cpt 1α）表达显著下调,提示miR-34a调控骨骼肌内脂肪的沉积机制可能是通过促进脂肪酸生成和抑制脂肪酸分解实现的。qRT-PCR和Western印迹证明,miR-34a可抑制Pgc-1β蛋白的表达。CoxⅡ/28S比例（线粒体定量指标）测定提示,注射miR-34a模拟物导致小鼠骨骼肌线粒体数目显著下调。生物信息分析显示,Pgc-1β mRNA的3′-UTR存在 miR-34a的潜在识别位点,因此miR-34a可能通过靶向识别Pgc-1β的3′-UTR抑制Pgc-1β表达,从而抑制线粒体生成。上述结果证明,miR-34a能通过靶向抑制PGC-1β表达,抑制线粒体生成,继而减少脂肪酸氧化分解,导致骨骼肌脂肪沉积增加。此外,上调脂肪酸合成酶也可能是miR-34a导致骨骼肌脂肪沉积增加的另一原因,其作用机制需进一步研究。     

HLA-Ⅱ类基因DRα及DRB_1~*0401~1转基因小鼠的制备

利用受精卵原核的显微注射技术,将人类HLA-Ⅱ类DRα及DRB1 *0401两种基因,显微注射至C57BL/6×DBA/1杂交小鼠受精卵中;并移植至假孕受体鼠的输卵管内.实验先后有8只鼠妊娠,稳定遗传五代,经PCR检测95只HLA-DRα、DRB1*0401阳性.α-32P-dCTP斑点杂交与Southem印迹杂交鉴定出68只整合含有DRα及DRB1 *0401混合型的转基因小鼠,有效率为20.9%.经Northern杂交…和RT-PCR检测,其HLA-DRα、DRB1 *0401基因在脾脏和肾脏中均有表达.结果说明,携带人类HLA-Ⅱ类DRα和DRB1* 0401基因的转基因动物模型构建成功.     

表达β-珠蛋白基因的安全性慢病毒载体的优化

目的:慢病毒载体(lentiviral vector,LVV)是一种有效的基因治疗导入系统。拟用已研发的携带人的β-珠蛋白基因自删除慢病毒载体,优化其表达有效性和提高其病毒颗粒数。方法:比较三款不同的启动子预测软件的分析结果,分别构建三种不同长度启动子的表达β-珠蛋白基因(β-globin)的LVV,并对其Ⅱ号内含子进行部分删减;用经优化的LVV转导β-地中海贫血(β-地贫)的小鼠诱导性多能性干细胞(induced pluripotent stem cells,iPSC)后,用此iPSC制备嵌合体小鼠模型;经RT-PCR、血涂片瑞氏吉姆萨染色等观察分析其功能性代偿的潜能。研究结果:经优化后的自删除慢病毒载体病毒对其病毒颗粒数的滴度影响不大(2.3×10~(11)LPs/ml),可在嵌合体小鼠模型体内检测到正常人β-珠蛋白基因的功能性表达。结论:优化了表达人β-珠蛋白基因的自删除LVV。     

The human growth hormone gene cluster locus control region supports position-independent pituitary- and placenta-specific expression in the transgenic mouse

The human growth hormone (hGH) cluster contains five genes. The hGH-N gene is predominantly expressed in pituitary somatotropes, whereas the remaining four genes, the chorionic somatomammotropin genes (hCS-L, hCS-A, and hCS-B) and hGH-V, are expressed selectively in the placenta. In contrast, the mouse genome contains a single pituitary-specific GH gene and lacks any GH-related CS genes. Activation of the hGH transgene in the mouse is dependent on its linkage to a previously described locus control region (LCR) located -15 to -32 kilobases upstream of the hGH cluster. The sporadic, nonreproducible expression of hCS transgenes lacking the LCR suggests that they may be dependent on hGH LCR activity as well. To determine whether the hCS genes could be expressed with appropriate placental specificity, a series of five transgenic mouse lines carrying an 87-kilobase human genomic insert encompassing the majority of the hGH gene cluster and the entire contiguous LCR was established. All of the hGH cluster genes were appropriately expressed in each of these lines. High level expression of hGH was restricted to the pituitary and hCS to the labyrinthine layer of the placenta. The expression of the GH cluster genes in their respective tissues paralleled transgene copy numbers irrespective of the transgene insertion site in the host mouse genome. These studies have extended the utility of the transgenic mouse model for the analysis of the full spectrum of hGH gene cluster activation. Further, they support a role for the hGH LCR in placental hCS, as well as pituitary hGH gene activation, and expression.     

Human gamma- to beta-globin gene switching using a mini construct in transgenic mice.

The developmental regulation of the human globin genes involves a key switch from fetal (gamma-) to adult (beta-) globin gene expression. It is possible to study the mechanism of this switch by expressing the human globin genes in transgenic mice. Previous work has shown that high-level expression of the human globin genes in transgenic mice requires the presence of the locus control region (LCR) upstream of the genes in the beta-globin locus. High-level, correct developmental regulation of beta-globin gene expression in transgenic mice has previously been accomplished only in 30- to 40-kb genomic constructs containing the LCR and multiple genes from the locus. This suggests that either competition for LCR sequences by other globin genes or the presence of intergenic sequences from the beta-globin locus is required to silence the beta-globin gene in embryonic life. The results presented here clearly show that the presence of the gamma-globin gene (3.3 kb) alone is sufficient to down-regulate the beta-globin gene in embryonic transgenic mice made with an LCR-gamma-beta-globin mini construct. The results also show that the gamma-globin gene is down-regulated in adult mice from most transgenic lines made with LCR-gamma-globin constructs not including the beta-globin gene, i.e., that the gamma-globin gene can be autonomously regulated. Evidence presented here suggests that a region 3' of the gamma-globin gene may be important for down-regulation in the adult. The 5'HS2 gamma en beta construct described is a suitable model for further study of the mechanism of human gamma- to beta-globin gene switching in transgenic mice.     

Analysis of mice containing a targeted deletion of beta-globin locus control region 5' hypersensitive site 3.

To examine the function of murine beta-globin locus region (LCR) 5' hypersensitive site 3 (HS3) in its native chromosomal context, we deleted this site from the mouse germ line by using homologous recombination techniques. Previous experiments with human 5' HS3 in transgenic models suggested that this site independently contains at least 50% of total LCR activity and that it interacts preferentially with the human gamma-globin genes in embryonic erythroid cells. However, in this study, we demonstrate that deletion of murine 5' HS3 reduces expression of the linked embryonic epsilon y- and beta H 1-globin genes only minimally in yolk sac-derived erythroid cells and reduces output of the linked adult beta (beta major plus beta minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK-neo cassette was left within the HS3 region of the LCR, a much more severe phenotype was observed at all developmental stages, suggesting that PGK-neo interferes with LCR activity when it is retained within the LCR. Collectively, these results suggest that murine 5' HS3 is not required for globin gene switching; importantly, however, it is required for approximately 30% of the total LCR activity associated with adult beta-globin gene expression in adult erythrocytes.