基因功能研究的技术和方法

随着基因组计划的深入,越来越多的有生理意义的基因被成功克隆,对基因功能的研究显得日益重要。目前基因功能研究的主要方法有;基因转导,反义技术,核酶,基因重组,染色体转导技术等。     

外源性端粒酶基因对人脐静脉内皮细胞的影响

为了观察外源性端粒酶逆转录酶基因(hTERT)在人脐静脉血管内皮细胞(HUVEC)的表达及对细胞功能和生长的影响。采用逆转录病毒载体转导的方法,将hTERT基因转入HUVEC,检测基因转导后内皮细胞端粒酶的活性和生物学特性。结果发现hTERT转导后细胞端粒酶表达阳性,未转导的亲代细胞为阴性;转导细胞的体外生存时间延长但未永生化,而内皮细胞黏附分子表达的功能未受影响。     

细胞因子基因转导对人乳腺癌细胞MHC抗原及细胞膜糖蛋白表达的影响

为探讨细胞因子基因(人IL-2、IL-6)转导对于肿瘤细胞膜MHC抗原及细胞膜糖蛋白表达调控的影响,本文利用脂质体介导的方法,将含人IL-6、IL-2基因的逆转录病毒载体分别导入人乳腺癌细胞系MCF-7细胞中,采用间接免疫荧光染色流式细胞仪测定法,对基因转导的瘤细胞细胞膜糖蛋白及MHC抗原表达进行测定。结果表明经两种基因修饰的MCF-7细胞MHCⅠ型抗原表达均获得增强,此外,基因转导细胞可程度不同地表现出细胞膜多种糖蛋白表达的变化。提示肿瘤细胞膜抗原及糖蛋白表达的改变可能是细胞因子基因转导影响肿瘤细胞免疫原性的重要结构基础。     

病毒诱导的基因沉默技术及其在植物中的研究进展

病毒诱导的基因沉默(virus-induced gene silencing,VIGS)是近年来发现的一种转录后基因沉默现象,是植物抵抗病毒侵染的一种自然机制。现已被开发为快速鉴定植物基因功能的一种反向遗传学新技术。与传统的植物转基因技术相比,VIGS无需构建转基因植株,而且具有操作简便、获得表型快速等优点,目前已广泛应用于与植物抗病、逆境胁迫、细胞信号转导以及生长发育等相关基因功能的研究。该文就VIGS技术的作用机理、主要操作规程、在植物基因功能研究方面的应用以及存在的问题进行综述。     

微生物基因功能的研究策略与方法

微生物基因功能的研究对于揭示微生物生命活动的规律及其在食品发酵、医药卫生、工农业生产等领域的应用机制具有重要意义。经过数十年的发展,微生物基因功能的研究方法已经从传统的同源重组技术发展到基于核酸内切酶的高效打靶技术,将微生物基因功能的研究推向了新的高度。文章就微生物基因功能的研究策略及常用方法做一综述,主要包括生物信息学方法预测、基因表达谱分析、基因敲除技术、基因敲入技术、基因沉默技术和基因编辑技术等。     

MicroRNA与细胞信号转导通路研究进展

成熟的microRNA(miRNA)是一种长约22 nt的非编码RNA,通过与靶基因的3′非翻译区(3′UTR)结合来调控靶基因的表达。直至目前,在不同物种中发现的miRNA达6 397个。miRNA的发现为基因表达调控研究打开了新的窗口。目前研究者不仅证实miRNA在生物体生长、发育和疾病发生等过程中发挥着重要的作用,而且开始进一步探寻其发挥作用的分子机理。综述了miRNA与细胞信号转导途径之间的关系,从而有助于从基因水平上理解疾病的发生机制,为疾病的诊断、治疗提供依据。     

蛋白质转导--外源物质进入细胞的新工具

相对于常规的基因转移技术,蛋白质直接跨过细胞膜进入细胞(protein transduction,蛋白质转导)还比较新颖。蛋白质转导结构坟(protein transduction doraain,FTD)最早发现于HIV的TAT蛋白,之后证明多种天然的和人工合成的蛋白质和多肽都具有蛋白质转导能力。PTD有相似的结构特征,但是其转导的详细机制仍不清楚,很多研究结果倾向于静电相互作用模式。大量研究证明,PTD具肓转导能力强、无细咆特异性、能够穿过血脑屏障等特点,这为癌症、AIDS、神经系统疾病等的基础研究和治疗提供了新的工具。     

三种基因转导方法在不同代龄复制性衰老细胞中的比较研究

以增强型绿色荧光蛋白 (EGFP)为报告基因 ,检测腺病毒、逆转录病毒和脂质体对 2 0代和 33代WI 38细胞的转导效率、EGFP的表达强度、对细胞增殖能力和生物学行为影响 ,比较病毒及非病毒方法对不同代龄成纤维细胞 (WI 38)细胞基因转导的特性 .结果显示 :对 2 0代和 33代WI 38细胞,腺病毒转染效率均最高 ,逆转录病毒其次 ;EGFP表达强度腺病毒最高 ,逆转录病毒最低 ;各方法2 0代细胞转导效率及表达强度均高于 33代细胞 (P <0 0 5 ) .逆转录病毒对细胞繁殖和SA β gal染色没有影响 ,腺病毒和脂质体均能导致细胞增殖能力下降 ,SA β gal染色阳性率上升 ,尤以脂质体明显 (P <0 0 5 ) .表明逆转录病毒介导基因对WI 38细胞衰老进程几乎没有影响 ,且转导效率较高 ,适于衰老细胞转基因研究     

玉米花药培养和单倍体育种的研究新进展

利用花药培养获得单倍体,从而加速育种进程,是一顶新兴的生物技术,目前在玉米育种中广泛应用,本文综合近几年来国内外玉米的花药培养、单位体育种以及基因工程等方面的研究进展,重点对影响玉米花药培养效率的诸多因素进行了详细论述,并讨论了利用单7保体植株进行基因转导的潜力。     

核衣壳蛋白VP39融合TAT转导肽对杆状病毒转导哺乳动物细胞的影响

为了提高昆虫杆状病毒在哺乳动物细胞中转导基因的效率,构建了重组杆状病毒AcRed-tat和AcRed。两者都能在哺乳动物细胞内表达红色荧光蛋白作为报告基因。同时,AcRed-tat带有HIV-1Tat转导肽、病毒主要衣壳蛋白基因vp39及增强型绿色荧光蛋白(egfp)三者的融合基因,并由杆状病毒多角体启动子表达,能够在昆虫细胞中表达该Tat融合蛋白,并掺入子代病毒粒子。而AcRed作为相应的对照病毒,带有多角体启动子表达vp39和egfp的融合基因。2株病毒分别转导哺乳动物细胞后,利用流式细胞仪检测报告基因的表达水平,发现在CHO和Vero细胞中AcRed-Tat介导的报告基因表达水平明显高于AcRed,而在HEK293细胞中2株病毒介导的报告基因表达水平差异不显著。结果表明Tat转导肽可以提高杆状病毒对一部分哺乳动物细胞的转导效率,为改进杆状病毒-哺乳动物细胞转导载体提供了新的思路。     

应用基因芯片筛选鼻咽癌信号转导相关基因

目的：建立具有组织特异性的鼻咽癌基因表达谱,筛选鼻咽癌中信号转导相关基因。方法：采用深圳微芯公司基于玻片的包含8046个人类基因的基因芯片,检测7例鼻咽癌组织及1例鼻咽炎组织,初步获得鼻咽癌异常表达基因;结合GO分类从异常表达的基因中筛选信号转导相关基因,以Biocarta信号通路数据库查询筛选基因相关转导信号通路信息。结果：在鼻咽癌组织独得1241个异常用表达基因,其中高表达基因871个,低表达基因343个。发现28个差异表达基因与细胞的信号转导相关,其中表达上调的21个,表达下调的7个。结论：成功建立了具有组织特异性的鼻咽癌基因表达谱,初步获得了鼻咽癌信号转导相关基因。     

Microbubble-enhanced sonoporation: efficient gene transduction technique for chick embryos

The gene transduction technique is a useful method to study gene functions that underlie vertebrate embryogenesis. In this study, a new gene transduction technique is reported using microbubble-enhanced sonoporation (hereafter referred to as sonoporation) to achieve ectopic and transient gene expression for several embryonic organs including embryonic chick limb bud mesenchymes. The technique proposed in this study has the advantages of 1) relatively simple gene transduction procedures, and 2) efficient exogenous gene transduction and expression with lower damages to embryos. Green fluorescent protein (GFP) or LacZ was misexpressed in limb bud mesenchymes by sonoporation, with the introduced expression transiently detected in the injected sites. Most of the transduced chick embryos survived without showing significant embryonic abnormalities or cell death after sonoporation. To demonstrate its efficacy for assessing the effect of transient gene transduction, the Shh (sonic hedgehog) was transduced into the developing chick limb bud. The transduced limb bud displayed limb malformations including partial digit duplication. Advantages and possible future applications in relation to this method are discussed.     

外源基因导入技术在主要农作物育种上的应用进展

以国内科技期刊上发表的文献研究结果为依据,综述了农杆菌介导技术、基因枪导入技术、花粉管通道技术与激光微束穿刺技术在小麦、水稻、棉花、大豆、油菜等主要农作物育种上的应用进展。同时,还简要介绍了其中主要外源基因导入技术的基本原理,并评价了其优缺点。     

Deficiency of oncoretrovirally transduced hematopoietic stem cells and correction through ex vivo expansion

BACKGROUND: Extensive efforts to develop hematopoietic stem cell (HSC) based gene therapy have been hampered by low gene marking. Major emphasis has so far been directed at improving gene transfer efficiency, but low gene marking in transplanted recipients might equally well reflect compromised repopulating activity of transduced cells, competing for reconstitution with endogenous and unmanipulated stem cells. METHODS: The autologous settings of clinical gene therapy protocols preclude evaluation of changes in repopulating ability following transduction; however, using a congenic mouse model, allowing for direct evaluation of gene marking of lympho-myeloid progeny, we show here that these issues can be accurately addressed. RESULTS: We demonstrate that conditions supporting in vitro stem cell self-renewal efficiently promote oncoretroviral-mediated gene transfer to multipotent adult bone marrow stem cells, without prior in vivo conditioning. Despite using optimized culture conditions, transduction resulted in striking losses of repopulating activity, translating into low numbers of gene marked cells in competitively repopulated mice. Subjecting transduced HSCs to an ex vivo expansion protocol following the transduction procedure could partially reverse this loss. CONCLUSIONS: These studies suggest that loss of repopulating ability of transduced HSCs rather than low gene transfer efficiency might be the main problem in clinical gene therapy protocols, and that a clinically feasible ex vivo expansion approach post-transduction can markedly improve reconstitution with gene marked stem cells.     

Investigation of optimal transduction conditions for baculovirus-mediated gene delivery into mammalian cells

Although baculovirus-mediated gene delivery into mammalian cells has been documented in a wealth of the literature, systematic investigation of the optimal transduction conditions remains unavailable. In this work, a transduction protocol using unconcentrated baculovirus is proposed for simple and efficient gene delivery into HeLa cells. We found that approximately 75-85% of the cells could be readily transduced and express the reporter protein when virus transduction occurred for 4 h at 25 degrees C using Dulbecco's phosphate-buffered saline (D-PBS) as the surrounding solution. This method contrasts with previous protocols in which transduction occurs for 1 h at 37 degrees C using growth medium (e.g., DMEM) as the surrounding solution. Investigation of the physical parameters led to the findings that: 1) baculovirus uptake by HeLa cells continued for at least 4 h in the event of high virus dosage, which led to higher gene expression; 2) the half-life of baculovirus dramatically decreased at 37 degrees C; 3) EGTA pretreatment did not apparently facilitate the gene delivery when the cells grew to multilayers; and 4) lower transduction efficiency and gene expression were obtained when DMEM was used (in comparison with D-PBS and TNM-FH), suggesting that DMEM contains certain inhibitory factors for baculovirus transduction. Our data uncovered several aspects that were not investigated before and the optimized transduction conditions allowed for gene delivery as efficient as that by the protocols commonly employed by others, but eliminated the need for virus ultracentrifugation. The protocol not only represented a simpler approach, but also considerably reduced possible virus inactivation during ultracentrifugation, thus making it easier to convert the baculovirus/mammalian cell system to a tool for eukaryotic protein production on a larger scale.     

Baculovirus-mediated gene transfer is attenuated by sodium bicarbonate

BACKGROUND: Baculovirus transduction of cultured mammalian cells is typically performed by incubating the cells with virus using culture medium (e.g. Dulbecco's modified Eagle's medium (DMEM)) as the surrounding solution. However, we previously uncovered that DMEM hinders the baculovirus-mediated gene transfer. METHODS: In this study, we systematically explored the influences of promoter and medium constituents on the transduction efficiency by using different recombinant viruses and surrounding solutions for transduction, followed by flow cytometric analyses. Whether the key medium component impeded baculovirus binding to the cells and subsequent virus entry was investigated by immunofluorescence/confocal microscopy and quantitative real-time polymerase chain reaction (Q-PCR). RESULTS: We demonstrated that the poorer transduction by using DMEM as the surrounding solution is independent of the promoter. Examination of the medium constituents group by group revealed that the balanced salt solution suppresses the baculovirus transduction. By omitting individual salt species in the balanced salt solution, we surprisingly uncovered that NaHCO(3), a common buffering agent, exerts the inhibitory effects in a concentration-dependent manner. Intriguingly, NaHCO(3) did not debilitate the baculovirus, nor did it inhibit virus binding to the cells. Instead, NaHCO(3) inhibited baculovirus transduction by reducing the intracellular virus number. CONCLUSIONS: To our best knowledge, this is the first report unraveling the significance of NaHCO(3) in gene transfer. Our finding suggests that baculovirus-mediated gene transfer can be readily enhanced by omitting NaHCO(3) from the medium during the transduction period.     

Evaluation of optimal concentration and exposure duration of valproic acid alone or in combination with ViraDuctin to augment adenovirus transduction in human adipose stem cells

Background aimsRecombinant adenoviruses have tremendous potential in both gene therapy research and therapeutic applications. Mesenchymal stromal cells have a set of several properties that make them ideally suited for both regenerative medicine and gene and drug delivery. A limitation of adenoviral-mediated gene transfer is indeed the poor transduction rate of cells with low or no levels of the specific adenoviral cell surface receptor coxsackie virus and adenovirus receptor (CAR), such as human mesenchymal stromal cells. In the present work, we tried to increase the adenovirus transduction level and mediated gene delivery of human adipose stem cells with the use of valproic acid (VPA) and determined the proper concentration and duration of treatment alone or in combination with ViraDuctin adenovirus transduction reagent.MethodsGreen fluorescent protein–expressing recombinant adenovirus was propagated. The effects of various doses and exposure periods of VPA on CAR expression in human adipose stem cells were speculated by quantitative real-time polymerase chain reaction and adenoviral transduction rate by flow cytometry in different doses and time intervals of VPA and in combination with ViraDuctin transduction reagent.ResultsCAR messenger RNA upregulation through VPA was observed in human adipose stem cells; it was a dependent factor of dose and exposure time. Consequently, adenoviral transduction level of human adipose stem cells treated with VPA was increased, and co-administration of VPA and ViraDuctin further enhanced the transduction rate.ConclusionsThese results confirm that addition of VPA to hASCs alone or in combination with ViraDuctin has enhancing effects on adenoviral transduction rate, which can be auspicious in adenoviral-mediated gene therapy.     

人白介素－3基因表达调节的研究

报导了ｈ－ＩＬ－３基因表达调节研究的结果：（１）人静止的外周血淋巴细胞几乎不表达ＩＬ－３ｍＲＮＡ,但受丝裂原ＰＨＡ的刺激后则诱导ＩＬ－３ｍＲＮＡ表达,ＴＰＡ与ＰＨＡ联合处理,使ＩＬ－３ｍＲＮＡ的蓄积进一步增加,但ＴＰＡ单独不足以诱导ＩＬ－３ｍＲＮＡ蓄积;（２）Ａ２３１８７／ＴＰＡ能代替ＰＨＡ／ＴＰＡ的刺激,并直接诱导ＩＬ－３ｍＲＮＡ表达;（３）ＴＲＥＯＤＮ处理则显著抑制ＰＨＡ／ＴＰＡ诱导的ＩＬ－３ｍＲＮＡ表达。这些结果揭示：ｈ－ＩＬ－３基因的表达在转录及转录后水平被调节,而且是可诱导的,诱导ｈ－ＩＬ－３基因表达、需要Ｃａ２＋依赖及ＰＫＣ依赖的两个信息转导系统,Ｆｏｓ蛋白是反式激活ＩＬ－３基因表达的转录因子,ＰＫＣ依赖的转导系统,可能与ＩＬ－３ｍＲＮＡ的稳定性有关。     

植物抗脱水胁迫的分子机制

主要介绍植物在脱水胁迫下,逆激基因产物的功能和胁迫信号的转导过程.逆激基因产物的功能可分为两类：一类起“保护”作用,另一类起“调节”作用.在脱水胁迫起始信号和基因表达之间至少存在四条信号转导通路,两条依赖脱落酸(ABA),两条不依赖ABA,依赖ABA的途径中有1条必须有蛋白质合成.不依赖ABA的途径中有1条与低温胁迫应答有共同的信号转导通路.     

Highly efficient baculovirus-mediated gene transfer into rat chondrocytes

To explore the potential of baculovirus serving as a gene delivery vector in tissue engineering of articular cartilage, the efficiencies of baculovirus-mediated gene delivery into primary rat chondrocytes were evaluated and the transduction protocol commonly employed by others (using concentrated virus at multiplicity of infection [MOI] 200 for 1 h) was found to be ineffective (<1%). Therefore, a modified protocol was adopted, which markedly enhanced the efficiency (68%). Optimization of the transduction parameters, such as incubation time (8 h), temperature (25 degrees C), and surrounding solutions (PBS), further increased the efficiency to 88% and prolonged the duration of expression to 21 days, suggesting that the cells previously considered nonpermissive to baculovirus transduction may be reexamined for their permissiveness using alternative transduction protocols. The elevated efficiency correlated well with increased virus uptake upon extended incubation time, as demonstrated by quantitative real-time polymerase chain reaction (Q-PCR). The Q-PCR also revealed the degradation of viral DNA over culture time. Although the virus transduction somewhat hindered the cell proliferation, growth rate could be restored in the long-term culture. More importantly, transduced cells could secrete articular cartilage-specific type II collagen and glycosaminoglycan as well as mock-transduced cells, confirming that normal differentiation state of rat chondrocytes is retained upon baculovirus transduction. Taken together, these data indicate that baculovirus is a safe and highly efficient gene delivery vehicle into rat chondrocytes.