同源重组构建表皮葡萄球菌纤维蛋白原结合基因缺失突变菌株

目的构建表皮葡萄球菌（表葡菌）纤维蛋白原结合基因（fbe基因）缺失突变菌株，为fbe基因功能研究提供实验工具。方法采用同源基因重组技术。构建质粒△pBT2（含fbe：：ermB基因），将其电转化进入金葡菌RN4220，再次抽提后电转化进入fbe基因阳性表葡菌（HB）中。将含有质粒△pBT2的表葡菌HB在5mg／L红霉素平板42℃经多次传代，使质粒ApBT2裂解，fbe：：ermB基因同源重组到表葡菌HB的染色体上，通过红霉素耐药、氯霉素敏感等特性筛选出含有fbe：：ermB基因的新表葡菌HB-ermB。结果经酶切鉴定后确认质粒△pBT2构建成功以及成功转导入金葡菌RN4220和表葡菌HB中，经PCR方法以及测序确认表葡菌HB的fbe基因缺失突变菌株HB-ermB构建成功。结论采用同源重组的方法完成了表葡菌HB的fbe基因缺失突变菌株的构建。     

Oxidative stress-induced homologous recombination as a novel mechanism for phenytoin-initiated toxicity

Although the mechanism(s) of phenytoin-initiated toxicity is unknown, phenytoin can be enzymatically bioactivated to a reactive intermediate leading to increased formation of reactive oxygen species, which can damage essential macromolecules, including DNA. The oxidation of DNA can induce DNA double-strand breaks (DSBs), which may be repaired through homologous recombination. Increased levels of DSBs may induce hyper-recombination, leading to deleterious genetic changes. We hypothesize that these genetic changes mediate phenytoin-initiated toxicity. To investigate this hypothesis we used a Chinese hamster ovary cell line containing a neo direct repeat recombination substrate to determine whether phenytoin-initiated DNA oxidation increases homologous recombination. Cells were treated with 0 to 800 microM phenytoin for 5 or 24 h, and homologous recombination frequencies and recombinant product structures were determined. Phenytoin-initiated DNA oxidation was determined by measuring the formation of 8-hydroxy-2'-deoxyguanosine. We demonstrate that phenytoin increases both DNA oxidation and homologous recombination in a concentration- and time-dependent manner. All recombination products analyzed arose via gene conversion without associated crossover. Our data demonstrate that phenytoin-initiated DNA damage can induce homologous recombination, which may be a novel mechanism mediating phenytoin-initiated toxicity.     

A role for homologous recombination and abnormal cell-cycle progression in radioresistance of glioma-initiating cells

Glioblastoma multiforme (GBM) is the most common form of brain tumor with a poor prognosis and resistance to radiotherapy. Recent evidence suggests that glioma-initiating cells play a central role in radioresistance through DNA damage checkpoint activation and enhanced DNA repair. To investigate this in more detail, we compared the DNA damage response in nontumor forming neural progenitor cells (NPC) and glioma-initiating cells isolated from GBM patient specimens. As observed for GBM tumors, initial characterization showed that glioma-initiating cells have long-term self-renewal capacity. They express markers identical to NPCs and have the ability to form tumors in an animal model. In addition, these cells are radioresistant to varying degrees, which could not be explained by enhanced nonhomologous end joining (NHEJ). Indeed, NHEJ in glioma-initiating cells was equivalent, or in some cases reduced, as compared with NPCs. However, there was evidence for more efficient homologous recombination repair in glioma-initiating cells. We did not observe a prolonged cell cycle nor enhanced basal activation of checkpoint proteins as reported previously. Rather, cell-cycle defects in the G(1)-S and S-phase checkpoints were observed by determining entry into S-phase and radioresistant DNA synthesis following irradiation. These data suggest that homologous recombination and cell-cycle checkpoint abnormalities may contribute to the radioresistance of glioma-initiating cells and that both processes may be suitable targets for therapy. Mol Cancer Ther; 11(9); 1863-72. ?2012 AACR.     

A new method (GOREC) for directed mutagenesis and gene repair by homologous recombination

Directed mutagenesis in mammalian cells has been the focus of intense research because of its promising application for gene correction and engineering. Both natural and modified oligonucleotides (ODN), RNA-DNA chimeric oligonucleotide (RDO) and small fragment DNA (SFHR), as well as vector DNA were used for promoting homologous replacement with varying success. It was recently shown that a triple helix-forming oligonucleotide (TFO) tethered to an oligonucleotide (donor DNA) can enhance mutagenesis by homologous recombination in cells. The basic idea is to accelerate homology search by oligonucleotide-directed triple helix formation in the vicinity of the target site for donor DNA. Here we describe a new method named GOREC (guided homologous recombination) which shares similar gene targeting, but has notable difference in the concept with the previous method. It is made of a homing device (TFO) and a donor DNA for effecting distinct functions. They are linked together by non-covalent or covalent interaction. This modular concept allows guidance of either an oligonucleotide (ODN, RDO) or a small DNA fragment to the target site for homologous replacement. Therefore, the triple helix site can be hundreds of base pairs away from the target site. An episomal assay for proof-of-principle study will be presented and discussed.     

Gene capture and random amplification for quantitative recovery of homologous genes

The polymerase chain reaction (PCR) is instrumental in molecular analysis of microorganisms, allowing for the selective amplification of nucleic acids directly from clinical and environmental samples. However, the principles that allow for targeted amplification of DNA become a hindrance when attempting to simultaneously discriminate and quantify complex mixtures of homologous genes. Here we present a simple solution to the quantitative problem by separating the enrichment and amplification aspects of a conventional PCR reaction. In this assay, genes are enriched using a DNA oligonucleotide capture probe and subsequently amplified in a two-step random amplification protocol. In order to evaluate the quantitative aspects of the gene capture assay, we used real-time quantitative-PCR to measure initial and final concentrations of homologous genes from constructed mixtures of genomes. Upon sampling for the universal DNA-dependent RNA polymerase gene, rpoC, we were able to demonstrate quantitative recoveries from a mixed DNA sample despite differences in gene copy number ranging up to 4 orders of magnitude. This suggests that minority populations as low as 0.01% of the total community are represented as accurately as populations at higher abundance. These results offer new possibilities for accurately and quantitatively monitoring diverse mixtures of microorganisms.     

基因治疗与心力衰竭

目的:探讨基因治疗方案的有效性和安全性。资料来源:应用计算机检索PUBMED1980-01/2006-10和EMCC 1994-10/2006-10与心力衰竭的基因治疗相关文章,检索词“Gen Therapy,Heart Failure”,并限定文章语言种类为“English”;同时计算机检索CMCC 1994-01/2006-10的相关文章,限定文章语言种类为中文,检索词“基因治疗、心力衰竭”。资料选择:对资料进行初审,选取试验包括上述治疗组和对照组的文献,然后筛除明显不随机临床试验的研究,对剩余的文献开始查找全文,进一步判断是否为RCT。纳入标准为:①试验包含平行对照组,即一般药物治疗。②治疗组为转基因治疗。资料提炼:共收集到49篇相关的随机和未随机试验,31个试验符合纳入标准。其余的排除。资料综合:31个试验包括478例动物模型,分别对应用不同靶点的转基因或基因敲除等方案进行治疗者予以评价。31篇文章中有10篇未达到预期的治疗目标,主要是在目的基因的选择及转染载体的选择方面出现了偏差。结论:虽动物试验取得了一定的成果,但尚无人体基因治疗的文献报道,随着基因技术的进步,基因治疗有望成为治疗心力衰竭的新途径。     

Single nucleotide polymorphisms in the homologous recombination repair genes and breast cancer risk in Polish women

Genetic polymorphisms in homologous recombination repair genes that can lead to protein haploinsufficiency are generally associated with increased cancer risk. The aim of the present study was to evaluate associations between the risk of breast cancer and single nucleotide polymorphisms in the genes, encoding three key proteins of the homologous recombination repair: RAD51 (the human homologue of the E. coli RecA protein), X-ray repair cross-complementing group (XRCC) 2 and XRCC3. The polymorphisms studied were G135C of the RAD51 gene (c. -98 G>C; rs1801320), Arg188His of the XRCC2 gene (c. 563 G>A; rs3218536), and Thr241Met of the XRCC3 gene (c. 722 C>T; rs861539). Each polymorphism was genotyped by the PCR-RFLP (restriction fragment-length polymorphism) method in 700 Polish female patients with sporadic breast cancer and in 708 cancer-free women, who served as controls. In the present study, we showed the association between RAD51 G135C polymorphism and the incidence of breast cancer (p < 0.0001), but found no significant association with XRCC2 Arg188His or XRCC3 Thr241Met polymorphism. Instead, significant association was identified between XRCC2 Arg188His or XRCC3 Thr241Met polymorphism and breast cancer progression, assessed by the histological grading. However, each of these three polymorphisms was not associated with the tumor size or the lymph node metastases. This study provides evidence that links single nucleotide polymorphisms of RAD51 and XRCC2/3 genes with the risk of breast cancer in Polish women. In conclusion, RAD51 G135C, XRCC2 Arg188His and XRCC3 Thr241Met polymorphisms may be regarded as predictive factors of sporadic breast cancer in female population.     

Gene therapy of mesothelioma

Despite improved diagnostic skills and new chemotherapeutic regimens, malignant mesothelioma (MM) remains a pathological disease with survival expectations after diagnosis remaining < 1 year. As the incidence of this disease has yet to peak, there is a pressing need for new therapeutic approaches. One such approach is gene therapy, which inserts 'therapeutic' genes into (generally) tumour cells seeking to induce tumour regression via a number of different theoretical mechanisms. This approach may be particularly relevent for mesothelioma as it is localised to body cavities and is readily accessible for biopsy sampling or for gene delivery. Furthermore, as MM patients rarely die from distant metastases, treating the primary tumour site may result in significant symptomatic and survival benefit. Herein, the paper discusses past, present and future views on gene therapy in the treatment of MM.