靶向Midkine基因的siRNA真核表达载体的构建及鉴定

目的 构建靶向midkine基因的siRNA 的表达载体,为研究midkine在肿瘤中作用提供一个新的方向.方法 根据基因库中midkine cDNA 序列设计和合成针对人midkine 基因的siRNA 寡核苷酸,定向克隆入质粒载体PGCsiU6,对该重组表达载体进行酶切鉴定及DNA 测序.结果 酶切鉴定、DNA 测序证实表达质粒构建成功,无碱基突变.结论 成功构建了靶向midkine 基因表达的siRNA 干扰质粒载体PGCsiU6/midkine,为进一步运用RNA干扰技术进行midkine 基因功能研究奠定了基础.     

反向遗传学策略及其在寄生虫研究中的应用

由基因结构认识基因功能的科学称为反向遗传学。随着越来越多生物的基因组序列被阐明,研究基因功能的反向遗传学已成为生物学领域的热点。反向遗传学所运用的技术也在不断地发展与成熟,如基因敲除、反义核酸等均对基因功能鉴定起到一定的作用。尤其是近五、六年内兴起的RNA干扰技术,其作为一种简单有效的代替基因敲除的反向遗传学的研究工具,在后基因组时代寄生虫的大规模基因功能研究中,具有广阔的应用前景。此外,选择适合的模式生物研究反向遗传学技术,也是研究的一个重要方面。     

反义DNA甲基化酶3b基因片断真核表达载体的构建及鉴定

目的构建反义DNA甲基化酶3b（DNMT3b）基因片断真核表达载体,为研究DNMT3b基因功能提供工具。方法根据DNMT3b基因cDNA序列中编码序列设计PCR引物,在上下游引物5′端分别添加XbaⅠ和KpnⅠ酶切位点,RT—PCR从胆管癌细胞QBC-939中获得485bp的DNA片断;将该片断反向插入真核表达载体pcDNA3．1（＋）的多克隆位点,构建反义DNMT3b基因片断真核表达载体,并用PCR、酶切法和DNA测序鉴定。结果PCR鉴定得到467bp特异条带,双酶切鉴定得到471bp片断和5．4kb载体片断,DNA测序说明插入片断序列正确。结论本研究构建的反义DNMT3b基因片断真核表达载体可为进一步研究DNMT3b基因功能提供实验工具。     

反向遗传学策略及其在寄生虫研究中的应用

由基因结构认识基因功能的科学称为反向遗传学。随着越来越多生物的基因组序列被阐明，研究基因功能的反向遗传学已成为生物学领域的热点。反向遗传学所运用的技术也在不断地发展与成熟，如基因敲除、反义核酸等均对基因功能鉴定起到一定的作用。尤其是近五、六年内兴起的RNA干扰技术，其作为一种简单有效的代替基因敲除的反向遗传学的研究工具，在后基因组时代寄生虫的大规模基因功能研究中，具有广阔的应用前景。此外，选择适合的模式生物研究反向遗传学技术，也是研究的一个重要方面。     

乙型肝炎病毒前S2-ENV融合基因在NIH3T3细胞中的表达和初步鉴定

HBV是严重威胁人类健康的病毒之一，在我国约有1．2亿HBV携带者，但目前对乙型肝炎的治疗仍停留在对症治疗及调节机体免疫功能等阶段，尚无直接抑制HBV复制的有效方法。反义技术是依赖反义寡核苷酸（ODN）、反义RNA和核酶（ribozyme）等手段进行基因治疗、基因调控研究的方法。它在基因水平上考虑选择性关闭或修饰靶基因，使其失活而达到对疾病的特异性治疗，或研究特定基因功能及基因调控机制等。HBV在复制过程中经历了从前基因组mRNA逆转录到DNA的过程，利用反义技术能直接抑制病毒复制。     

RNA干扰技术在真核细胞型病原体研究中的应用

RNA干扰是由与靶基因序列同源的双链RNA介导的序列特异性的靶基因沉默过程,具有高特异性、高效性、可扩散性和可遗传性等特点,能相对快速、简便地提供基因功能的信息,为原虫和真菌基因功能的研究提供强有力的工具。该文综述了RNA干扰技术在原虫及机会致病真菌等真核细胞型病原体研究中的应用现状与进展。     

RNAi技术在胰腺癌研究中的应用进展

在后基因组时代，基因功能的研究以前所未有的速度快速发展，各种研究基因功能的新技术不断涌现。RNA干扰（RNA interference，RNAI），一种新的、强有力的研究工具，在功能基因组学领域具有巨大的应用潜力。所谓RNAi就是利用双链RNA（double—stranded RNA，dsRNA）高效、特异地阻断体内特定基因表达，促使mRNA降解，使细胞表现出特定基因缺失表型的过程，即诱导序列特异的转录后基因沉默（PTGS）。RNAi技术作为一种简单、有效的代替基因敲除的工具，迅速成为基因功能研究的有力工具以及最有潜力的基因干预治疗方法，     

病原菌新基因功能的研究策略

随着高通量测序技术和蛋白组学技术的发展,越来越多的病原菌新基因被发现,研究病原菌新基因有助于更好的防控由该病原菌引起的疾病的传播。本文从生物信息学角度、新基因功能获得与缺失、与新基因编码产物互作蛋白的分析等方面综述了适用于病原菌新基因功能研究的策略,为基因分子生物学等相关领域提供参考。此外,本文还结合常规的新基因研究方法,将芯片技术,2D-DIGE技术渗透入病原菌新基因研究策略中,使研究者能更快捷准确的找出新基因功能研究的切入点,进而制定切实可行的新基因功能的研究方案。     

DNA甲基化异常的机制及其在肿瘤中的作用

恶性肿瘤的发生是一个多因素、多阶段的复杂过程，涉及多种基因功能异常。导致这些基因功能异常的原因包括基因突变、基因缺失、基因过表达和基因表型(epigenetic)改变等。基因表型改变有DNA甲基化、组蛋白乙酰化状态异常等形式。自肿瘤发生早期，DNA甲基化状态就开始发生异常，并通过多条途径参与肿瘤发生发展。九十年代以来，对DNA甲基化异常认识的深入，使我们对恶性肿瘤的发生机理有了更深刻、更全面的认识。     

RNA干扰技术在寄生虫基因功能研究中的应用

RNA干扰（RNAi）技术是近几年发现的一种基因沉默技术，已成为分析寄生虫基因功能的有效手段，它为基因治疗、药物阻断剂候选基因的筛选等方面的研究提供了新方法。该文对RNAi技术的发现、作用机制、分子特征及其在寄生虫基因功能研究中的最新进展作一综述。     

Epigenetic inactivation of the CIP/KIP cell-cycle control pathway in acute leukemias

Dysregulation of the cell cycle is important in oncogenesis. We analyzed the potential inactivation of the CIP/KIP family of the cyclin E/CDK/RB pathway by gene promoter hypermethylation in leukemias. The methylation-specific polymerase chain reaction (MSP) with primers for methylated (M-MSP) and unmethylated (U-MSP) alleles of the p21, p27, and p57 genes was used to study five leukemic cell lines, 50 acute myeloid leukemia (AML) samples, and 25 acute lymphoblastic leukemia (ALL) samples. p21 was hemizygously methylated in Raji and Jurkat but remained unmethylated in U937, HL60, and NB4. p27 was hemizygously methylated in Raji but unmethylated in the other cell lines. p57 was completely methylated in Raji and NB4, hemizygously methylated in U937, and unmethylated in HL60 and Jurkat. At diagnosis, p21 methylation was not detected in any case of AML or ALL. p27 methylation occurred in 2 (4%) AML patients and in 1 (4%) ALL patient. p57 methylation occurred in 1 (2%) AML patient and in 1 (4%) ALL patient. Therefore, methylation inactivation of the INK4/CDK/RB pathway in leukemia is infrequent. A review of the literature showed a marked variation in the frequencies of methylation of these genes, which might be attributable to difference in methodologies used to detect gene methylation.     

SOCS1基因与肝细胞癌的关系研究进展

SOCS1基因位于16p12-p13.1,它编码蛋白SOCS1并属于SOCS蛋白家族,通过抑制JAK—STAT信号转导通路发挥作用,其失活机制主要是甲基化和杂合性缺失。SOCS1在肝细胞癌中广泛甲基化和表达明显降低,提示SOCS1可能是抑癌基因,在肝细胞癌的发生发展中起十分重要的作用。     

Methylation is an inactivating mechanism of the p16 gene in multiple myeloma associated with high plasma cell proliferation and short survival

In order to gain further insights into the role of the p16 gene in cell cycle regulation and the prognostic implications of its inactivation, we investigated the methylation status of the p16 gene in 98 untreated patients using a polymerase chain reaction assay based on the inability of some restriction enzymes to digest methylated sequences. Forty-one patients showed a p16 methylated gene (42%). The percentage of S-phase plasma cells (PC) in these patients was almost three times higher than in those with an unmethylated p16 gene (4.16% +/- 3.37%vs 1.5% +/- 1.41%, P < 0.001). The presence of p16 methylation also correlated with both elevated beta2-microglobulin serum levels and high C-reactive protein values. Patients with a p16 methylated gene had shorter overall and progression-free survival than those patients without p16 methylation. However, this feature did not retain independent prognostic influence on multivariate analysis, probably due to its association with the S-phase PC, which had more potent statistical significance in the Cox model. These findings showed methylation of the p16 gene was a frequent event inMM patients at diagnosis, and was associated with an increased proliferative rate of plasma cells and a poor prognosis, indicating an important role for p16 gene in the cell cycle regulation of multiple myeloma tumour cells, and thus in the clinical outcome of the disease.     

Promoter hypermethylation of cancer-related genes: a strong independent prognostic factor in acute lymphoblastic leukemia

Promoter hypermethylation plays an important role in the inactivation of cancer-related genes. This abnormality occurs early in leukemogenesis and seems to be associated with poor prognosis in acute lymphoblastic leukemia (ALL). To determine the extent of hypermethylation in ALL, we analyzed the methylation status of the CDH1, p73, p16, p15, p57, NES-1, DKK-3, CDH13, p14, TMS-1, APAF-1, DAPK, PARKIN, LATS-1, and PTEN genes in 251 consecutive ALL patients. A total of 77.3% of samples had at least 1 gene methylated, whereas 35.9% of cases had 4 or more genes methylated. Clinical features and complete remission rate did not differ among patients without methylated genes, patients with 1 to 3 methylated genes (methylated group A), or patients with more than 3 methylated genes (methylated group B). Estimated disease-free survival (DFS) and overall survival (OS) at 11 years were 75.5% and 66.1%, respectively, for the nonmethylated group; 37.2% and 45.5% for methylated group A; and 9.4% and 7.8% for methylated group B (P < .0001 and P = .0004, respectively). Multivariate analysis demonstrated that the methylation profile was an independent prognostic factor in predicting DFS (P < .0001) and OS (P = .003). Our results suggest that the methylation profile may be a potential new biomarker of risk prediction in ALL.     

A DNA insulator prevents repression of a targeted X-linked transgene but not its random or imprinted X inactivation

Some genes on the inactive X chromosome escape silencing. One possible escape mechanism is that heterochromatization during X inactivation can be blocked by boundary elements. DNA insulators are candidates for blocking because they shield genes from influences of their chromosomal environment. To test whether DNA insulators can act as boundaries on the X chromosome, we inserted into the mouse X-linked Hprt locus a GFP transgene flanked with zero, one, or two copies of a prototypic vertebrate insulator from the chicken beta-globin locus, chicken hypersensitive site 4, which contains CCCTC binding factor binding sites. On the active X chromosome the insulators blocked repression of the transgene, which commences during early development and persists in adults, in a copy number-dependent manner. CpG methylation of the transgene correlated inversely with expression, but the insulators on the active X chromosome were not methylated. On the inactive X chromosome, insulators did not block random or imprinted X inactivation of the transgene, and both the insulator and transgene were almost completely methylated. Thus, the chicken hypersensitive site 4 DNA insulator is sufficient to protect an X-linked gene from repression during development but not from X inactivation.     

The X chromosome in development in mouse and man

Summary In mammals, dosage compensation for X-linked genes between males and females is achieved by the inactivation of one of the X chromosomes in females. The inactivation event occurs early in development in all cells of the female mouse embryo and is stable and heritable in somatic cells. However, in the primordial germ cells, reactivation occurs around the time of meiosis. Owing to random inactivation in somatic cells, all female mice and humans are mosaic for X-linked gene function. Variable mosaicism can result in expression of disease in human females heterozygous for an X-linked gene defect.In the extra-embryonic lineages of female mouse embryos, and in the somatic cells of female marsupials, the paternally inherited X chromosome is preferentially inactivated. The X chromosomes in the egg and sperm must be differentially marked or imprinted, so that they are distinguished by the inactivation mechanism in these tissues.Initiation of inactivation of an entire X chromosome appears to spread from a single X-inactivation centre and may involve the recently discovered gene,XIST, which is expressed only from the inactive X chromosome. The maintenance of inactivation of certain household genes on the inactive X chromosome involves methylation of CpG islands in their 5' regions. Critical CpG sites are methylated at, or very close to, the time of inactivation in development.The mouse and the human X chromosomes carry the same genes but their arrangement is different and there are some genes in the pairing segment and elsewhere on the human X chromosome which can escape inactivation. Regions of homology between the mouse and human X chromosomes allow prediction of the map positions of homologous genes and provide mouse models of genetic disease in the human.