Effect of HS2 and HS3 elements on erythroid-specific expression in transgenic mice

The expression plasmids CMV/GFP, HS2ALL, HS3ALL and HS23ALL were selected to investigate the effect of HS2 and HS3 element on erythroid-specific expression in transgenic mice. These plasmids were digested with restriction enzymes and purified. And five DNA fragments, CMV/GFP, HS2/GFP, CMV/HS2/GFP, HS23/GFP and HS3/GFP were obtained. After purification, the above DNA fragments were microinjected into the pre-nuclei of the mice fertilized eggs and transgenic mice were generated, with an integration rate of 10.89%. The green fluorescence protein(GFP) expression in many transgenic mouse tissues was determined by FACS analysis. The results showed that the HS2 and 1.7 kb of β-globin gene promoter were sufficient for the erythroid-specific expression of β-globin gene. The GFP expression of different recombinant constructs was also analyzed in blood of all the transgenic mice with FACS. The results indicated that HS2 and HS3 had the same enhancement activity on the regulation of β-globin gene expression. Moreover, these two elements showed a significant synergistic effect on gene expression at the transgenic mouse level, although the GFP expression varied largely among different transgenic mouse litters.     

人endostatin基因重组逆转录病毒载体的构建及其在NIH3T3细胞中表达

为探讨转人血管内皮抑制基因（endostatin）在转染细胞中的表达，利用逆转录病毒载体构建人endostatin基因的重组质粒，通过脂质体（lipofectamine）将重组质粒导入包装细胞PA317,制备重组病毒液。用重组病毒液感染NIH3T3细胞，经G418筛选获得转入endostatin基因细胞株NIH3T3-endo。同法制备对照细胞株NIH3T3-pLncx.PCR检测NIH3T3-endo细胞基因组，在扩增产物中一份550bp人endostatin基因特异性片段，对照组为阴性。免疫组化测定示仅NIH3T3-endo细胞中有外源性endostatin蛋白的表达，说明人endostatin基因已被成功导入NIH3T3细胞，并获得稳定表达。     

Effects of point mutation C→T at - 64 of human δ globin gene promoter on DNA binding proteins

By electrophoretic mobility shift assay (EMSA), the effect of point mutation C→T at - 64 of human δ-globin gene on its binding proteins has been studied. Two segments of 36 bp from - 83- - 48 bp of the 6 globin gene promoter, named WOG and MOG, were synthesized. WOG includes wild type CAAT-like box (CCAAC), while MOG includes the mutant CAAT-like box (CCAAT, -64 C→T). Results indicate that: ( i ) in erythroid cell lines MEL, K562 and Hemin induced K562, the affinity of MOG with CCAAT binding protein (CBF) and GATA-1 was greatly increased; ( ii ) in Hemin induced K562 cell line, there were another two novel specific DNA binding proteins, named C and D temporarily, besides the above two factors. The former was combined with WOG and MOG, likely indicating its relation with the increased gene expression after induction. The latter was only combined with MOG, which had possible relationship with the point mutation of - 64 C→T; ( iii ) EMSA also indicates that the suppression mechanisms of the expression of 6 globin gene is different in various periods of human developments. The result evidently supports the hypothesis that the defect CAAT-like box in human 6 globin gene contributes the main reason of its low level expression. The defect c/s-acting element CAAT-like box affects gene expression by its combination with the frans-acting element CBF and GATA-1.     

Interactions between the nuclear matrix proteins and the 5'-flanking cis-acting elements of the human ε-globin gene

An erythroid-specific nuclear matrix protein (termed ε-NMPk) in K562 cells, which can specifically bind to the positive stage-specific regulatory element (ε-PRE Ⅱ , - 446- - 419 bp) upstream of the human ε-globin gene, has been identified by using gel mobility shift assay. Meanwhile, Southwestern blotting assay showed that the nuclear matrix protein ε-NMPk in K562, cells may be composed of two polypeptides ( ～ 40 ku). In addition, it is observed in the gel mobility shift assay that the nuclear matrix proteins from K562, HEL and Raji cells can bind to the silencer DNA ( - 392- - 177 bp) in the 5'-flanking sequence of human ε-globin gene respectively. However, the shift band K detected in K562 cells is different from shift band H/R in HEL and Raji cells, suggesting that a common nuclear matrix protein may exist in HEL and Raji cells. Results show that the nuclear matrix protein may play an important role in the regulation of the human ε-globin gene expression.     

Expression of a transfected human c-myc oncogene inhibits differentiation of a mouse erythroleukaemia cell line

The Friend-virus-derived mouse erythroleukaemia (MEL) cell lines represent transformed early erythroid precursors that can be induced to differentiate into more mature erythroid cells by a variety of agents including dimethyl sulphoxide (DMSO). There is a latent period of 12 hours after inducer is added, when 80-90% of the cells become irreversibly committed to the differentiation programme, undergoing several rounds of cell division before permanently ceasing to replicate. After DMSO induction, a biphasic decline in steady-state levels of c-myc and c-myb messenger RNAs occurs. Following the initial decrease in c-myc mRNA expression, the subsequent increase occurs in, and is restricted to, the G1 phase of the cell cycle. We sought to determine whether the down-regulation is a necessary step in chemically induced differentiation. Experiments reported here indicate that expression in MEL cells of a transfected human c-myc gene inhibits the terminal differentiation process.     

Human gamma-globin genes silenced independently of other genes in the beta-globin locus.

Erythropoiesis during human development is characterized by switches in expression of beta-like globin genes during the transition from the embryonic through fetal to adult stages. Activation and high-level expression of the genes is directed by the locus control region (LCR), located 5' to the epsilon gene. The location of the LCR and its role in directing high-level expression of the globin genes has led to the suggestion that competition from the beta gene for interaction with the LCR has a major role in silencing the fetal gamma genes during adult life. We have now constructed lines of transgenic mice containing the human A gamma globin gene linked to the LCR. We observe high-level expression of the transgene in the embryonic stages but silencing of the gene in adult animals. We conclude that the gamma gene is not deregulated by the presence of the LCR and that competition from the beta gene is not required for silencing of the gamma genes in adult life. The silencing is therefore likely to be mediated by stage-specific factors binding to sequences immediately flanking the genes.     

以精子载体法制备转基因小鼠的初步研究

将外源融合基因BaLA-HI与DOSPER脂质体等比较混合,加入获能精子悬液中,37度,5％CO2共培养0．5h,以这种处理精子作为转基因载体乾鼠的体外受精及胚胎移植,在获得的40只移植后代后,经PCR特异片段扩增和Southern杂交,共检测出2只呈相性的转基因小鼠,证明人胰 基因已在小鼠染色体上实现了整合,基因整合率为5％。     

Interactions between the nuclear matrix proteins and the 5′-flankingcis-acting elements of the human ε-globin gene

An erythroid-specific nuclear matrix protein (termed ε-NMP_k) in K562 cells, which can specifically bind to the positive stage-specific regulatory element (ε-PRE II, - 446 - 419 bp) upstream of the human ε-globin gene, has been identified by using gel mobility shift assay. Meanwhile, Southwestern blotting assay showed that the nuclear matrix protein ε-NMP_k in K562, cells may be composed of two polypeptides (∼ 40 ku). In addition, it is observed in the gel mobility shift assay that the nuclear matrix proteins from K562, HEL and Raji cells can bind to the silencer DNA (- 392 - 177 bp) in the 5′-flanking sequence of human ε-globin gene respectively. However, the shift band K detected in K562 cells is different from shift band H/R in HEL and Raji cells, suggesting that a common nuclear matrix protein may exist in HEL and Raji cells. Results show that the nuclear matrix protein may play an important role in the regulation of the human ε-globin gene expression.     

不同血清型腺相关病毒在大鼠 DRG神经元转染效果的比较

摘要:目的 比较不同血清型腺相关病毒( AAV5、AAV6、AAV8 和 AAV9) 对背根神经节( DRG) 初级感觉神经元的转导效能。 方法 将 24 只雄性 SD 大鼠随机分为 8 组,分别通过 DRG 直接注射不同血清型腺相关病毒( AAV5、AAV6、AAV8 和 AAV9)载体,各组大鼠分别在转染后第 3 周和第 4 周取材,并在共聚焦显微镜下观测对大鼠 DRG初级感觉神经元的转染效果。 结果 组织切片的荧光结果显示,腺相关病毒各亚型的转染效果如下:转染 3 周,AAV9> AAV6> AAV8 > AAV5;转染 4 周,AAV9> AAV6> AAV8> AAV5。 其中,AAV9 载体对大鼠 DRG 神经元的转导效果较好。 结论 DRG 注射不同血清型腺相关病毒( AAV5、AAV6、AAV8 和 AAV9) 载体,AAV9 对 DRG 神经元的转导效能最佳。     

靶向TTF-1的siRNA腺相关病毒载体组装验证

为了解决靶向TTF-1的siRNA导入肺腺癌细胞的问题,设计了3条靶向NCI-H1975细胞TTF-1基因的siRNA,并将其分别构建到腺相关病毒上.在293细胞中装配病毒,收获病毒原初液后进行超滤浓缩、层析柱纯化,测定病毒滴度.重组病毒感染肺腺癌细胞系NCI-H1975,通过Western-Blot实验检测siRNA效果,利用凋亡实验验证细胞生物学效应.Western-Blot实验测得siRNA有较好的干扰效果,凋亡实验测得肺腺癌细胞NCI-H1975产生了凋亡,从而证明具有感染性的靶向TTF-1的siRNA腺相关病毒载体组装成功.     

人MiTERF3基因启动子的克隆及其荧光素酶报告基因载体的构建

人MiTERF3基因编码线粒体基因转录和能量代谢的负调控因子。采用PCR技术对人MiTERF3基因5''侧翼上游1251 bp启动子序列进行扩增,并将其克隆至荧光素酶表达载体pGL6-TA,构建人MiTERF3基因启动子荧光素酶报告基因质粒。经酶切、测序鉴定后,将其用脂质体转染体外培养的HEK293细胞株,利用双荧光素酶测定系统检测其表达活性。研究结果表明,克隆获得的1251 bp DNA序列与GenBank报道的一致,且插入方向正确。含人MiTERF3基因启动子的报告基因荧光素酶的表达活性显著提高（P<0.05）,约为对照组（空载体pGL6-TA）的9.8倍。本研究通过对人MiTERF3基因启动子的克隆及其荧光素酶表达载体构建与表达活性的测定,为进一步阐明人MiTERF3基因表达的调控机制奠定实验基础。     

人白介素-3乳腺表达载体的构建

人白介素-3是一种造血系统和免疫系统调节剂,在治疗造血系统疾病、肿瘤、先天或获得性免疫功能缺陷等方面发挥着重要作用.应用牛乳腺生物反应器生产人白介素-3的研究具有重要的临床应用和经济价值.研究选用具有红色荧光蛋白报告基因(R ed2)和新霉素抗性基因(neor)表达框架的pD sR ed2-1质粒为骨架构建人白介素-3乳腺表达载体.通过PCR方法分别扩增牛β-酪蛋白基因5′端上游调控序列、人IL-3基因以及CM V启动子序列,将它们按先后顺序分别定向克隆于质粒pD sR ed2-1的多克隆位点内,使牛β-酪蛋白基因调控序列位于人IL-3基因的上游,指导人IL-3基因在乳腺组织中特异性表达,而CM V启动子位于红荧光蛋白基因的上游,指导红荧光蛋白基因在所有的组织中非特异性表达.限制性酶切片段分析及部分DNA序列鉴定结果表明,所构建载体结构正确.     

Expression of c-myc changes during differentiation of mouse erythroleukaemia cells

The transforming gene of avian myelocytomatosis virus MC29, v-myc, causes a variety of malignancies in chickens. A cellular homologue, c-myc, has been implicated in B-cell malignancies in mice and humans but is also expressed in many normal cell types and may be important in the control of normal cell proliferation. c-myc is highly conserved in vertebrates. We have been investigating the relationship between c-myc expression and the terminal differentiation of cultured mouse erythroleukaemia (MEL) cells. We find that the level of c-myc messenger RNA shows a rapid biphasic change in MEL cells induced to differentiate by dimethyl sulphoxide or hypoxanthine. The changes occur during the first few hours of the differentiation programme and require active protein synthesis. These data suggest that changes in c-myc expression may be important in the irreversible commitment of MEL cells to terminal erythroid differentiation.     

胰岛自身抗原GAD65片段和胰岛素B链基因共表达DNA疫苗的构建与表达

构建两种胰岛自身抗原谷氨酸脱羧酶（ glutamic acid decarboxylase, GAD） 65片段与胰岛素B链基因共表达DNA疫苗,并检测其在体外COS-7细胞中的表达。PCR方法从GAD65质粒中扩增出GAD190-385和GAD490-570两个片段的cDNA,overlap法再分别与信号肽基因进行拼接,将拼接后的融合基因SGAD190-515和SGAD490-570分别与胰岛素B链基因依次克隆入双启动子真核表达载体pBudCFA．1中。重组质粒经酶切和测序鉴定后,脂质体体外转染COS-7细胞,Western blot方法检测目的基因在该细胞中的表达。结果表明核酸序列测定克隆的融合基因和胰岛素B链基因序列与报告序列一致,开放阅读框正确,Western blot显示转染了DNA疫苗的COS-7细胞中均可检测两个目的基因的表达。两种GAD65片段与胰岛素B链基因共表达DNA疫苗均成功构建,为自身免疫糖尿病的免疫干预研究奠定了实验基础。