Construction of recombinant adeno-associated viral vectors in human neurenergen-3 gene

BACKGROUND： Research of transgene brings hope for gene therapy of various diseases; in addition, some projects have been tested in clinic. Recently, the focus has been to find an ideal vehicle and a suitable therapeutic gene. OBJECTIVE： To explore an effective way to construct recombinant adeno-associated viral vectors expression in human neurnnergen-3 gene. DESIGN： Gene directed cloning. SETTING： Central Laboratory of Northern China Coal Medical College. MATERIALS： DH5a competent bacillus coli strain was provided by Capital Medical University; pCDNA3-NT-3 by professor Chen from Bengbu Medical College; pAAV-Laze, pAAV-Helper, pAAV-RC and pAAV-MCS plasmids by Capital Medical University; HEK293 cells by Cell Center of Basic Medical College of Tongji Medical University. METHODS： NT-3 genes which were selected from pCDNA3-NT-3 plasmids were cloned in pAAV-MCS to form a recombinant adeno-associated viral plasmid （pAAV-NT-3）. pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were extracted, purified and subjected to enzyme-shearing evaluation. In addition, pAAV-NT-3 and pAAV-LacZ were cotransfected with pHelper and pAAV-RC, respectively into AVV-293 cells with DNA mediated by calcium superphosphate transfection gene; and then, AVV-293 cells were packed into recombinant adeno-associated viral rAAV-NT-3 and rAAV-LacZ. After collection of viral particles, rAAV-LacZ viral stock solution was diluted based on ratio of 10：1 and the mixture was used to infect HT 1080 cells. X-gal stain was used to measure virus titer. MAIN OUTCOME MEASURES： Size of targeted gene fragments, validity of vehicle construction and virus titer. RESULTS： Targeted gene NT-3 was successfully inserted into the relative vehicle pAAV and pAAV-NT-3 was correctly recongnized by enzyme-shearing evaluation. Enzyme-shearing electrophoresis demonstrated that pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were successfully extracted and purified. β-galactoside staining in situ indicated that LacZ genes were expressed in human fibrosarcoma cells （HT1080） and the recombinant virus titer was measured as 1 ×10^12 virus particles per milliliter. CONCLUSION： Total-length cDNA fragment of NT-3 gene, which is obtained from pCDNA3-NT-3 plasmids, is closely matched to polyclone enzyme-shearing sites of adeno-associated viral vectors, while the combination can be used to construct recombinant adeno-associated viral vectors expression in hNT-3 gene.

人c－fos原癌基因cDNA克隆及核酸序列分析

本研究应用逆转录ＰＣＲ方法从Ｊｕｒｋａｔ传代细胞株获得一３８０ｂｐ的人ｃ一ｆｏｓ原癌基因ｃＤＮＡ片段，并将其克隆到具有双向转录启动子的载体质粒ｐＧＥＭ３Ｚｆ（＋）中。核酸序列分析表明，所克隆的ｃＤＮＡ片段包括人ｃ－ｆｏｓ基因外显子２的大部分，外显子３的全部及外显子４的一部分。该重组质粒可分别用于制备ｃ－ｆｏｓｃＤＮＡ探针和ＲＮＡ探针，为ｃ－ｆｏｓ基因结构、表达调控等方面的研究奠定了工作基础。

Copper—Controllable,Site—Specific DNA Excision in Transgenic Plants

A copper-inducible, Cre-loxP recombination-mediated DNA excision system has been developed in transgenic tobacco plants. The copper inducible system derived from yeast was used for the control of the expression of the Cre recombinase. Upon copper induction, the GVS reporter gene expression unit flanked by two direct lox sites was excised from the transgenic tobacco genome. Quantitative fluorometric GUS assays, Northern blot and PCR analyses showed a high-efficient, copper-dependent and Cre-loxP mediated DNA recombination in all the tested transgenic lines. The copper inducible foreign gene excision might be of great potential in genetic control of transgenic crops.

Construction of Eukaryotic Expression Vector pBlacz and Its Expression Both in Vitro and In Vivo

A novel eukaryotic expression vector pBlacZ was constructed, which was transfected into the cell lines of NIH/3T3, COS-1, CHO and the primary culture of murine dermatic fibroblasts in vitro, and also into the murine subcutaneous layer and skeletal muscles of rats in vivo. It was detected that the gene expression vector could encode the E. Coli β-galactosidase effectively in all these histocytes. The results suggested that pBlacZ, as a novel expression vector, might have certain value of application.

不用接头进行异末端DNA重组的方法

用过渡载体通过两次重组进行不同粘性末端之间的ＤＮＡ重组．该方法不需要替换接头，从而省去昂贵的试剂和平末端连接这种较难的操作，既经济，简单又有效，易于操作．

Molecular evolutionary analysis of gene families encoding DNA recombination and repair proteins and histone demethylases,and their functional implications

Many eukaryotic genes are members of multi-gene families due to gene duplications, which generate new copies that allow functional divergence. However, the relationship between

DNA End Resection:Facts and Mechanisms

DNA double-strand breaks（DSBs）,which arise following exposure to a number of endogenous and exogenous agents,can be repaired by either the homologous recombination（HR）or non-homologous end-joining（NHEJ） pathways in eukaryotic cells.A vital step in HR repair is DNA end resection,which generates a long 30single-stranded DNA（ss DNA） tail that can invade the homologous DNA strand.The generation of 30 ss DNA is not only essential for HR repair,but also promotes activation of the ataxia telangiectasia and Rad3-related protein（ATR）.Multiple factors,including the MRN/X complex,C-terminal-binding protein interacting protein（Ct IP）/Sae2,exonuclease 1（EXO1）,Bloom syndrome protein（BLM）/Sgs1,DNA2 nuclease/helicase,and several chromatin remodelers,cooperate to complete the process of end resection.Here we review the basic machinery involved in DNA end resection in eukaryotic cells.

A novel treatment for weight reduction by the recombinant "Pichia pastoris" yeast expressing the hybrid protein of "irisin-furin-transferrin"

Obesity is a major health problem across the world, but there are few ways to effectively treat or manage it in the long term. Researchers are searching for more convenient, cost-effective and noninvasive therapies for overweight and obese people. Recent studies have illustrated that the microbiome of the body＇s different organs can be used as a vehicle for in-situ gene therapy. We suggest that the recombinant form of ＂Pichia pastoris＂ yeast expressing the hybrid protein of ＂irisin-furin-transferrin＂ under the control of the enolase 1 promoter is a new nutraceutical strategy to absorb fewer calories from intestinal nutrients, and induce a higher metabolic rate to expend more calories, similar to that from engaging in physical activity. By comparison, this method can be a long-term, noninvasive treatment and can be used for obese patients who have movement limitations.

Multi-functions of exonuclease 1 in DNA damage response and cancer susceptibility

Exonuclease 1(EXO1)can catalyze nucleotide chain excision with its conserved N-terminal domain of 5′ to 3′ exonuclease activity,enabling it to influence diverse biological processes facing the challenges of genotoxic environmental factors such as ionizing radiation.This nuclease activity enables EXO1 to maintain replication forks and telomeres length,to facilitate post-replication DNA repair and to process the end resection step of homologous recombination of DNA double-strand breaks-induced by ionizing radiation.When DNA replication is disrupted or blocked,EXO1 can cleave the broken DNA ends to form 3’ssDNA,leading to repair pathways activation.Excess EXO1-mediated nucleotide excision,however,can introduce an abundance of single-stranded DNA that can cause mutation and recombination via micro-homology-mediated end joining or single-strand annealing mechanisms,contributing to a loss of genetic information.EXO1 activity must therefore be carefully regulated within healthy cells.The mutations and dysregulations of EXO1 can increase the sensitivity of cells to radiation injury and risk of oncogenic transformation,limit the adoption of specific treatments in a range of human diseases.As such,EXO1 represents a promising target for the treatment and prevention of cancer.In the present review,we delineate the structural properties and functional characteristics of EXO1,discuss the relationship between this exonuclease and cancer susceptibility as well as the second cancers related to radiotherapy.

Applications of 3D printed chimeric DNA biomaterials

The influence of genetic engineering on daily life is prominent,from genetically modified food to transgenic,antibiotic resistant plants.The direct manipulation of an organism’s genotype has opened the door to a myriad of applications in an effort to treat chronic diseases.This paper proposes the unification of chimeric DNA technology and three-dimensional bioprinting to spark the development of new therapies.While studies of chimeric DNA,i.e.recombinant DNA,have been conducted since 1970,bioprinting is a budding method for tissue engineering and regenerative medicine.Both technologies are further described in the background section of this paper,and followed by a detailed analysis into current research,benefits,and limitations of 3D printed chimeric biomate-rials.Major benefits include low-cost and effective treatments for cancer,bio-morphological nanostructures for targeted drug delivery,personalized medicine with the use of stem cells,and a significantly reduced rate of addi-tional surgeries and transplant rejection after implantation.However,there are several shortcomings with current chimeric DNA applications,e.g.CAR-T cell therapies,and ethical dilemmas regarding the creation and regulation of human-animal chimeras.This review then presents future directions,in which inks made from chimeric DNA and live cells can be printed into bioactive engineered tissue,or biodegradable vehicles for targeted delivery of hiPSCs or CAR T-cells.Finally,this review concludes with a reaffirmation of its main points and the authors’thoughts on the potential of 3D printed chimeric biomaterials.

The archaeal KEOPS complex possesses a functional Gon7 homolog and has an essential function independent of the cellular t^(6)A modification level

Kinase,putative Endopeptidase,and Other Proteins of Small size(KEOPS)is a multisubunit protein complex conserved in eukaryotes and archaea.It is composed of Pcc1,Kae1,Bud32,Cgi121,and Gon7 in eukaryotes and is primarily involved in N^(6)-threonylcarbamoyl adenosine(t^(6)A)modification of transfer RNAs(tRNAs).Recently,it was reported that KEOPS participates in homologous recombination(HR)repair in yeast.To characterize the KEOPS in archaea(aKEOPS),we conducted genetic and biochemical analyses of its encoding genes in the hyperthermophilic archaeon Saccharolobus islandicus.We show that aKEOPS also possesses five subunits,Pcc1,Kae1,Bud32,Cgi121,and Pcc1-like(or Gon7-like),just like eukaryotic KEOPS.Pcc1-like has physical interactions with Kae1 and Pcc1 and can mediate the monomerization of the dimeric subcomplex(Kae1-Pcc1-Pcc1-Kae1),suggesting that Pcc1-like is a functional homolog of the eukaryotic Gon7 subunit.Strikingly,none of the genes encoding aKEOPS subunits,including Pcc1 and Pcc1-like,can be deleted in the wild type and in a t^(6)A modification complementary strain named TsaKI,implying that the aKEOPS complex is essential for an additional cellular process in this archaeon.Knock-down of the Cgi121 subunit leads to severe growth retardance in the wild type that is partially rescued in TsaKI.These results suggest that aKEOPS plays an essential role independent of the cellular t^(6)A modification level.In addition,archaeal Cgi121 possesses dsDNA-binding activity that relies on its tRNA 3ʹCCA tail binding module.Our study clarifies the subunit organization of archaeal KEOPS and suggests an origin of eukaryotic Gon7.The study also reveals a possible link between the function in t^(6)A modification and the additional function,presumably HR.

Expression of TIMP-3 Gene by Construction of a Eukaryotic Cell Expression Vector and Its Role in Reduction of Metastasis in a Human Breast Cancer Cell Line

The present study is aimed at studying the gene for TIMP-3,a mammalian tissue inhibitor,by constructing a recombinant eukaryotic cell vector for gene therapy in human breast cancer.We obtained the TIMP-3 gene from the human placent by RT-PCR.TIMP-3 gene was subcloned into pcDNA3.1 vetor from pMD18T vector by means of gene cloning to construct pcDNA3.1 recombinant vector.Human breast cancer cell line MDA-MB-453 was transfected with pcDNA3.1-TIMP3 recombinant vector using lipofectamine reagent.Then the expression of TIMP-3 and the effect on the metastasis of MDA-MB-453 were examined.The correct construction of pcDNA-TIMP3 was identified by means of restriction enzyme analysis,PCR amplication and nucleotide sequencing.Western blotting showed that the transfected cells were able to express TIMP-3, indicating that our construction of the pcDNA-TIMP3 eukaryotic expression vector was constructed successfully.Our experiments further indicated that the potential of metastasis was significantly reduced for the transfected cell line MDA-MB-453.Cellular & Molecular Immunology.2004;1(4):308-310.

重组变应原在特异性免疫治疗中的应用

免疫治疗是治疗变应性疾病的根本措施,重组变应原就是利用重组DNA技术对已明确致敏蛋白组分的变应原构建变异体,其特性就是保持变应原的免疫特性,降低IgE结合活性.目前,已有几百种变应原有了重组变应原,重组变应原在临床诊断和评价患者的致敏状况方面已成为一种很好的方法;对用于皮下和舌下免疫治疗的适宜性还有待于进行评估.本文就重组变应原的优点、基本特性、低变应原性、单一变应原制剂和复合变应原制剂的临床研究进展、个性化免疫治疗进行综述.

Regulatory issues for genetically modified animals

Precision genetics and breeding have the potential to meet the agricultural needs and goals of the world in the 21 st century.These needs include increasing the efficiency of production of animals and improving their products with minimal impact on the environment.The USA is the major innovator in genomic science and the acknowledged leader in formulating policies to regulate genetic applications in medicine and agriculture.However,governments worldwide have been exceedingly reluctant to support the introduction of genetically modified(GM)animals into agriculture.Regulatory policies have stagnated due to legal guidelines that could not anticipate the needs and solutions that are evident today.This must change if we are to maintain planetary integrity.I propose a new,market-based regulatory model for GM livestock that has both a strong scientific foundation and has worked for 10000 years.The model is similar to that for information technology in which specific algorithms drive computer and cell phone applications.Genome engineers write genetic algorithms that drive the traits in biological organisms.Accordingly,GM products should be viewed in terms of their use and public benefit rather than by limitations to the genetic programing coming from a few highly vocal groups.Genetic algorithms(Genapps)of the 21st century will include not only introduction of synthetic genes,but also complete natural and synthetic biochemical pathways to produce agricultural products that are maximally efficient,healthy to humans and animals,and sustainable in an era of changing climates while avoiding environmental degradation.