Lentivirus Mediated Gene Manipulation in Trophectoderm of Porcine Embryos

Development of tools that can manipulate gene expression specifically and efficiently in the trophectoderm（TE） lineage would greatly aid understanding the roles of different genetic pathways in TE versus embryonic lineages. Here, we showed first time that short-term lentivirus infection of porcine blastocysts could lead to rapid expression of transgene specifically in TE cells. Efficient TE-specific gene knockdown could also be achieved by lentivirus-mediated pol III-driven short hairpin RNA（shRNA） and TE-specific gene expression could be temporal controlled efficiently by combining this system with Tet-On system. This lentivirus lineage-specific infection system would facilitate gene function studies in porcine pre-implatation embryos by specifically knockdown or overexpression of these genes in TE.

The road toward Cd-safe rice:From mass selection to marker-assisted selection and genetic manipulation

Rice is an important dietary source of the toxic mineral cadmium(Cd) for populations in which rice is the main staple food.When grown in agricultural soils that are contaminated with Cd,rice often accumulates excessive Cd into the grains,which is a serious threat to agricultural sustainability and human health.To limit Cd accumulation in rice grains,studies on the genetic basis of Cd accumulation in rice have been carried out extensively,and some low-Cd rice varieties have also been developed in recent years.However,the challenges in low-Cd rice breeding still exist because the outcomes of the current genetic improvements for low-Cd rice cannot fully meet the requirements for the development of Cd-safe rice at present.In this review,we outline the progress in understanding the physiological mechanisms and the genetic nature of Cd accumulation in rice and summarize the strategies and outcomes of low-Cd rice breeding over the past decade.By graphing the physiological mechanism of Cd transport in the rice plant,three key steps and some underlying genes are summarized and discussed.Also,two genetic features of the natural variation in rice grain-Cd accumulation,the phenotypic plasticity and subspecies divergence,and the potential genetic explanations for these features are also discussed.Finally,we summarize and discuss current progress and the potential issues in low-Cd rice breeding using different breeding strategies.We hope to propose strategies for future success in the breeding of low-Cd rice varieties over the next decade.

Employing pH-responsive RNA triplex to control CRISPR/Cas9-mediated gene manipulation in mammalian cells

The clustered regularly interspersed short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)system is an RNA-guided platform for highly efficient and specific genome targeting in diverse organisms,which has been exploited for various applications in gene manipulation.Compared with the constantly active CRISPR/Cas9 function,conditional control of its activity can improve the performance of the system with reduced side effects and high spatiotemporal precision.The pH-responsive triplex RNA was successful used in CRISPR-derived RNA/trans-activating crRNA(crRNA/tracrRNA)of CRISPR/Cas9,thus affecting RNA/dead Cas9(dCas9)complex to target DNA in vitro and in vivo.This design of triplex RNA opens a new window towards the broad involvement of eukaryotic cells for conditional control of CRISPR/Cas9function.?2024 Published by Elsevier B.V.on behalf of Chinese Chemical Society and Institute of Materia Medica,Chinese Academy of Medical Sciences.

Immunological treatment of liver tumors

Although multiple options for the treatment of liver tumors have often been described in the past, including liver resection, radiofrequency ablation with or without hepatic pump insertion, laparoscopic liver resection and the use of chemotherapy, the potential of immunotherapy and gene manipulation is still largely unexplored.Immunological therapy by gene manipulation is based on the interaction between virus-based gene delivery systems and dendritic cells. Using viruses as vectors, it is possible to transduce dendritic cells with genes encoding tumor-associated antigens, thus inducing strong humoral and cellular immunity against the antigens themselves.Both chemotherapy and radiation therapy have the disadvantage of destroying healthy cells, thus causing severe side-effects. We need more precisely targeted therapies capable of killing cancer cells while sparing healthy cells. Our goal is to establish a new treatment for solid liver tumors based on the concept of cytoreduction,and propose an innovative algorithm.

Switching On/Off of Guide RNA by Photoinduced Strand Displacement for Functional Control of CRISPR/Cas9

Functional control of CRISPR/Cas9 is essential for precise gene manipulation.Chemical engineering of guide RNA(gRNA)provides diverse approaches for conditional control of CRISPR/Cas9 function with a variety of chemical reactive groups.However,previous investigations into chemically engineering gRNA only unidirectionally regulated the CRISPR/Cas9 function via stimuli-induced caging/decaging processes.Herein,we propose a combinatory strategy to engineer the dynamics of gRNA in which photocontrolled strand-displacement reactions coupled with sequence designs of gRNA can achieve lightinduced switching-on/off control of CRISPR/Cas9 function.Biochemical analysis and cellular gene regulation indicate this approach is capable of both activating and deactivating CRISPR/Cas9 activities using light irradiation.Moreover,photocontrolled multiplex modulations of gene expression for opposite regulatory effects have also been achieved simultaneously under the same cellular context.This work establishes an essential principle for construction of stimuli-induced switching-on/off modulations of gRNA that can greatly enrich the versatility of conditional control for a variety of CRISPR/Cas9-based applications.

A Review on Genetically Modified Plants Designed to Phytoremediate Polluted Soils： Biochemical Responses and International Regulation

In recent years,there has been an increasing interest in finding sustainable strategies for the efficient removal of contaminants from soils.The objective of this review is to examine the biochemical principles of specific genetic modifications in plants,their applications in the field for specific contaminants as phytotechnologies,and their international regulation.In addition,the review presents some biological aspects of rhizosphere-related phenomena,the interactions of organic and inorganic pollutants with plants,and the performance of the phytotechnologies across the continents.During the last few decades,at least eight genera of genetically modified plants(GMPs)have been tested and used for soil remediation with outstanding results.Arabidopsis,Nicotiana,and Oryza are the plant genera most widely studied.Specific plant genes such as metal transporters,chelators,metallothioneins,phytochelatins,and oxygenases have been transferred to plants to improve the elimination of contaminants in soil.We discuss some important aspects of gene manipulation and its application for removal of diverse contaminants.A key challenge faced by phytotechnologies is the final disposal of the generated biomass,from a safety aspect.We argue that the commercial success of phytotechnologies depends on the generation of valuable biomass on contaminated land and its use for bioenergy generation.The use of such technologies would promote a broader understanding of the importance of plants,especially GMPs,in the environment and their contribution to environmental sustainability.

Using a novel approach -- recombineering- to generate odf2 null alleles

This article uses a real-life example to illustrate the concept and methodology of recombineering, arevolutionary genetic engineering technique based on phage-mediated homologous recombination. A step-by-step approach is presented along with a flow diagram, from obtaining gene-harboring BACs to the in vitro generation of a conditional null allele. This method can be used to target any gene at any position, without the knowledge or use of any restriction site. The extensive applicability of recombineering to gene manipulation is discussed.

Molecular mechanisms of plant adaptation to acid soils: A review

Acid soils are widespread and limit global plant production.Aluminum(Al)/manganese(Mn)toxicity and phosphorus(P)deficiency are the major limiting factors affecting plant growth and productivity on acid soils.Plants,however,have evolved various strategies to adapt to these stresses.These strategies include using both external and internal mechanisms to adapt to Al toxicity,regulating Mn uptake,translocation,and distribution to avoid Mn toxicity,and orchestrating a set of P transport mechanisms to efficiently take up P from the soil.Here,we review the current state of knowledge about the molecular mechanisms of plant adaptation to these constraints in acid soils,focusing on the roles of transporters involved in Al/Mn tolerance and P efficiency.Gene manipulation combined with other biotechnology will contribute to the development of novel strategies to improve plant adaptation to acid soils.The molecular mechanisms of plant coadaptation to multiple stresses in acid soils are largely unknown and require further investigation.