Enhancement of RecET-mediated in vivo linear DNA assembly by a xonA mutation

Assembly of intact, replicating plasmids from linear DNA fragments introduced into bacterial cells, i.e. in vivo cloning, is a facile genetic engineering technology that avoids many of the problems associated with standard in vitro cloning. Here we report characterization of various parameters of in vivo linear DNA assembly mediated by either the RecET recombination system or the bacteriophage λ Red recombination system. As previously observed, RecET is superior to Red for this reaction when the terminal homology is 50 bases. Deletion of the E. coli xonA gene, encoding Exonuclease I, a 3′→5′ single-strand DNA exonuclease, substantially improves the efficiency of in vivo linear DNA assembly for both systems. Deletion of ExoI function allowed robust RecET assembly of six DNA segments to create a functional plasmid. The linear DNAs are joined accurately with very few errors. This discovery provides a significant improvement to previously reported in vivo linear DNA assembly technologies.

Native and Engineered Probiotics: Promising Agents against Related Systemic and Intestinal Diseases

Intestinal homeostasis is a dynamic balance involving the interaction between the host intestinal mucosa, immune barrier, intestinal microecology, nutrients, and metabolites. Once homeostasis is out of balance, it will increase the risk of intestinal diseases and is also closely associated with some systemic diseases. Probiotics (Escherichia coli Nissle 1917, Akkermansia muciniphila, Clostridium butyricum, lactic acid bacteria and Bifidobacterium spp.), maintaining the gut homeostasis through direct interaction with the intestine, can also exist as a specific agent to prevent, alleviate, or cure intestinal-related diseases. With genetic engineering technology advancing, probiotics can also show targeted therapeutic properties. The aims of this review are to summarize the roles of potential native and engineered probiotics in oncology, inflammatory bowel disease, and obesity, discussing the therapeutic applications of these probiotics.

Highly efficient CRISPR/Cas9 system in Plasmodium falciparum using Cas9-expressing parasites and a linear donor template

The CRISPR/Cas9 system is a powerful genetic engineering technology for Plasmodium falciparum. We here report further improvement of the CRISPR/Cas9 system by combining the Cas9-expressing parasite with a liner donor template DNA. The Cas9-expressing parasite was generated by inserting the cas9 gene in the genome by double crossover recombination. The site-directed mutagenesis and the fusion of fluorescence protein was achieved within two weeks with high efficiency (> 85%), by transfecting the schizonts of the Cas9-expressing parasite with the liner donor template and the plasmid carrying the sgRNAs. Notably, there were neither off-target mutations in the resultant transgenic parasites nor unexpected recombination, that are the technical problems of the current CRISPR/Cas9 system. Furthermore, with our system, two genes on different chromosomes were successfully modified in single transfection. Because of its high efficiency and robustness, our improved CRISPR/Cas9 system will become a standard technique for genetic engineering of P. falciparum, which dramatically advances future studies of this parasite.

Delivery of miR-26a Using an Exosomes-Based Nanosystem Inhibited Proliferation of Hepatocellular Carcinoma

Background: MicroRNA (abbreviated miRNA)-based treatment holds great promise for application as clinical antitumor therapy, but good carriers for delivery of the miRNA drug are lacking. Exosomes secreted by mesenchymal stem cells (MSCs) have proved to be safe, and exogenously modified exosomes may potentially represent an excellent drug delivery vehicle.Methods: In this study, we designed a delivery nano system using single-stranded variable fragment (scFv)-modified exosomes derived from human cord blood MSCs. Genetic engineering technology was used to obtain anti-Glypican 3 (GPC3) scFv-modified exosomes, which were then loaded with miR-26a mimics through electroporation.Results: Results of electron microscopy and dynamic light scattering indicated that the diameter of the drug-carrying exosomes was about 160 nm. Furthermore, anti-GPC3 scFv-modified exosomes effectively delivered miR-26a to GPC3-positive hepatocellular carcinoma cells, thereby inhibiting cell proliferation and migration by regulating the expression of downstream target genes of miR-26a. The exosomes-based nano system displayed favorable anti-tumor effect in vivo with no obvious side effects.Conclusion: Our data provided a new perspective for the use of exosome delivery systems for miRNA-based antitumor therapy.

Oncolytic Adenovirus: Prospects for Cancer Immunotherapy

Immunotherapy has moved to the forefront of modern oncologic treatment in the past few decades. Various forms of immunotherapy currently are emerging, including oncolytic viruses. In this therapy, viruses are engineered to selectively propagate in tumor cells and reduce toxicity for non-neoplastic tissues. Adenovirus is one of the most frequently employed oncolytic viruses because of its capacity in tumor cell lysis and immune response stimulation. Upregulation of immunostimulatory signals induced by oncolytic adenoviruses (OAds) might significantly remove local immune suppression and amplify antitumor immune responses. Existing genetic engineering technology allows us to design OAds with increasingly better tumor tropism, selectivity, and antitumor efficacy. Several promising strategies to modify the genome of OAds have been applied: capsid modifications, small deletions in the pivotal viral genes, insertion of tumor-specific promoters, and addition of immunostimulatory transgenes. OAds armed with tumor-associated antigen (TAA) transgenes as cancer vaccines provide additional therapeutic strategies to trigger tumor-specific immunity. Furthermore, the combination of OAds and immune checkpoint inhibitors (ICIs) increases clinical benefit as evidence shown in completed and ongoing clinical trials, especially in the combination of OAds with antiprogrammed death 1/programed death ligand 1 (PD-1/PD-L1) therapy. Despite remarkable antitumor potency, oncolytic adenovirus immunotherapy is confronted with tough challenges such as antiviral immune response and obstruction of tumor microenvironment (TME). In this review, we focus on genomic modification strategies of oncolytic adenoviruses and applications of OAds in cancer immunotherapy.

Functional Characterization of the EMBRYONIC FLOWER 2 Gene Involved in Flowering in Ginkgo biloba

Ginkgo biloba has edible, medicinal, and ornamental value. However, the long juvenile phase prevents the development of the G. biloba industry, and there are few reports on the identification and functional analysis of genes regulating the flowering time of G. biloba. EMBRYONIC FLOWER 2 (EMF), an important protein in flower development, functions to promote vegetative growth and repress flowering. In this study, a novel EMF gene (GbEMF2) was cloned and characterized from G. biloba. GbEMF2 contains a 2,193 bp open reading frame (ORF) encoding 730 amino acids. GbEMF2 harbors conserved VEFS-Box domain by the plant EMF protein. The phylogenic analysis showed that GbEMF2 originated from a polycomb-group (Pc-G) protein ancestor and was a member of the EMF2 protein. The quantitative real-time PCR (qRT-PCR) analysis revealed that GbEMF2 was expressed in all detected organs, and it showed a significantly higher level in ovulating strobilus and microstrobilus than in other organs. Compared with emf2 mutant plants, overexpression of GbEMF2 driven by the CaMV 35S promoter in emf2 mutant Arabidopsis plants delayed flowering but earlier than wild-type (WT) plants. This result indicated that GbEMF2 repressed flowering in G. biloba. Moreover, the RNA-seq analysis of GbEMF2 transgenic Arabidopsis plants (GbEMF2-OE/emf2), WT plants, and emf2 mutants screened out 227 differentially expressed genes (DEGs). Among these DEGs, FLC, MAF5, and MAF5-1 genes were related to flower organ development and regulated by GbEMF2. In addition, some genes participating in sugar metabolism, such as Alpha-amylase 1 (AMY1), BAM1, and Sucrose synthase 3 (SUS3) genes, were also controlled by GbEMF2. Overall, our results suggested that GbEMF2 negatively regulates flowering development in G. biloba. This finding provided a foundation and target gene for shortening the Ginkgo juvenile period by genetic engineering technology.

PRODUK MAKANAN UBAH SUAI GENETIK (GMF) DALAM PERSPEKTIF KONSEP HALALAN TOYYIBAN BERDASARKAN PENILAIAN MAQASID HIFZ AN NAFS

in English: Nowadays, research and production of new food products based on genetic engineering technology are carried out to give benefit to the community. However, the production of food products through this technology has raised concerns in the determination of the legal status and the safety of the food products to be eaten by the consumer. Therefore, this article discusses genetically modified food (GMF) in the concept of halalan toyyiban based on the evaluation of maqasid hifz an nafs. This article uses qualitative design through the documentation study method by comparing and analyzing important information from selected sources such as al-Quran, fiqh books, scientific articles, fatwas, and journals from previous research. The results obtained showed that the GMF products are considered to meet the halalan toyyiban concept if the products follow the six principles in the concept which are halal, healthy, accepted by fitrah, safe, clean, and free from syubhah. This study also found that GMF products can be evaluated through three main maslahah (benefit) in maqasid syariah which is dharuriyyat (necessity), hajiyyat (will), and tahsiniyyat (perfection). Abstract in Bahasa Malaysia: Pada masa kini, penyelidikan serta penghasilan produk makanan baru yang berasaskan teknologi kejuruteraan genetik dilaksanakan bagi memberi manfaat kepada masyarakat. Namun, penghasilan makanan melalui teknologi ini secara tidak langsung telah menimbulkan kemusykilan dalam penentuan status hukum dan keselamatan produk makanan yang akan dimakan oleh pengguna. Oleh itu, artikel ini membincangkan mengenai produk makanan ubah suai genetik (GMF) dalam konsep halalan toyyiban berlandaskan penilaian maqasid hifz an nafs. Artikel ini menggunakan reka bentuk kualitatif iaitu melalui kaedah kajian dokumentasi dengan membandingkan serta menganalisis maklumat penting daripada sumber yang dipilih seperti kitab suci al-Quran, kitab-kitab fiqh, artikel-artikel ilmiah, fatwa-fatwa serta jurnal dari penyelidikan terdahulu. Hasil kajian yang diperolehi menunjukkan produk GMF dianggap menepati konsep halalan toyyiban sekiranya ia mengikut keenam-enam prinsip konsep tersebut iaitu prinsip halal, berkhasiat, diterima fitrah jiwa sejahtera, selamat, bersih dan bebas syubhah. Kajian ini juga mendapati bahawa produk GMF dapat dinilai melalui tiga maslahah utama dalam maqasid syariah iaitu dharuriyyat (keperluan), hajiyyat(kehendak) dan tahsiniyyat (kesempurnaan).

Genome-wide identification of the Tubby-Like Protein (TLPs) family in medicinal model plant Salvia miltiorrhiza

Tubby-Like Proteins (TLPs) are important transcription factors with many functions and are found in both animals and plants. In plants, TLPs are thought to be involved in the abiotic stress response. To reveal the potential function of TLPs in the medicinal model plant Salvia miltiorrhiza, we identified 12 S. miltiorrhiza TLPs (SmTLPs) and conducted a comprehensive analysis. We examined SmTLP gene structure, protein structure, phylogenetics, and expression analysis. Our results show that all SmTLPs, except SmTLP11, have a complete typical Tub domain. Promoter analysis revealed that most SmTLPs are involved in hormone and abiotic stress responses. Expression analysis revealed that the 12 SmTLPs could be divided into three categories: those specifically expressed in roots, those specifically expressed in stems, and those specifically expressed in leaves. Additional studies have shown that SmTLP10 may play an important role in the plant cold resistance, while SmTLP12 may be involved in the S. miltiorrhiza ABA metabolic pathway. Our study represents the first comprehensive investigation of TLPs in S. miltiorrhiza. These data may provide useful clues for future studies and may support the hypotheses regarding the role of TLPs in plant abiotic stress process. All in all, we may provide a reference for improving S. miltiorrhiza quality using genetic engineering technology.

Genetic Engineering, Globalization and the Future of Ecology: An Ecocritical Study of Margaret Atwood’s The Year of the Flood

Applying ecocritical perspectives, this study examines and analyzes the impacts of genetic engineering under the dominance of corporate organizations in the era of globalization in Margaret Atwood’s fiction The Year of the Flood. The intrusion of genetic engineering in the age of globalization has been problematic because of the anthropocentric values of the corporate houses. In this context, this study argues that genetic engineering technology, if goes uncontrolled, is manipulated for corporate profit only, and raises serious risks to global biodiversity by promoting monoculture flora and fauna. Similarly, the study proves that the intense profit-making desire of the corporate world leads to global pandemic threatening the existence of natural organisms including humans. Members of the ‘God’s Gardeners’ in The Year of the Flood prepare themselves to be safe from the ‘Waterless Flood,’ a global pandemic that has been the result of uncontrolled experimentation of genetic engineering on food, animals and drug for corporate houses. The text, however, offers the possibilities of saving lives if genetic engineering is used from humanitarian perspectives. This research helps in understanding the role of economic activities in disturbing the global biodiversity. For the purpose of textual analysis, the study applies ecocritical perspectives of Vandana Shiva, Jeffery M. Smith, Claire Hope Cummings, and others with especial focus on “bioimperialism” as discussed by Shiva.