Tools for large-scale genetic manipulation of yeast genome / 生物工程学报

Large-scale genetic manipulation of the genome refers to the genetic modification of large fragments of DNA using knockout, integration and translocation. Compared to small-scale gene editing, large-scale genetic manipulation of the genome allows for the simultaneous modification of more genetic information, which is important for understanding the complex mechanisms such as multigene interactions. At the same time, large-scale genetic manipulation of the genome allows for larger-scale design and reconstruction of the genome, and even the creation of entirely new genomes, with great potential in reconstructing complex functions. Yeast is an important eukaryotic model organism that is widely used because of its safety and easiness of manipulation. This paper systematically summarizes the toolkit for large-scale genetic manipulation of the yeast genome, including recombinase-mediated large-scale manipulation, nuclease-mediated large-scale manipulation, de novo synthesis of large DNA fragments and other large-scale manipulation tools, and introduces their basic working principles and typical application cases. Finally, the challenges and developments in large-scale genetic manipulation are presented.

MicroRNAs as potential targets for improving rice yield via plant architecture modulation: Recent studies and future perspectives

Ensuring agricultural food security is a major concern for the future world, and being the second mostconsumed crop, rice yield needs an urgent upliftment. Grain yield is a pleiotropic trait that employs a plethoraof genes functioning in complex signalling cascades. The yield related genes are controlled by variousregulatory factors including the microRNAs (miRNAs), the small 20–22 nucleotide (nt) non-coding RNAs,which have emerged as the master ribo-regulators of eukaryotic genes. Plant miRNAs can bind to highlycomplementary sequences in the target messenger RNAs (mRNAs) and negatively regulate gene expression tocoordinate the various biological processes involved in plant development. In rice, an ideal plant architecture(IPA) has been regarded as the key to attain high yield and several miRNAs have been deciphered to playimportant roles in orchestrating vital regulatory procedures for achieving optimum plant morphological yieldrelated traits like less unproductive tillers, more panicle branches and heavier grains. In this review, we presentand discuss the various genetic engineering strategies undertaken to manipulate the miRNA-mRNA expressionlevels in order to achieve improved grain output by modulation of rice plant architecture and recent advancesmade in this regard

Small Interfering RNA (siRNA) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR): Emerging Molecular Tools for Genetic Manipulation

@#Gene manipulation tools have transformed biomedical research and improved the possibilities of their uses for therapeutic purposes. These tools have aided effective genomic modification in many organisms and have been successfully applied in biomedical engineering, biotechnology and biomedicine. They also shown a potential for therapeutic applications to alleviate genetic and non-genetic diseases. Small interfering RNA (siRNA) and clustered regularly inter-spaced short-palindromic repeat/associated-protein system (CRISPR/Cas) are two of the tools applied in genetic manipulation. This review aims to evaluate the molecular influence of siRNA and CRISPR/Cas as novel tools for genetic manipulations. This review discusses the molecular mechanism of siRNA and CRISPR/Cas, and the advantages and disadvantages of siRNA and CRISPR/Cas. This review also presents comparison between siRNA and CRISPR/Cas as potential tools for gene therapy. siRNA therapeutic applications occur through protein knockout without causing damage to cells. siRNA knocks down gene expression at the mRNA level, whereas CRISPR/Cas knocks out gene permanently at the DNA level. Inconclusion, gene manipulation tools have potential for applications that improve therapeutic strategies and plant-derived products, but ethical standards must be established before the clinical application of gene editing.

Productive infection and transduction by bacteriophage P1 in the species Salmonella bongori

Background: Horizontal gene transfer (HGT) is the most important mechanism in the evolution of new genetic capabilities in bacteria, including specific degradative pathways, virulence factors, and resistance to antibiotics. Among the processes involved in HGT, transduction is noteworthy. This is a mechanism for gene transmission mediated by a bacteriophage that functions both as a reservoir and as a vector of exogenous genes, which remain protected from environmental effects in the bacteriophage's capsid. Within this context, this investigation aimed to evaluate the ability of the generalized transducing bacteriophage P1 to productively infect and transduce in the bacterial species Salmonella bongori. Results: We could establish that a derivative of bacteriophage P1, P1Cm, infects strains of S. bongori with frequencies of lysogenization in the order of ~10−2 lysogens/UFP. Through thermal induction, infective viral progeny was obtained, and we could show that P1Cm readily formed plaques on S. bongori lawns, a phenomenon thus far not reported for other members of the genus Salmonella. Finally, we showed P1Cm-mediated transduction of the model plasmid RP4 at frequencies of ~10−7 transductants/donor. Conclusion: Therefore, bacteriophage P1 can be used as a tool for the genetic manipulation in the species S. bongori.

Methods for the genetic manipulation of marine bacteria

Genetic manipulation of bacteria is a procedure necessary to obtain new strains that express peculiar and defined genetic determinants or to introduce genetic variants responsible for phenotypic modifications. This procedure can be applied to explore the biotechnological potential associated with environmental bacteria and to utilize the functional properties of specific genes when inserted into an appropriate host. In the past years, marine bacteria have received increasing attention because they represent a fascinating reservoir of genetic and functional diversity that can be utilized to fuel the bioeconomy sector. However, there is an urgent need for an in-depth investigation and improvement of the genetic manipulation tools applicable to marine strains because of the paucity of knowledge regarding this. This review aims to describe the genetic manipulation methods hitherto used in marine bacteria, thus highlighting the limiting factors of the different techniques available today to increase manipulation efficiency. In particular, we focus on methods of natural and artificial transformations (especially electroporation) and conjugation because they have been successfully applied to several marine strains. Finally, we emphasize that, to avoid failure, future work should be carried out to establish tailored methodologies for marine bacteria.

RecET recombination system driving chromosomal target gene replacement in Zymomonas mobilis

Background: Zymomonas mobilis is a Gram-negative microaerophilic bacterium with excellent ethanol-producing capabilities. The RecET recombination system provides an efficient tool for direct targeting of genes in the bacterial chromosome by PCR fragments. Results: The plasmids pSUZM2a-RecET and pSUZM2a-RecE588T were first developed to co-express RecE or RecE588 and RecT for homologous recombination. Thereafter, the PCR fragments of the tetracycline resistance marker gene flanked by 60 bp of adhA (alcohol dehydrogenase I) or adhB (alcohol dehydrogenase II) homologous sequences were electroporated directly into ZM4 cells harboring pSUZM2a-RecET or pSUZM2a-RecE588T. Both adhA and adhB were replaced by the tetracycline resistance gene in ZM4, yielding two mutant strains, Z. mobilis ZM4 ΔadhA and Z. mobilis ZM4 ΔadhB. These two mutants showed varying extent of reduction in ethanol production, biomass generation, and glucose metabolism. Furthermore, enzyme activity of alcohol dehydrogenase II in Z. mobilis ZM4 ΔadhB exhibited a significant reduction compared to that of wild-type ZM4. Conclusion: This approach provided a simple and useful method for introducing mutations and heterologous genes in the Z. mobilis genome.

Molecular mechanism of artemisinin biosynthesis and regulation in Artemisia annua / 中国中药杂志

Artemisinin-based combination therapy (ACT) is the best available treatment, particularly for Plasmodium falciparum malaria. Artemisinin, whose main source is Artemisia annua, has large demand and shortsupply every year.Artemisininis synthesized,stored, and secreted by the glandular secretory trichomes of A. annua(AaGSTs).In general, the population and morphology of AaGSTs are often positively correlated with artemisinin content.This review article introduces the molecular mechanism of biosynthesis and regulation of artemisininin A. annua. Furthermore, this article will refresh the classification of trichomes in A. annua and provide anoverview of the recent achievements regarding AaGSTs and artemisinin.These will shed light on exploring the method for increasing plant-derived artemisinin.

Genetic manipulation in zebrafish / 生物工程学报

The increasing number of genetic manipulation approaches and high-resolution live imaging technique applied in zebrafish have propelled the rise of this organism as a mainstream model for developmental biology and human diseases studies. Zebrafish has many advantages for functional genomics analysis, allowing for easy, cheap and fast functional characterization of novel genes in the vertebrate genome. Here we provide an overview of the principles of genetic manipulation in zebrafish, such as Ethylnitrosourea (ENU) mutagenesis, insertional mutagenesis, gene trapping mutagenesis, Morpholino mediated gene knockdown, targeting induced local lesions in genomes (TILLING), genome editing with engineered nucleases ZFN (Zinc finger nuclease), TALEN (Transcription activator-like effector nuclease) and CRISPR/Cas9 system, and transgenic methods used in zebrafish.

New research progress on the epidemiology of age - related macular degeneration / 国际眼科杂志(Guoji Yanke Zazhi)

Age-related macular degeneration ( AMD ) is a kind of age-related blinding degenerative fundus lesions, totally about 30 million patients suffering from AMD all over the world, with about 500 000 people blind for it yearly. As the development of economy and the aging of the population intensified, incidence of AMD indicates a trend of rising year by year, being the third major cause of blindness in our country. At present, the pathogenesis of AMD is not fully clear, as reported it may be related to oxidative stress, inflammatory immune response, VEGF and genetic manipulation. Clinical treatments mainly include photodynamic therapy, drug therapy, radiation therapy, laser photocoagulaory operation, the pupil warm treatments, Chinese medicine and intravitreous injection VEGF antagonists such as Ranibizumab, Conbercept and so on. ln this issue, we mainly expound on the progress in the epidemiological studies of AMD, especially elaborate the progress made on genetic manipulation in recent years.

Ficção científica e o Admirável mundo novo: previsões concretizadas no atual século e considerações bioéticas / Science fiction and the Brave New World: predictions fulfilled in our century and bioethical considerations

A celeridade com que a ciência gera resultados na sociedade moderna torna necessária uma reflexão sobre os limites da aplicação do progresso científico. Essa é a discussão de base de Admirável mundo novo, livro de Aldous Huxley publicado em 1932, que retrata uma futura sociedade tecnológica nos moldes do fordismo. Este artigo estabelece uma relação entre a sociedade tecnocrata atual e a sociedade descrita por Huxley, discutindo a viabilidade dos aspectos técnicos e biológicos das manipulações narradas à luz dos conhecimentos atuais. São também elaboradas algumas considerações bioéticas sobre os procedimentos 'inventados' pelo autor e que já são, ou poderiam ser, desenvolvidos na sociedade moderna.

The speed with which science generates results in modern society requires reflection on the limits of scientific progress. This is the foundation of Brave New World, a book published by Aldous Huxley in 1932 that portrays a future technological society along the lines of Fordism. This article establishes a relationship between our current technocratic society and that described by Huxley, discussing the viability of the technical and biological aspects of the manipulations narrated in the book in light of current knowledge. Some bioethical considerations with respect to the procedures 'invented' by the author - and which are already or could be developed in modern society - will also be addressed.

The use of genes for performance enhancement: doping or therapy?

Recent biotechnological advances have permitted the manipulation of genetic sequences to treat several diseases in a process called gene therapy. However, the advance of gene therapy has opened the door to the possibility of using genetic manipulation (GM) to enhance athletic performance. In such ‘gene doping’, exogenous genetic sequences are inserted into a specific tissue, altering cellular gene activity or leading to the expression of a protein product. The exogenous genes most likely to be utilized for gene doping include erythropoietin (EPO), vascular endothelial growth factor (VEGF), insulin-like growth factor type 1 (IGF-1), myostatin antagonists, and endorphin. However, many other genes could also be used, such as those involved in glucose metabolic pathways. Because gene doping would be very difficult to detect, it is inherently very attractive for those involved in sports who are prepared to cheat. Moreover, the field of gene therapy is constantly and rapidly progressing, and this is likely to generate many new possibilities for gene doping. Thus, as part of the general fight against all forms of doping, it will be necessary to develop and continually improve means of detecting exogenous gene sequences (or their products) in athletes. Nevertheless, some bioethicists have argued for a liberal approach to gene doping.

Terapia génicay principios éticos / Gen therapy and ethical principles / A terapia gênica e os princípios éticos

La terapia génica constituye una forma de manipulación genética que trata de corregir o disminuir los efectos que ocasionan enfermedades de origen genético. Existen grandes esperanzas en el desarrollo de este tipo de terapia, sobre todo para enfermedades somáticas de origen genético que no tienen curación. Pero hay también numerosas dificultades técnicas que no están del todo resueltas. No se ha demostrado todavía eficacia clínica y se pueden producir daños irreversibles en el organismo. Por una parte, existe la necesidad de regular por medio de protocolos que deben ser rigurosamente analizados por comités de evaluación ética y científica y, por otra, hace falta un diálogo entre países, con el fin de evitar una presentación exitista de la técnica en el mercado sin mencionar sus dificultades inherentes. Una de las preocupaciones mayores es que la aceptación paulatina de este tipo de terapia, y la eliminación de restricciones, permita el uso de la técnica para terapia génica germinal y para terapia génica de mejoría, cuya validez ética es cuestionada. Usar la terapia génica en células germinales conlleva el riesgo de introducir daños genéticos en generaciones posteriores.

Gene therapy is a form of genetic manipulation which tries to correct or to diminish the effects caused by genetic diseases. There are great hopes for the development of this type of therapy, specially for somatic genetic diseases that has no cure, but there are many technical difficulties which are not yet solved. It has not yet been shown clinical efficacy and the technique may cause irreversible damages in the organism. On one hand, it is considered necessary to establish regulatory mechanisms to avoid possible abuse and the research protocols must be strictly analyzed by ethical and scientific review committees. On the other hand, at the international level, it is necessary a dialogue between nations in order not to present gene therapy in the market as a successful technique, without mentioning all the technical difficulties. One of the major concerns consists in that the gradual acceptance of this technique and the removal of restrictions may lead to the use of germ gene therapy and genetic enhancement, whose ethical validity is questionable. The use of germ gene therapy carries the risk of introducing genetic damages in posterior generations.

A terapia gênica constitui uma forma de manipulação genética que trata de corrigir ou diminuir os efeitos que ocasionam enfermidades de origem genética. Existem grandes esperanças no desenvolvimento deste tipo de terapia, sobretudo para doenças somáticas de origem genética que não tem cura. Existem, não obstante isso numerosos dificuldades técnicas que não estão totalmente resolvidas. Não se demostrou ainda a eficácia clínica e se pode causar danos irreversíveis no organismo. De um lado, existe a necessidade de regular por meio de protocolos que devem ser rigorosamente analisados por comitês de avaliação ética e científica, por outra, faz falta um diálogo entre países, para se evitar uma apresentação unilateral exitosa da técnica da técnica no mercado sem mencionar suas dificuldades inerentes. Uma das preocupações maiores é que a aceitação paulatina deste tipo de terapia, e a eliminação de restrições, permite o uso da técnica para terapia gênica germinal e para terapia gênica de melhora, cuja validade ética é questionada. Usar a terapia gênica em células germinais traz o risco de introduzir danos genéticos em gerações posteriores.