蓝藻基因转移系统的选择与建立

蓝藻是一类进行光合放氧的原核生物 ,因其结构的特殊性 ,已成为表达外源基因的理想宿主之一。然而外源基因转化系统的选择与建立一直影响着蓝藻基因工程的快速发展。总结了各类蓝藻基因转移系统的特点、影响因素、各系统间的优缺点、以及不同蓝藻株系最适基因转移系统的选择等 ,为利用蓝藻进行遗传操作提供可能 ,为蓝藻基因工程发展提供信息。     

Gene transfer into mammalian somatic cells in vivo.

Direct gene transfer into mammalian somatic tissues in vivo is a developing technology with potential application for human gene therapy. During the past 2 years, extensive progress and numerous breakthroughs have been made in this area of research. Genetically engineered retroviral vectors have been used successfully to infect live animals, effecting foreign gene expression in liver, blood vessels, and mammary tissues. Recombinant adenovirus and herpes simplex virus vectors have been utilized effectively for in vivo gene transfer into lung and brain tissues, respectively. Direct injection or particle bombardment of DNA has been demonstrated to provide a physical means for in situ gene transfer, while carrier-mediated DNA delivery techniques have been extended to target specific organs for gene expression. These technological developments in conjunction with the initiation of the NIH human gene therapy trials have marked a milestone in developing new medical treatments for various genetic diseases and cancer. Various in vivo gene transfer techniques should also provide new tools for basic research in molecular and developmental genetics.     

Gene transfer into eukaryotic cells using activated polyamidoamine dendrimers

The development of efficient methods to transfer genes into eukaryotic cells is important for molecular biotechnology. A number of different technologies to mediate gene transfer have been developed over the last 35 years, but most have drawbacks such as cytotoxicity, low efficiency and/or restricted applicability. Activated polyamidoamine (PAMAM)-dendrimers provide a new technology for gene transfer that offers significant advantages over classical methods. Reagents based on this technology provide high gene transfer efficiencies, minimal cytotoxicity, and can be used with a broad range of cell types. This technology could also be useful for in vivo gene transfer in gene therapy applications.     

Intergeneric transfer of ribosomal genes between two fungi

Background  

Horizontal gene transfer, also called lateral gene transfer, frequently occurs among prokaryotic organisms, and is considered an important force in their evolution. However, there are relatively few reports of transfer to or from fungi, with some notable exceptions in the acquisition of prokaryotic genes. Some fungal species have been found to contain sequences resembling those of bacterial genes, and with such sequences absent in other fungal species, this has been interpreted as horizontal gene transfer. Similarly, a few fungi have been found to contain genes absent in close relatives but present in more distantly related taxa, and horizontal gene transfer has been invoked as a parsimonious explanation. There is a paucity of direct experimental evidence demonstrating the occurrence of horizontal gene transfer in fungi.     

Efficient c-kit receptor-targeted gene transfer to primary human CD34-selected hematopoietic stem cells

We have previously reported effective gene transfer with a targeted molecular conjugate adenovirus vector through the c-kit receptor in hematopoietic progenitor cell lines. However, a c-kit-targeted recombinant retroviral vector failed to transduce cells, indicating the existence of significant differences for c-kit target gene transfer between these two viruses. Here we demonstrate that conjugation of an adenovirus to a c-kit-retargeted retrovirus vector enables retroviral transduction. This finding suggests the requirement of endosomalysis for successful c-kit-targeted gene transfer. Furthermore, we show efficient gene transfer to, and high transgene expression (66%) in, CD34-selected, c-kit(+) human peripheral blood stem cells using a c-kit-targeted adenovirus vector. These findings may have important implications for future vector development in c-kit-targeted stem cell gene transfer.     

The use of retroviral vectors for gene therapy-what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery

Retroviral vector-mediated gene transfer has been central to the development of gene therapy. Retroviruses have several distinct advantages over other vectors, especially when permanent gene transfer is the preferred outcome. The most important advantage that retroviral vectors offer is their ability to transform their single stranded RNA genome into a double stranded DNA molecule that stably integrates into the target cell genome. This means that retroviral vectors can be used to permanently modify the host cell nuclear genome. Recently, retroviral vector-mediated gene transfer, as well as the broader gene therapy field, has been re-invigorated with the development of a new class of retroviral vectors which are derived from lentiviruses. These have the unique ability amongst retroviruses of being able to infect non-cycling cells. Vectors derived from lentiviruses have provided a quantum leap in technology and seemingly offer the means to achieve significant levels of gene transfer in vivo.     

基因治疗心肌缺血的载体应用新进展

近年来,随着基因治疗技术的不断进步,为心肌缺血的治疗开辟了一条全新的途径,并取得了一些令人鼓舞的进展。基因治疗主要包括治疗基因、基因转移载体以及基因导入途径三个方面。基因转移载体又在治疗基因和基因表达之间起着桥梁作用,因此,发展安全、高效的基因转移系统是基因治疗的关键之一。目前用于基因治疗心肌缺血基因转移的载体主要有病毒载体和非病毒载体。下面将就不同载体在心肌缺血的基因治疗中的应用进展进行简要的总结。     

Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer

Retroviral vectors have become an important tool for gene transfer in vitro and in vivo. Classical Moloney murine leukemia virus (MLV) based retroviral vectors have been used for over 20 years to transfer genes into dividing cells. Cell lines for production of retroviral vectors have become commonly available and modifications in retroviral vector design and use of envelope proteins have made the production of high titer, helper-free, infectious virus stocks relatively easy. More recently, lentiviral vectors, another class of retroviruses, have been modified for in vitro and in vivo gene transfer. The ability of lentiviral vectors to transduce non-dividing cells has made them especially attractive for in vivo gene transfer into differentiated, non-dividing tissues. Several improvements in helper plasmids and vectors have made lentivirus a safe vector system for ex vivo and in vivo gene transfer. This review will briefly summarize the background of these vector systems and provide some common protocols available for the preparation of MLV based retroviral vectors and HIV-1 based lentiviral vectors.     

In vivo gene gun-mediated DNA delivery into rodent brain tissue

Various types of gene transfer into live tissues have been tried. However, in vivo gene transfer into brain tissue or neuronal cells without virus vector has required a great effort. Particle-mediated gene transfer into live brain tissue was thought to be impossible because of its fragility and the mechanical problem of a previous type of gene gun. In addition, particle-mediated DNA transfer into monolayer-cultured cells without mechanical damage has been difficult. We successfully transferred DNA into rodent live brain tissue and also into monolayer-cultured cells without mechanical damage by using a new type of gene gun and also confirmed gene expression in the brain. This new method represents another variation of gene transfer into the brain.     

基因治疗心肌缺血的载体应用新进展

近年来,随着基因治疗技术的不断进步,为心肌缺血的治疗开辟了一条全新的途径,并取得了一些令人鼓舞的进展。基因治疗主要包括治疗基因、基因转移载体以及基因导入途径三个方面。基因转移载体又在治疗基因和基因表达之间起着桥梁作用,因此,发展安全、高效的基因转移系统是基因治疗的关键之一。目前用于基因治疗心肌缺血基因转移的载体主要有病毒载体和非病毒载体。下面将就不同载体在心肌缺血的基因治疗中的应用进展进行简要的总结。     

The molecular evolution of catalatic hydroperoxidases: evidence for multiple lateral transfer of genes between prokaryota and from bacteria into eukaryota

The past decade has produced an increasing number of reports on horizontal gene transfer between prokaryotic organisms. Only recently, with the flood of available whole genome sequence data and a renewed intensity of the debate about the universal tree of life, a very few reports on lateral gene transfer (LGT) from prokaryotes into the Eukaryota have been published. We have investigated and report here on the molecular evolution of the gene families that encode catalatic hydroperoxidases. We have found that this process included not only frequent horizontal gene transfer among prokaryotes but also several lateral gene transfer events between bacteria and fungi and between bacteria and the protistan ancestor of the alga/plant lineage.     

Environmental Factors Influencing Gene Transfer Agent (GTA) Mediated Transduction in the Subtropical Ocean

Microbial genomic sequence analyses have indicated widespread horizontal gene transfer (HGT). However, an adequate mechanism accounting for the ubiquity of HGT has been lacking. Recently, high frequencies of interspecific gene transfer have been documented, catalyzed by Gene Transfer Agents (GTAs) of marine α-Proteobacteria. It has been proposed that the presence of bacterial genes in highly purified viral metagenomes may be due to GTAs. However, factors influencing GTA-mediated gene transfer in the environment have not yet been determined. Several genomically sequenced strains containing complete GTA sequences similar to Rhodobacter capsulatus (RcGTA, type strain) were screened to ascertain if they produced putative GTAs, and at what abundance. Five of nine marine strains screened to date spontaneously produced virus-like particles (VLP's) in stationary phase. Three of these strains have demonstrated gene transfer activity, two of which were documented by this lab. These two strains Roseovarius nubinhibens ISM and Nitratireductor 44B9s, were utilized to produce GTAs designated RnGTA and NrGTA and gene transfer activity was verified in culture. Cell-free preparations of purified RnGTA and NrGTA particles from marked donor strains were incubated with natural microbial assemblages to determine the level of GTA-mediated gene transfer. In conjunction, several ambient environmental parameters were measured including lysogeny indicated by prophage induction. GTA production in culture systems indicated that approximately half of the strains produced GTA-like particles and maximal GTA counts ranged from 10-30% of host abundance. Modeling of GTA-mediated gene transfer frequencies in natural samples, along with other measured environmental variables, indicated a strong relationship between GTA mediated gene transfer and the combined factors of salinity, multiplicity of infection (MOI) and ambient bacterial abundance. These results indicate that GTA-mediated HGT in the marine environment with the strains examined is favored during times of elevated bacterial and GTA abundance as well as in areas of higher salinity.     

Progress in gene transfer by germ cells in mammals

Use of germ cells as vectors for transgenesis in mammals has been well developed and offers exciting prospects for experimental and applied biology,agricultural and medical sciences.Such approach is referred to as either male germ cell mediated gene transfer(MGCMGT) or female germ cell mediated gene transfer(FGCMGT) technique.Sperm-mediated gene transfer(SMGT),including its alternative method,testis-mediated gene transfer(TMGT),becomes an established and reliable method for transgenesis.They have been exten...     

Progress in gene transfer by germ cells in mammals

Use of germ cells as vectors for transgenesis in mammals has been well developed and offers exciting prospects for experimental and applied biology, agricultural and medical sciences.Such approach is referred to as either male germ cell mediated gene transfer (MGCMGT)or female germ cell mediated gene transfer(FGCMGT)technique.Sperm-mediated gene transfer (SMGT),including its alternative method,testis-mediated gene transfer(TMGT),becomes an established and reliable method for transgenesis.They have been extensively used for producing transgenic animals.The newly developed approach of FGCMGT,ovary-mediated gene transfer(OMGT) is also a novel and useful tool for efficient transgenesis.This review highlights an overview of the recent progress in germ cell mediated gene transfer techniques,methods developed and mechanisms of nucleic acid uptake by germ cells.     

Evolution of mercuric reductase (merA) gene: a case of horizontal gene transfer

In the present study the role of horizontal gene transfer events in providing the mercury resistance is depicted. merA is key gene in mer operon and has been used for this study. Phylogenetic analysis of aligned merA sequences shows broad similarities to the established 16S rRNA phylogeny. But there is no separation of bacterial merA from archael merA which suggests that merA gene in both these groups share considerable sequence homology. However, inconsistencies between merA and 16S rRNA gene phylogenetic trees are apparent for some taxa. These discrepancies in the phylogenetic trees for merA gene and 16S rRNA gene have lead to the suggestion that horizontal gene transfer (HGT) is a major contributor for its evolution. The close association among members of different groups in merA gene tree, as supported by high bootstrap values, deviations in GC content and codon usage pattern indicate the possibility that horizontal gene transfer events might have taken place during the evolution of this gene.     

Techniques for gene transfer into neurons

To illuminate the function of the thousands of genes that make up the complexity of the nervous system, it is critical to be able to introduce and express DNA in neurons. Over the past two decades, many gene transfer methods have been developed, including viral vectors, liposomes and electroporation. Although the perfect gene transfer technique for every application has not yet been developed, recent technical advances have facilitated the ease of neuronal gene transfer and have increased the accessibility of these techniques to all laboratories. In order to select a transfection method for any particular experiment, the specific advantages and disadvantages of each technique must be considered.     

DNA-mediated gene transfer without carrier DNA

DNA-mediated gene transfer is a procedure which uses purified DNA to introduce new genetic elements into cells in culture. The standard DNA-mediated gene transfer procedure involves the use of whole cell DNA as carrier DNA for the transfer. We have modified the standard DNA-mediated gene transfer procedure to transfer the Herpes simplex virus type 1 thymidine kinase gene (TK) into TK- murine recipient cells in the absence of whole cell carrier DNA. The majority (8/10) of carrier-free transformant lines expressed the TK+ phenotype stably, in sharp contrast to our results with carrier-containing DNA-mediated gene transfer. There was a wide range in donor DNA content among independent transformants. Further analysis on one transformant line using DNA restriction digests and in situ hybridization provided evidence that, in the absence of whole cell carrier DNA, multiple donor DNA sequences became integrated at a single chromosomal site.     

Genetic engineering in marine cyanobacteria

Many species of microalgae producing useful materials have been isolated from marine environments. For their industrial application, widely applicable and stable gene expression is required. It is necessary to establish gene transfer methods as an essential first step in genetic manipulation. Although gene transfer techniques for cyanobacteria have been developed, only naturally transformable strains have been used. Here, we describe recent progress made in developing gene transfer methods for marine cyanobacteria. The following are covered: (1) transformation, (2) electroporation, (3) conjugation, (4) particle gun. A plasmid from the marine cyanobacterium, Synechococcus sp., whose copy number is dependent on salinity, was characterized. This plasmid is being used to develop a stable and controllable gene expression system.     

Effect of serum on in vitro electrically mediated gene delivery and expression in mammalian cells

In many cell systems, electric pulses can efficiently mediate gene transfer with a high level of expression in vitro. In vivo results have been reported where decrease in efficiency was obtained. The mechanisms involved in the process are unknown. Since, in vivo, the efficiency of non-viral methods of gene transfer is generally limited by the presence of serum, we report here the effect of serum on in vitro electrically mediated chinese hamster ovary cell membrane permeabilization, viability, gene transfer and expression. The results indicate that permeabilization and gene transfer are not inhibited by serum. By acting as a protector of cell viability, serum indeed increases gene transfer and expression.