用DNA介导的基因转移法提高XP细胞对紫外辐射损伤的修复能力

本文用两种DNA介导的基因转移法(DNA—磷酸钙共沉淀法和电脉冲刺激法),将具有切除修复功能的人HeLaS3细胞的DNA,植入切除修复缺陷的着色性干皮症(XP)细胞中。实验结果表明:植入HeLaS3 DNA后,可以部份恢复XP细胞DNA切除修复的功能,提高其对紫外线辐射损伤的抗性。表现为转化细胞在UV_(254)照射后存活率的显著升高和非周期DNA合成能力的增强。     

正义、反义MCP-1基因逆转录病毒载体重组质粒的构建和包装

为构建含单核细胞趋化蛋白-1(Monocytechemoattractantprotein-1,MCP-1)基因的重组逆转录病毒pLXSN/MCP-1质粒.用RT-PCR技术从大鼠系膜细胞中扩增出MCP-1全长DNA,将其与Pgem-TE连接,用限制性内切酶EcoRⅠ对pTE-MCP-1和逆转录病毒质粒pLXSN分别进行酶切.在T4连接酶的作用下,构建重组逆转录病毒质粒pLXSN/MCP-1.经BglⅡ,XhoⅠ酶切鉴定MCP-1在质粒中的方向,用脂质体介导的方法把重组质粒DNA转染进入包装细胞PA317中,经过G418筛选出抗性克隆.通过NIH3T3细胞测定病毒液的病毒滴度.结果证实经过RT-PCR技术从大鼠系膜细胞中扩增出MCP-1全长DNA与所需的大小一致.重组质粒经酶切分析与预期的结果一致.G418筛选出抗性克隆,能稳定合成并分泌重组逆转录病毒颗粒.NIH3T3细4胞测定病毒滴度为17×10cfu/mL.     

腺病毒介导的Her2基因RNAi载体的构建和鉴定

目的:构建腺病毒介导的Her2基因RNAi栽体.方法:构建含有Her2基因片断的siRNA质粒Her2-pSuppressor,通过特异性酶切将目的基因与穿梭载体pshuttle相连,再通过特异性酶切位点将目的片断与腺病毒DNA相连,并用PCR和酶切鉴定方法进行筛选和鉴定,获得重组腺病毒DNA.以Pac Ⅰ酶切线性化后转染包装含有腺病毒E1的HEK293细胞.以软琼脂平板上的空斑数量计算重组腺病毒的的滴度.结果:Xba Ⅰ Mlu Ⅰ双酶切鉴定Her2a-RNAi/pShuttle和Her2b.RNAi/pShuttle阳性重组子获得304bp的目的片段,重组腺病毒载体Adeno-Her2a-RNAi和Adeno-Her2b-RNAi经PCR扩增出同样大小片断,经线性化后用脂质体法转染293细胞,观察到细胞病变效应.病毒滴度达1.2× 108PFU/mL.结论:成功构建了Adeno-Her2-RNAi,为肿瘤的基因治疗奠定了良好的基础.     

利用基因捕获技术建立稳定表达HBV的细胞模型

pGEM-HBV1.3质粒经HindIII限制性内切酶消化,将HBV1.3全长DNA切下,与同样经HindIII限制性内切酶降解过的PU21连接,得到PU21-HBV重组质粒。将该重组质粒采用电击转染方法导入HepG2细胞中,G418筛选阳性克隆并以X-gal染色,RT-PCR、Southern blot等方法验证HBV DNA的插入和表达。 PU21-HBV重组质粒经测序证明HBV1.3全长DNA正确与PU21载体连接,该重组质粒转染HepG2细胞后经G418筛选,得到一系列阳性克隆, Southern blot证实HepG2细胞基因组中含HBV DNA,RT-PCR结果表明HBV DNA在HepG2细胞中有功能基因的转录。HBV1.3已被整合在HepG2细胞染色体中并能稳定表达其RNA。稳定的HBV表达细胞模型构建成功。HBV表达细胞模型的建立,为进一步研究相关基因对HBV的转录、复制、转录后调节以及HBV各种蛋白的表达机理研究提供实验材料。     

人Stathmin基因siRNA腺病毒载体的构建及病毒鉴定

目的:为了寻求解决非特异性杀伤作用的星形细胞瘤的基因治疗问题的新途径.本研究构建带有Stathmin siRNA的复制缺陷腺病毒载体并包装病毒.方法:(1)两个目标siRNA基因片段合成并分别插入pSilencer4.1-CMV载体中从而构建两个重组真核表达栽体:pSilencer4.1-CMV-S1和pSilencer4.1-CMV-S2.(2)用EcoR Ⅰ和BamH Ⅲ双酶切pSilencer4.1-CMV-S1和pSilencer4.1-CMV-S2,将目的片段分别装进穿梭质粒,从而构建pShuttle-CMV neo-S1和pShuttle-CMV neo-S2.以独特的限制性内切酶PI-Sce Ⅰ合I-Ceu Ⅰ双酶切这两个穿梭质粒并重组至骨架Adeno-XTM Viral DNA上.并以PCR方法来鉴定.(3)线性化Ad-pShuttle-CMV neo-S1和Ad-pShuttle-CMV neo-s2并转染入HEK293细胞,出毒后进行PCR毒种鉴定和滴度分析.结果:(1)酶切分析和DNA测序表明,小干扰RNA片段的成功连接到pSilencer4.1-CMV载体上分别.(2)酶切分析表明两个目的基因片段,都分别成功连接入穿梭载体pShuttle.PCR表明pShuttle-CMV neo-S1和pShuttle-CMV neo-S2分别与骨架重组成功.(3)腺病毒DNA进行PCR鉴定后表明成功地在体外重组并生产出腺病毒.且冻融产毒细胞保证了较高的重组腺病毒滴度.结论:Adeno-XTM系统是一个能简单而有效生产能表达目的基因腺病毒的系统.我们构建的腺病毒有望成为星形细胞瘤新的高效而安全的治疗方法.     

一种简单的哺乳动物细胞附着体质粒CaCl2转化方法

目的:建立一种简单经济的哺乳动物细胞附着体质粒还原实验方法。方法:构建附着体载体,转染中国卵巢仓鼠(CHO)细胞和小鼠脑神经瘤细胞Neuro-2a,利用改良的赫特裂解法提取附着体质粒,CaCl2法转化附着质粒至宿主菌大肠杆菌DH5α,再次从DH5α中提取质粒,将转染前后质粒用KpnⅠ/BamHⅠ双酶切和BamHⅠ单酶切,并将转染前后的质粒进行DNA测序分析。结果:与最初转染的质粒相比,还原质粒双酶切和单酶切后的条带大小一致;DNA测序分析表明,转染前后质粒中的插入序列相同。结论:建立了可用于质粒还原实验的简单的CaCl2转化方法。     

结核分枝杆菌furA基因真核表达质粒的构建及表达

以BamH Ⅰ和Hind Ⅲ双酶切pRSET-furA,获得结核分枝杆苗铁调控基因furA,将其克隆入真核表达载体pcDNA3．1（-）,构建了重组质粒pcDNA-furA,经酶切鉴定正确后,将重组质粒以阳离子聚合物转染CHO细胞。经RT—PCR分析表明,furA可在CHO细胞中转录;用间接免疫荧光检测,表达有FurA蛋白的细胞着染。以上结果表明,通过构建结核分枝杆菌furA基因的真核表达载体pcDNA-furA,使知以基因可以在CHO细胞中表达。     

大鼠胎脑神经干细胞HPRT基因的敲除

根据已知大鼠次黄嘌呤鸟嘌呤磷酸核糖转移酶 (HypoxanthineGuaninePhosphoribosylTransferase ,HPRT)基因的外显子序列 ,从大鼠HPRT基因组DNA序列的细菌人工染色体 (BacterialArtificialChromosome ,BAC)中用酶切和PCR方法分别分离得到用于构建基因敲除载体的 3 0kb的 5′长臂 (LongArm ,LA)和 1 7kb的 3′短臂 (ShortArm ,SA) ,并分别克隆到pSL1180和pCR2 1中。进一步构建大鼠HPRT基因打靶载体———pKO HPRT ,经酶切鉴定后的大鼠HPRT基因敲除载体用NotⅠ酶切使其线性化 ,经溴乙锭、正丁醇、酚、酚 /氯仿提纯后 ,将终浓度调至 1μg μl。在FuGene 6转染试剂的作用下转染培养 2 4h的第二代大鼠胎脑神经干细胞 (RatFetalNeuralStemCells,rFNSCs)。转染后的细胞用 80 μg mlG4 18和 0 2 μmol L的Ganc全培养液筛选 ,2w后将存活细胞进行悬浮培养 ,使细胞形成球形物 ,挑选单个的球形物进行单克隆增殖 ,其中一部分细胞 (约 2～ 3× 10 3)用裂解液处理 ,取上清用于PCR检测 ,大部分细胞 (5× 10 7)用于DNA和RNA的提取 ,进行Southernbolt和RT PCR检测 ,剩余细胞冷冻保存。最后 1次实验共分离培养了 32个rFNSCs单克隆 ,其中 3个单克隆 (9 3% )经PCR、Southernbolt和RT PCR证实HPRT基因已被敲除     

瞬时外向钾通道Kv4.3真核表达载体的构建

[目的]构建Kv4.3真核表达载体,观察其在真核细胞中表达情况。[方法]提取小鼠前额叶皮质RNA,通过RT-PCR和常规PCR技术扩增获得Kv4.3目的基因,利用EcoRⅠ和XbaⅠ限制性内切酶和T4 DNA连接酶将其克隆在pcDNA3.1载体中,并将构建好的重组质粒转染至HEK293细胞中,免疫印迹方法检测Kv4.3蛋白表达。[结果]成功扩增了Kv4.3基因(2 000 bp处有明显条带),先后经酶切和酶连后的重组体转化培养,提取质粒,再通过双酶切观察到2 000 bp处有Kv4.3目的条带和5 400 bp处有pcDNA3.1载体条带。将此重组质粒送去测序,结果显示基因测序结果与GenBank中Kv4.3序列一致。免疫印迹结果发现在HEK293细胞中,转染的重组质粒能够表达Kv4.3蛋白。[结论]成功构建了Kv4.3真核表达载体,其在HEK293细胞中可以稳定表达。     

Excess processing of oxidative damaged bases causes hypersensitivity to oxidative stress and low dose rate irradiation

Abstract

Ionizing radiations such as X-ray and γ-ray can directly or indirectly produce clustered or multiple damages in DNA. Previous studies have reported that overexpression of DNA glycosylases in Escherichia coli (E. coli) and human lymphoblast cells caused increased sensitivity to γ-ray and X-ray irradiation. However, the effects and the mechanisms of other radiation, such as low dose rate radiation, heavy-ion beams, or hydrogen peroxide (H₂O₂), are still poorly understood. In the present study, we constructed a stable HeLaS3 cell line overexpressing human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) protein. We determined the survival of HeLaS3 and HeLaS3/hOGG1 cells exposed to UV, heavy-ion beams, γ-rays, and H₂O₂. The results showed that HeLaS3 cells overexpressing hOGG1 were more sensitive to γ-rays, OH^?, and H₂O₂, but not to UV or heavy-ion beams, than control HeLaS3. We further determined the levels of 8-oxoG foci and of chromosomal double-strand breaks (DSBs) by detecting γ-H2AX foci formation in DNA. The results demonstrated that both γ-rays and H₂O₂ induced 8-oxoguanine (8-oxoG) foci formation in HeLaS3 cells. hOGG1-overexpressing cells had increased amounts of γ-H2AX foci and decreased amounts of 8-oxoG foci compared with HeLaS3 control cells. These results suggest that excess hOGG1 removes the oxidatively damaged 8-oxoG in DNA more efficiently and therefore generates more DSBs. Micronucleus formation also supported this conclusion. Low dose-rate γ-ray effects were also investigated. We first found that overexpression of hOGG1 also caused increased sensitivity to low dose rate γ-ray irradiation. The rate of micronucleus formation supported the notion that low dose rate irradiation increased genome instability.     

DNA methylation in xeroderma pigmentosum

DNA methylation was examined in xeroderma pigmentosum (XP) cells. The amount of 5-methylcytosine (mC) in DNA from XP cells was about 70% of that in DNA from normal controls. Southern hybridization analysis showed that the HLA-DR alpha gene in XP lymphocyte B cells was differently methylated from normals, but its expression was apparently unaffected. The methylation of dihydrofolate reductase, a housekeeping gene, was the same as in controls. The revertants to UV resistance from XP fibroblasts recovered a methylation level close to that of normal cells. Results suggested that XP DNA was undermethylated non-randomly, and that DNA methylation might be associated with DNA repair function.     

Retrovirus-mediated DNA repair gene transfer into xeroderma pigmentosum cells: Perspectives for a gene therapy

The rare hereditary disease xeroderma pigmentosum (XP) is clinically characterized by extreme sun sensitivity and an increased predisposition for developing skin cancer. Cultured cells from XP patients exhibit hypersensitivity to ultraviolet (UV) radiation due to the defect in nucleotide excision repair (NER), and other cellular abnormalities. Seven genes identified in the classical XP forms, XPA to XPG, are involved in the NER pathway. In view of developing a strategy of gene therapy for XP, we devised recombinant retrovirus-carrying DNA repair genes for transfer and stable expression of these genes in cells from XP patients. Results showed that these retroviruses are efficient tools for transducing XP fibroblasts and correcting repair-defective cellular phenotypes by recovering normal UV survival, unscheduled DNA synthesis, and RNA synthesis after UV irradiation, and also other cellular abnormalities resulting from NER defects. These results imply that the first step of cellular gene therapy might be accomplished successfully.     

Signaling of DNA damage is not sufficient to induce p53 response: (re)activation of wt p53 protein strongly depends on cellular context

It is generally accepted that exposure of cells to a variety of DNA-damaging agents leads to up-regulation and activation of wild-type (wt) p53 protein. We investigated the (re)-activation of p53 protein in two human cancer cell lines in which the gene for this tumor suppressor is not mutated: HeLaS(3) cervix carcinoma and MCF-7 breast cancer cells, by induction via different genotoxic and cytotoxic stimuli. Treatment of human cells with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or different anti-cancer drugs resulted in a strong DNA damage as evidenced by Comet assay and a marked increase in site-specific phosphorylation of H2AX. Unlike in MCF-7 cells, in HeLaS(3) cells the expression of p53 protein did not increase after MNNG treatment despite a strong DNA damage. However, other agents for example doxorubicin markedly induced p53 response in HeLaS(3) cells. After exposure of these cells to MNNG, the ATM-dependent effector proteins Chk2 and NBS1 were phosphorylated, thereby evidencing that MNNG-induced DNA breakage was recognized and properly signaled. In HeLaS(3) cells wt p53 protein is not functional due to E6-mediated targeting for accelerated ubiquitylation and degradation. Therefore, the activation of a p53 response to genotoxic stress in HeLaS(3) cells seems to depend on the status of E6 oncoprotein. Indeed, the induction of p53 protein in HeLaS(3) cells in response to distinct agents inversely correlates with the cellular level of E6 oncoprotein. This implicates that the capability of different agents to activate p53 in HeLaS(3) cells primarily depends on their inhibitory effect on expression of E6 oncoprotein.     

Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells.

Xeroderma pigmentosum (XP) is an autosomal recessive human disease, characterized by an extreme sensitivity to sunlight, caused by the inability of cells to repair UV light-induced damage to DNA. Cell fusion was used to transfer fragments of Chinese hamster ovary (CHO) chromosomes into XP cells. The hybrid cells exhibited UV resistance and DNA repair characteristics comparable to those expressed by CHO cells, and their DNA had greater homology with CHO DNA than did the DNA from XP cells. Control experiments consisted of fusion of irradiated and unirradiated XP cells and repeated exposure of unfused XP cells to UV doses used for hybrid selection. These treatments did not result in an increase in UV resistance, repair capability, or homology with CHO DNA. The hybrid cell lines do not, therefore, appear to be XP revertants. The establishment of these stable hybrid cell lines is an initial step toward identifying and cloning CHO DNA repair genes that complement the XP defect in human cells. The method should also be applicable to cloning genes for other diseases, such as ataxia-telangiectasia and Fanconi's anemia.     

Differential features of sister-chromatid exchange responses to ultraviolet radiation and caffeine in xeroderma pigmentosum lymphoblastoid cell lines

Sister-chromatid exchange (SCE) induced by ultraviolet (UV) irradiation and viability after UV irradiation were studied in lymphoblastoid cell lines derived from 7 patients with xeroderma pigmentosum (XP) and 6 normal donors. UV irradiation caused significant increases of SCEs in both XP and normal cells. In 3 XP cell lines, which were deficient in unscheduled DNA synthesis (UDS) and sensitive to the killing effect of UV, very high SCE frequencies were observed after UV irradiation. Cells from a patient with the De Sanctis-Cacchione syndrome were the most sensitive to UV in terms of both SCE induction and cell killing. In 2 of 4 UDS-proficient XP cell lines tested, the incidences of UV-induced SCEs were similar to those in normal cell lines, but in 2 other UDS-proficient lines from 2 XP patients with skin cancer, the frequencies of UV-induced SCEs were significantly higher than in normal cells.Continuous post-UV treatment with 1 mM caffeine markedly enhanced UV-induced SCEs in 3 of 4 UDS-proficient XP cell lines but had only slight effects on cells from the 4th UDS-proficient XP patient and from normal individuals.     

The oxidative DNA lesion 8,5'-(S)-cyclo-2'-deoxyadenosine is repaired by the nucleotide excision repair pathway and blocks gene expression in mammalian cells

Xeroderma pigmentosum (XP) patients with inherited defects in nucleotide excision repair (NER) are unable to excise from their DNA bulky photoproducts induced by UV radiation and therefore develop accelerated actinic damage, including cancer, on sun-exposed tissue. Some XP patients also develop a characteristic neurodegeneration believed to result from their inability to repair neuronal DNA damaged by endogenous metabolites since the harmful UV radiation in sunlight does not reach neurons. Free radicals, which are abundant in neurons, induce DNA lesions that, if unrepaired, might cause the XP neurodegeneration. Searching for such a lesion, we developed a synthesis for 8,5'-(S)-cyclo-2'-deoxyadenosine (cyclo-dA), a free radical-induced bulky lesion, and incorporated it into DNA to test its repair in mammalian cell extracts and living cells. Using extracts of normal and mutant Chinese hamster ovary (CHO) cells to test for NER and adult rat brain extracts to test for base excision repair, we found that cyclo-dA is repaired by NER and not by base excision repair. We measured host cell reactivation, which reflects a cell's capacity for NER, by transfecting CHO and XP cells with DNA constructs containing a single cyclo-dA or a cyclobutane thymine dimer at a specific site on the transcribed strand of a luciferase reporter gene. We found that, like the cyclobutane thymine dimer, cyclo-dA is a strong block to gene expression in CHO and human cells. Cyclo-dA was repaired extremely poorly in NER-deficient CHO cells and in cells from patients in XP complementation group A with neurodegeneration. Based on these findings, we propose that cyclo-dA is a candidate for an endogenous DNA lesion that might contribute to neurodegeneration in XP.