Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 576
Filter
1.
Chinese Journal of Biotechnology ; (12): 2465-2484, 2023.
Article in Chinese | WPRIM | ID: wpr-981212

ABSTRACT

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.


Subject(s)
DNA , Gene Editing , Genetic Engineering , Saccharomyces cerevisiae/genetics , Translocation, Genetic
2.
Article in Chinese | WPRIM | ID: wpr-970355

ABSTRACT

Viruses are powerful tools for the study of modern neurosciences. Most of the research on the connection and function of neurons were done by using recombinant viruses, among which neurotropic herpesvirus is one of the most important tools. With the continuous development of genetic engineering and molecular biology techniques, several recombinant neurophilic herpesviruses have been engineered into different viral tools for neuroscience research. This review describes and discusses several common and widely used neurophilic herpesviruses as nerve conduction tracers, viral vectors for neurological diseases, and lytic viruses for neuro-oncology applications, which provides a reference for further exploring the function of neurophilic herpesviruses.


Subject(s)
Herpesviridae/genetics , Neurosciences , Genetic Vectors/genetics , Genetic Engineering , Neurons
3.
Chinese Journal of Biotechnology ; (12): 3125-3142, 2023.
Article in Chinese | WPRIM | ID: wpr-1007949

ABSTRACT

C1 gases including CO, CO2 and CH4, are mainly derived from terrestrial biological activities, industrial waste gas and gasification syngas. Particularly, CO2 and CH4 are two of the most important greenhouse gases contributing to climate change. Bioconversion of C1 gases is not only a promising solution to addressing the problem of waste gases emission, but also a novel route to produce fuels or chemicals. In the past few years, C1-gas-utilizing microorganisms have drawn much attention and a variety of gene-editing technologies have been applied to improve their product yields or to expand product portfolios. This article reviewed the biological characteristics, aerobic or anaerobic metabolic pathways as well as the metabolic products of methanotrophs, autotrophic acetogens, and carboxydotrophic bacteria. In addition, gene-editing technologies (e.g. gene interruption technology using homologous recombination, group Ⅱ intron ClosTron technology, CRISPR/Cas gene editing and phage recombinase-mediated efficient integration of large DNA fragments) and their application in these C1-gas-utilizing microorganisms were also summarized.


Subject(s)
Gene Editing , Gases , Carbon Dioxide , Genetic Engineering , Cloning, Molecular
4.
Acta bioeth ; 28(1): 149-156, jun. 2022.
Article in English | LILACS | ID: biblio-1383281

ABSTRACT

Abstract: The invention and widely use of organ allotransplantation provides effective treatment of some originally fetal diseases such as liver/kidney failure and has saved million of lives around the globe. However, the scarcity of human organs has caused many patients, who could have been treated, to die while waiting for suitable organs around the world. Pig-to human xenotransplantation provides a potential solution to solve this tough problem. Pig organs have been considered as major sources of xenotransplantation because of the sufficient number of donors, the sizes of organs, and physiologically structural similarities. However, xenotransplantation also has some problems, such as the possibility of spreading animal diseases to human, the interspecies immunological barrier, organs of animal origin challenging human nature, and potential informed consent issues. This article will discuss these potential issues and to see whether it is the suitable time to conduct clinical xenotransplantation trials in humans.


Resumen: La invención y el amplio uso de trasplantes alógenos proporciona tratamiento efectivo de algunas enfermedades de origen fetal, como la insuficiencia renal y hepática, y ha salvado a millones de pacientes en el mundo. Sin embargo, la escasez de órganos humanos ha causado que muchos pacientes en el mundo, que podrían haber sido tratados, murieran por esperar un órgano adecuado. El xenotrasplante del cerdo al humano proporciona una solución potencial para resolver este difícil problema. Los órganos de cerdo han sido considerados como fuentes mayores para xenotrasplantes debido al suficiente número de donantes, el tamaño de los órganos y estructuras fisiológicas similares. No obstante, el xenotrasplante también tiene algunos problemas, como la posibilidad de expandir enfermedades animales a humanos, la barrera inmunológica entre especies, el desafío para la naturaleza humana de tener órganos de origen animal y problemas potenciales de consentimiento informado. Este artículo discute estos temas potenciales y plantea si estamos en un momento apropiado para realizar ensayos clínicos de xenotrasplantes en humanos.


Resumo: A invenção e amplo uso de alotransplante de órgãos propicia tratamento efetivo para algumas doenças originalmente fetais tais como falência hepática/renal e tem salvo milhões de vidas em todo o globo. Entretanto, a escassez de órgãos humanos tem causado a morte de muitos pacientes que poderiam ter sido tratados - aguardando por órgãos apropriados em todo o globo. Xenotransplante porco-para-humanos propicia uma solução potencial para resolver este difícil problema. Órgãos de porco tem sido considerados como as principais fontes de xenotransplante por causa do número suficiente de doadores, do tamanho dos órgãos e de similaridades estruturais fisiológicas. Entretanto, xenotransplante também tem alguns problemas, tais como a possibilidade de disseminar doenças animais aos humanos, a barreira imunológica entre espécies, órgão de origem animal desafiando a natureza humana e aspectos potenciais de consentimento informado. Esse artigo discutirá esses aspectos potenciais e verificará se é o momento adequado para conduzir ensaios clínicos de xenotransplante em humanos.


Subject(s)
Humans , Animals , Swine , Transplantation, Heterologous/ethics , Clinical Trials as Topic , Transplantation, Heterologous/adverse effects , Transplantation, Heterologous/psychology , Zoonoses/etiology , Genetic Engineering , Informed Consent
5.
Protein & Cell ; (12): 476-489, 2022.
Article in English | WPRIM | ID: wpr-939867

ABSTRACT

Cell therapy approaches that employ engineered mammalian cells for on-demand production of therapeutic agents in the patient's body are moving beyond proof-of-concept in translational medicine. The therapeutic cells can be customized to sense user-defined signals, process them, and respond in a programmable and predictable way. In this paper, we introduce the available tools and strategies employed to design therapeutic cells. Then, various approaches to control cell behaviors, including open-loop and closed-loop systems, are discussed. We also highlight therapeutic applications of engineered cells for early diagnosis and treatment of various diseases in the clinic and in experimental disease models. Finally, we consider emerging technologies such as digital devices and their potential for incorporation into future cell-based therapies.


Subject(s)
Animals , Humans , Cell Engineering , Gene Regulatory Networks , Genetic Engineering , Mammals/genetics , Synthetic Biology
6.
Chinese Journal of Biotechnology ; (12): 1619-1630, 2022.
Article in Chinese | WPRIM | ID: wpr-927806

ABSTRACT

Synthetic Biology is one of the most promising fields of modern Biology and a frontier interdisciplinary subject in the 21st century. With the rapid development of synthetic biology, the International Genetically Engineered Machine (iGEM) competition has emerged. The iGEM competition, based on the subject foundation of Synthetic Biology, intends to solve the biological problems in our daily life by applying modern biological technology. In recent years, with the continuous increase of participating teams, the iGEM competition has received extensive attention and achieved great progress. On the basis of the development of Synthetic Biology, we analyzed the 2018-2020 award-winning projects of the iGEM competition and illustrated the role and significance of the iGEM competition in cultivating college students' innovative thinking and ability with the participation experience of the iGEM team of Southwest Jiaotong University as an example.


Subject(s)
Humans , Genetic Engineering , Students , Synthetic Biology , Universities
7.
Chinese Journal of Biotechnology ; (12): 4816-4826, 2022.
Article in Chinese | WPRIM | ID: wpr-970352

ABSTRACT

The international genetically engineered machine (iGEM) competition is a global top college academic competition in synthetic biology. The iGEM competition has exhibited extensive international influence and attracted teams from more than 40 countries and regions around the world to participate in. The annual iGEM outputs have attracted the attention of top academic journals or international media such as Science, Nature, Scientific American, The Economist, British Broadcasting Corporation (BBC), etc. High school teams participated in iGEM since 2011, and the number of high school teams has increased year by year. High school participants are increasingly becoming one of the most important forces to promote the development of iGEM and synthetic biology. IGEM competition has also become an important platform to foster the core literacy of high school students. This paper summarized the track rules, topic selection tendency and awards of high school teams based on data of 2017 to 2021 iGEM competition. In addition, we analyzed the significance of iGEM competition on fostering of high school students' core literacy and discussed the development trend of global high school teams, with the aim to provide a reference for high school team building in the future.


Subject(s)
Humans , Genetic Engineering , Students , Universities , Synthetic Biology
8.
Bol. malariol. salud ambient ; 62(4): 663-669, 2022.
Article in Spanish | LILACS, LIVECS | ID: biblio-1411936

ABSTRACT

La ineficacia de las estrategias actuales para el control químico de los mosquitos vectores plantea la necesidad de desarrollar enfoques novedosos, entre estos están las estrategias genéticas para reducir las poblaciones de mosquitos vectores o sustituirlos por aquellos que no son capaces de transmitir patógenos, esto se logra a través de herramientas moleculares que permiten la manipulación y transgénesis de genes. Las secuencias del genoma de los mosquitos y las bases de datos de marcadores de secuencias expresadas asociadas permiten investigaciones a gran escala para proporcionar nuevos conocimientos sobre las vías evolutivas, bioquímicas, genéticas, metabólicas y fisiológicas. Además, la genómica comparativa revela las bases de los mecanismos evolutivos con especial atención a las interacciones específicas entre vectores y patógenos. Se ha desarrollado tecnología de transgénesis para el mosquito de la fiebre amarilla y dengue, Aedes aegypti. Se ha logrado integración exitosa de ADN exógeno en la línea germinal de este mosquito con los elementos transponibles. La disponibilidad de múltiples elementos y genes marcadores proporciona un poderoso conjunto de herramientas para investigar las propiedades biológicas básicas de este insecto vector, así como los materiales para desarrollar nuevas estrategias de control genético de poblaciones de mosquitos basadas en la técnica del insecto estéril. Una de estas estrategias consiste en liberar a la población machos esterilizados por radiación; otro, de integrar un gen letal dominante bajo el control de un promotor específico en hembras inmaduras. El uso de esta técnica de modificación genética constituirá una herramienta importante para el manejo integrado de vectores(AU)


The ineffectiveness of current strategies for the chemical control of vector mosquitoes raises the need to develop novel approaches, among these are genetic strategies to reduce populations of vector mosquitoes or replace them with those that are not capable of transmitting pathogens, this is achieved through molecular tools that allow the manipulation and transgenesis of genes. Mosquito genome sequences and associated expressed sequence marker databases enable large-scale investigations to provide new insights into evolutionary, biochemical, genetic, metabolic, and physiological pathways. Furthermore, comparative genomics reveals the basis of evolutionary mechanisms with special attention to the specific interactions between vectors and pathogens. Transgenesis technology has been developed for the yellow fever and dengue mosquito, Aedes aegypti. Successful integration of exogenous DNA into the germ line of this mosquito with the transposable elements has been achieved. The availability of multiple elements and marker genes provides a powerful set of tools to investigate the basic biological properties of this insect vector, as well as the materials to develop new strategies for genetic control of mosquito populations based on the sterile insect technique. One of this strategy is to release radiation-sterilized males into the population; another, to integrate a dominant lethal gene under the control of a specific promoter in immature females. The use of this genetic modification technique will constitute an important tool for the integrated management of vectors(AU)


Subject(s)
Animals , Arboviruses , Genetic Engineering , Gene Transfer Techniques , Aedes , Arbovirus Infections , Health Strategies , Mosquito Vectors , Genetics
9.
Chinese Journal of Biotechnology ; (12): 2116-2126, 2021.
Article in Chinese | WPRIM | ID: wpr-887785

ABSTRACT

Carrimycin (CAM) is a new antibiotics with isovalerylspiramycins (ISP) as its major components. It is produced by Streptomyces spiramyceticus integrated with a heterogenous 4″-O-isovaleryltransferase gene (ist). However, the present CAM producing strain carries two resistant gene markers, which makes it difficult for further genetic manipulation. In addition, isovalerylation of spiramycin (SP) could be of low efficiency as the ist gene is located far from the SP biosynthesis gene cluster. In this study, ist and its positive regulatory gene acyB2 were inserted into the downstream of orf54 gene neighboring to SP biosynthetic gene cluster in Streptomyces spiramyceticus 1941 by using the CRISPR-Cas9 technique. Two new markerless CAM producing strains, 54IA-1 and 54IA-2, were obtained from the homologous recombination and plasmid drop-out. Interestingly, the yield of ISP in strain 54IA-2 was much higher than that in strain 54IA-1. Quantitative real-time PCR assay showed that the ist, acyB2 and some genes associated with SP biosynthesis exhibited higher expression levels in strain 54IA-2. Subsequently, strain 54IA-2 was subjected to rifampicin (RFP) resistance selection for obtaining high-yield CAM mutants by ribosome engineering. The yield of ISP in mutants resistant to 40 μg/mL RFP increased significantly, with the highest up to 842.9 μg/mL, which was about 6 times higher than that of strain 54IA-2. Analysis of the sequences of the rpoB gene of these 7 mutants revealed that the serine at position 576 was mutated to alanine existed in each sequenced mutant. Among the mutants carrying other missense mutations, strain RFP40-6-8 which carries a mutation of glutamine (424) to leucine showed the highest yield of ISP. In conclusion, two markerless novel CAM producing strains, 54IA-1 and 54IA-2, were successfully developed by using CRISPR-Cas9 technique. Furthermore, a novel CAM high-yielding strain RFP40-6-8 was obtained through ribosome engineering. This study thus demonstrated a useful combinatory approach for improving the production of CAM.


Subject(s)
CRISPR-Cas Systems/genetics , Genetic Engineering , Ribosomes , Spiramycin , Streptomyces/genetics
10.
Chinese Journal of Biotechnology ; (12): 2956-2966, 2021.
Article in Chinese | WPRIM | ID: wpr-887857

ABSTRACT

Teaching in experiments of biology is important for the cultivation of life science talents. In view of the rapid development of life science and the increasing demand for research-oriented talent training, teaching in experiments of biology should set up a variety of learning outcomes: to train experimental skill, to cultivate students' experimental design and operation abilities, and to improve students' scientific thinking and innovative consciousness. We have carried out an educational reform on experimental genetic engineering blended course. In this paper, we introduced our methods of organizing online materials, the curriculum design of the blended course, the implementation details, and a preliminary analysis of teaching effects. We found that experimental genetic engineering blended course could support students' active learning and a learning-centered teaching model. Moreover, it could facilitate students' achievement of improving experimental skills, cultivating a rigorous scientific attitude, professional research quality and academic innovation ability.


Subject(s)
Humans , Biological Science Disciplines , Curriculum , Genetic Engineering , Students
11.
Chinese Journal of Biotechnology ; (12): 1457-1463, 2021.
Article in Chinese | WPRIM | ID: wpr-878648

ABSTRACT

Starting from participating the high-level professional competition, our school has built a talent training system with the spirit of "biomaker" and an innovative practical ability training system. Such system takes the interest of student as the starting point, and relies on the strong scientific research and teaching infrastructure. The programme gives full play to students' initiatives and enhances the scientific research literacy and comprehensive ability of undergraduates majoring in biotechnology. It is an effective exploration of the traditional university education model and meets the urgent demand for innovative talents training in the era of rapid development of life sciences.


Subject(s)
Humans , Biological Science Disciplines , Biotechnology , Genetic Engineering , Students , Universities
12.
Gac. méd. Méx ; 156(1): 53-59, ene.-feb. 2020.
Article in Spanish | LILACS | ID: biblio-1249870

ABSTRACT

Resumen En este ensayo se analizan las implicaciones bioéticas de la reciente manipulación genética en embriones humanos con CRISPR-Cas9 para eliminar el gen CCR5 y el nacimiento de dos gemelas discordantes. El experimento se divulgó en medios sociales. Los principales problemas bioéticos identificados son la justificación del modelo, el proceso de consentimiento informado y la falta de declaración de evidentes conflictos de interés. No se evaluaron apropiadamente las consecuencias del experimento sobre la vida de las gemelas nacidas como la afectación a su autonomía, los supuestos beneficios por recibir y los riesgos futuros de daño durante su vida. Habiendo manipulado la línea celular germinal, no se consideraron los efectos sobre su descendencia futura. Este tipo de acciones tiene un impacto negativo en la forma como la sociedad concibe la ciencia. La ingeniería genética debe reservarse al contexto experimental básico o bien como investigación cínica para la corrección de enfermedades conocidas graves de origen genético, bajo estricta supervisión regulatoria y bioética y de manera gradualista de acuerdo con el progreso de las técnicas de edición genética.


Abstract In this essay, the bioethical implications of the recent genetic manipulation in human embryos with CRISPR-Cas9 to eliminate the CCR5 gene and the birth of a pair of discordant twin girls are analyzed. The experiment was disseminated via social media. The main bioethical flaws identified include the justification of the model, the informed consent process and the lack of disclosure of evident conflicts of interest. The consequences of the experiment on the life of the twins that were born were not properly evaluated, such as the impact on their autonomy, the alleged benefits to be received and the future risks of harm during their lifetime. Having manipulated the germ cell line, the effects on their future offspring were not considered. This type of actions negatively affects the way society conceives science. Genetic engineering should be reserved to the basic experimental context or as clinical research for the correction of known serious diseases of genetic origin under strict regulatory and bioethical supervision and using a gradualist approach in accordance with the advances of gene editing techniques.


Subject(s)
Humans , Female , Receptors, CCR5/genetics , CRISPR-Cas Systems , Gene Editing/ethics , Publishing/ethics , Research Design , Twins, Dizygotic , Genetic Engineering/classification , Genetic Engineering/ethics , Genome, Human , HIV Infections/prevention & control , China , Conflict of Interest , Sperm Injections, Intracytoplasmic , Bioethical Issues , Therapeutic Human Experimentation/ethics , Informed Consent/ethics
13.
Chinese Journal of Biotechnology ; (12): 1269-1276, 2020.
Article in Chinese | WPRIM | ID: wpr-826850

ABSTRACT

Human adenoviruses are widespread causative agent that induces respiratory diseases, epidemic keratoconjunctivitis and other related diseases. Adenoviruses are commonly used in experimental and clinical areas. It is one of the most commonly used virus vectors in gene therapy, and it has attracted a lot of attention and has a high research potential in tumor gene therapy and virus oncolytic. Here, we summarize the biological characteristics, epidemiology and current application of adenovirus, in order to provide reference for engineering application of adenovirus.


Subject(s)
Humans , Adenovirus Infections, Human , Epidemiology , Virology , Adenoviruses, Human , Genetics , Genetic Engineering , Methods , Genetic Vectors , Oncolytic Virotherapy , Oncolytic Viruses , Genetics , Virus Replication
14.
Chinese Journal of Biotechnology ; (12): 891-898, 2020.
Article in Chinese | WPRIM | ID: wpr-826887

ABSTRACT

Unnatural amino acid orthogonal translation machinery can insert unnatural amino acids at desired sites of protein through stop codon by means of foreign orthogonal translation system composed of aminoacyl-tRNA synthetase and orthogonal tRNA genes. This new genetic engineering technology is not only a new tool for biochemical researches of proteins, but also an epoch-making technology for the development of new-type live viral vaccines. The mutated virus containing premature termination codon in genes necessary for replication can be propagated in transgenic cells harboring unnatural amino acid orthogonal translation machinery in media with corresponding unnatural amino acid, but it cannot replicate in conventional host cells. This replication-deficient virus is a new-type of live viral vaccine that possesses advantages of high efficacy of traditional attenuated vaccine and high safety of killed vaccine. This article reviews the application and prospect of unnatural amino acid orthogonal translation machinery in the development of novel replication-deficient virus vaccines.


Subject(s)
Amino Acids , Genetics , Amino Acyl-tRNA Synthetases , Genetic Engineering , Protein Engineering , RNA, Transfer , Viral Vaccines
15.
Chinese Journal of Biotechnology ; (12): 678-692, 2020.
Article in Chinese | WPRIM | ID: wpr-827001

ABSTRACT

As water-soluble, natural pigments, anthocyanins are responsible for the red, purple and blue colors of many flowers, which attract pollinators to spread pollen. The colors of flowers are also essential for plants to survive in the nature and become one of the most significant characteristics of ornamental plants. In the booming floriculture industry, to produce various flower colors could increase the richness of natural colors, but it is still difficult to breed flowers with coveted blue color. The diversity of flower color is mainly determined by the types and contents of anthocyanins and their derivatives. The synthesis of delphinidin pigments is the key factor for breeding blue flowers. However, there are no structural genes in many plants to biosynthesize delphinidin pigments. Blue flowers are successfully created by genetic engineering in recent years. In this paper, using common ornamental plants as examples, we review the mechanism of plant flower coloration from the aspects of the key factors affecting the synthesis of delphinidin pigment and the production strategies of blue flowers based on the regulation of anthocyanin metabolism. Different strategies of molecular breeding could provide opportunities to improve colors of other floriculture plants and to develop anthocyanin-rich economic crops, such as colored cotton with blue fibers.


Subject(s)
Anthocyanins , Metabolism , Flowers , Gene Expression Regulation, Plant , Genetic Engineering , Pigmentation , Genetics
16.
Chinese Journal of Biotechnology ; (12): 2162-2170, 2020.
Article in Chinese | WPRIM | ID: wpr-878475

ABSTRACT

We constructed the CS1-targeted second- and third-generation CAR-T cells with genetic engineered 4-1BB or/and ICOS as a costimulatory signaling molecule by use of lentiviral platform. The CS1-targeted second-generation CAR-T cells with ICOS or 4-1BB had similar anti-neoplastic activity. When effector/target ratio was 1:1, the CAR-T cells with ICOS showed better killing effect on IM9-lucgfp cells than those with 4-1BB. However, The CS1-targeted third-generation CAR-T cells exihibited lower cytolytic capacity against IM9-lucgfp cells than the CS1-targeted second-generation CAR-T cells when the ratio of effector/target was 1:1, 2:1 or 5:1. When the ratio of effector/target was 10:1, the killing efficacy of both the second- and third-generation CAR-T cells against IM9-lucgfp cells was more than 85%, significantly higher than that of the control T cells. Taken together, both the CS1-targeted second- and third-generation CAR-T cells with ICOS or/and 4-1BB could efficiently kill CS1-positive multiple myeloma cells, but the CS1-targeted second-generation CAR-T cells had more potent killing effect on CS1-positive multiple myeloma cells than the CS1-targeted third-generation CAR-T cells.


Subject(s)
Humans , 4-1BB Ligand/metabolism , Cell Line, Tumor , Genetic Engineering , Inducible T-Cell Co-Stimulator Protein/metabolism , Multiple Myeloma/therapy , Signal Transduction , T-Lymphocytes/chemistry , Xenograft Model Antitumor Assays
17.
Chinese Journal of Biotechnology ; (12): 2193-2205, 2020.
Article in Chinese | WPRIM | ID: wpr-878478

ABSTRACT

Endoglucanase (EG) is an important component of cellulases and play an important role in cellulose degradation. However, its application is limited due to the low yield of endoglucanase from natural microorganisms. Efficient heterologous expression of endoglucanase is an effective way to solve this problem. To obtain the engineered Saccharomyces cerevisiae for high-yield endoglucanase, endoglucanase gene was cloned from Clostridium cellulovorans, with a total length of 1 996 bp, encoding 440 amino acids, and the complete expression cassette (PαEGC) was constructed with the PGK promoter sequence from Saccharomyces cerevisiae, α-signal peptide sequence from pPIC9K plasmid and CYC1 terminator sequence from pSH65 plasmid by gene splicing by overlap extension PCR (SOE PCR), and the expression vector of endoglucanase in Saccharomyces cerevisiae was constructed by rDNA integration. The relationship between copy number and protein expression was explored. Random multicopy expression of endoglucanase was performed in Saccharomyces cerevisiae. The copy number of endoglucanase was identified by Droplet Digital PCR and explore the relationship between copy number and protein expression.The engineered Saccharomyces cerevisiae of endoglucanase with copy numbers of 1, 3, 4, 7, 9, 11, 15, 16, 19, 21, 22 and 23 were obtained by rDNA integration, respectively. The results showed that when the copy number was 15, the enzyme activity was the highest, namely 351 U/mL. The engineered strain of Saccharomyces cerevisiae for endoglucanase was successfully constructed, which can provide reference for the heterologous expression of other industrial enzymes.


Subject(s)
Cellulase/genetics , Genetic Engineering , Industrial Microbiology , Plasmids/genetics , Saccharomyces cerevisiae/genetics
18.
Acta sci., Biol. sci ; 42: e52272, fev. 2020. tab, ilus, graf
Article in English | LILACS, VETINDEX | ID: biblio-1460946

ABSTRACT

Soybean loss due to pests and pathogens is a serious problem worldwide. Soybean producers have few options to manage diseases caused by general pathogens where major genes for full resistance have not been discovered. The innate defense of soybean plants could be enhanced by improving content and composition of lignin by genetic engineering of the phenylpropanoid pathway.We used a novel technique of germ-line genetic transformation of soybean plants via natural pollen tubes as vectors. This technique uses Agrobacterium tumefaciensto mediate transfer of genes of interest to the zygote to introduce the key lignification genes (PtMYB4, PAL5, F5H, CAD1) into soybean genome. We observed 5.6% average transformation efficiency in the first generation of transgenic plants and in the second generation the presence of the transgene constructs was confirmed in more than 50% (for CsVMV/PtMYB4sens, 35SVTM/PAL5, C4H/F5H, CsVMV/CAD1constructs) transgenic soybean lines. We confirmed the expression of the introduced genes at transcriptional level using RT-PCR and Northern blot. Functional analysis using lignin content determination and the activity of PAL5 and CAD1 enzymes demonstrated that the transgenes perform their function in planta. The proposed technique is effectiveand inexpensive and can be used to create novel stress and disease resistant soybean genotypes.


Subject(s)
Genetic Engineering , Genome , Metabolism , Whole Genome Sequencing
19.
Frontiers of Medicine ; (4): 69-82, 2019.
Article in English | WPRIM | ID: wpr-771259

ABSTRACT

Cytokine-activated T cells (CATs) can be easily expanded and are widely applied to cancer immunotherapy. However, the good efficacy of CATs is rarely reported in clinical applications because CATs have no or very low antigen specificity. The low-efficacy problem can be resolved using T cell antigen receptor-engineered CAT (TCR-CAT). Herein, we demonstrate that NY-ESO-1 HLA-A*02:01-specific high-affinity TCR (HAT)-transduced CATs can specifically kill cancer cells with good efficacy. With low micromolar range dissociation equilibrium constants, HAT-transduced CATs showed good specificity with no off-target killing. Furthermore, the high-affinity TCR-CATs delivered significantly better activation and cytotoxicity than the equivalent TCR-engineered T cells (TCR-Ts) in terms of interferon-γ and granzyme B production and in vitro cancer cell killing ability. TCR-CAT may be a very good alternative to the expensive TCR-T, which is considered an effective personalized cyto-immunotherapy.


Subject(s)
Humans , Cell Line, Tumor , Cytokines , Metabolism , Cytotoxicity, Immunologic , Genetic Engineering , HLA-A2 Antigen , Metabolism , Immunotherapy, Adoptive , Methods , Lymphocyte Activation , Receptors, Antigen, T-Cell , Genetics , Allergy and Immunology , T-Lymphocytes , Allergy and Immunology
20.
Chinese Journal of Biotechnology ; (12): 472-481, 2019.
Article in Chinese | WPRIM | ID: wpr-771360

ABSTRACT

Isovalerylspiramycin (ISP)Ⅰ, as a major component of bitespiramycin (BT), exhibits similar antimicrobial activities with BT and has advantages in quality control and dosage forms. It has been under preclinical studies. The existing ISPⅠ producing strain, undergoing three genetic modifications, carries two resistant gene markers. Thus, it is hard for further genetic manipulation. It is a time-consuming and unsuccessful work to construct a new ISPⅠ strain without resistant gene marker by means of the classical homologous recombination in our preliminary experiments. Fortunately, construction of the markerless ISPⅠ strain, in which the bsm4 (responsible for acylation at 3 of spiramycin) gene was replaced by the Isovaleryltansferase gene (ist) under control of the constitutive promoter ermEp*, was efficiently achieved by using the CRISPR-Cas9 gene editing system. The mutant of bsm4 deletion can only produce SPⅠ. Isovaleryltransferase coded by ist catalyzes the isovalerylation of the SPⅠat C-4" hydroxyl group to produce ISPⅠ. As anticipated, ISPⅠ was the sole ISP component of the resultant strain (ΔEI) when detected by HPLC and mass spectrometry. The ΔEI mutant is suitable for further genetic engineering to obtain improved strains by reusing CRISPR-Cas9 system.


Subject(s)
CRISPR-Cas Systems , Gene Editing , Genetic Engineering , Homologous Recombination
SELECTION OF CITATIONS
SEARCH DETAIL