Genetic Engineering Research Articles

Bibliometric analysis of dendritic cell-cytokine induced killers cell therapy

Objective: Dendritic cell-cytokine induced killers (DC-CIK) immunotherapy involves co-culturing dendritic cells (DCs) and cytokine-induced killer (CIK) cells to generate activated immune cells for reinfusion into patients, directly killing tumor cells while enhancing the body's immune response, aiming to inhibit tumor growth and recurrence. Here, we conducted a comprehensive bibliometric analysis of DC-CIK immunotherapy, to provide insights into the current state of DC-CIK therapy research and guide future directions in this promising immunotherapeutic approach. Methods: Data were collected from the Web of Science Core Collection (WOSCC) using the search terms "Dendritic cell-cytokine induced killers cell therapy" and "Immune cell therapy" for the period from 1997 to 2024. Bibliometric analysis was performed using VOSviewer to examine publication trends, citation networks, thematic focuses, and global collaboration networks. Results: The number of publications on DC-CIK therapy has shown a significant increase over the years, peaking in 2014 and fluctuating since then. China leads in the number of publications, followed by the United States. Sun Yat-sen University is the most prolific institution, and Ying Mu and Anqi Zhang are the most productive authors. Frontiers in immunology is the most productive journal, publishing 15 papers on DC-CIK therapy. Keyword analysis revealed a focus on cancer biology, immune modulation, and therapeutic strategies, with a particular emphasis on co-culture techniques, cytokines, immune checkpoint inhibitors, and genetic engineering. Conclusion: DC-CIK therapy represents a significant advancement in cancer treatment, integrating the unique properties of DCs and CIK cells to enhance anti-tumor immunity. Despite its promise, challenges remain in optimizing clinical efficacy and addressing translational hurdles. Future research should focus on refining co-culture strategies, improving therapeutic outcomes, and ensuring the safe and accessible application of DC-CIK therapy in clinical settings.

Current Advances in Viral Nanoparticles for Biomedicine.

Viral nanoparticles (VNPs) have emerged as crucial tools in the field of biomedicine. Leveraging their biological and physicochemical properties, VNPs exhibit significant advantages in the prevention, diagnosis, and treatment of human diseases. Through techniques such as chemical bioconjugation, infusion, genetic engineering, and encapsulation, these VNPs have been endowed with multifunctional capabilities, including the display of functional peptides or proteins, encapsulation of therapeutic drugs or inorganic particles, integration with imaging agents, and conjugation with bioactive molecules. This review provides an in-depth analysis of VNPs in biomedicine, elucidating their diverse types, distinctive features, production methods, and complex design principles behind multifunctional VNPs. It highlights recent innovative research and various applications, covering their roles in imaging, drug delivery, therapeutics, gene delivery, vaccines, immunotherapy, and tissue regeneration. Additionally, the review provides an assessment of their safety and biocompatibility and discusses challenges and future opportunities in the field, underscoring the vast potential and evolving nature of VNP research.

Principles and research progress of gene editing technology

Gene editing is a new genetic engineering technology. Given the quick development of molecular biology technology, genome editing tools have made great progress from the initial ZFNs and TALEN technology to CRISPR-Cas9 technology in recent years. Gene editing is the modification of specific targets in the genome of an organism to change its genetic information and phenotypic characteristics. This study analyzed the principle and research progress of gene editing technology through the data. For example, CRISPR-Cas9 can protect against secondary viral attacks, and ZFNs can be cleaved on any genomic sequence. Studies have found that there are still great challenges in the clinical field of genetic technology. Including the need for standardized safety protocols, regulatory transparency, and pricing frameworks, which increase the risk of further mutations. This article elaborates on the role and future development of gene editing in medicine, and provides a reference for the future field of gene editing technology.

Transgenic expression of SeCYP9A186 and PxFMO2 confers resistance to emamectin benzoate in Plutella xylostella.

The overexpression of metabolic enzymes constitutes a crucial mechanism for insects to detoxify xenobiotics and metabolic pesticides. A flavin-containing monooxygenase gene (PxFMO2) from Plutella xylostella and a P450 gene (SeCYP9A186, F116V mutant allele) from Spodoptera exigua have been reported to be involved in insecticide resistance. In this study, we aim to utilize transgenic technology to validate their in vivo detoxification functions in Plutella xylostella. We established two transgenic strains of Plutella xylostella with expressing an endogenous PxFMO2 gene from Plutella xylostella and an exogenous SeCYP9A186 gene from S. exigua, respectively. Bioassays demonstrated that the transgenic Plutella xylostella strain (IPP-FMO2) expressing PxFMO2 exhibited a 12-fold resistance to emamectin benzoate and a 6.4-fold resistance to chlorantraniliprole compared to the background strain (IPP-S). In contrast, the transgenic Plutella xylostella strain (IPP-9A186) expressing SeCYP9A186 displayed a 235-fold resistance to emamectin benzoate and a 115-fold resistance to abamectin. Moreover, resistance to emamectin benzoate in the IPP-9A186 strain of Plutella xylostella was inherited as an incompletely dominant trait and was genetically linked to the transgene locus. Our results not only elucidated the in vivo contribution of the PxFMO2 and SeCYP9A186 to the insecticide resistance phenotype in Plutella xylostella, but also provided a genetic engineering toolkit to manipulate resistance pathways. These insights and methodologies could further aid in developing sustainable pest management strategies in Plutella xylostella. © 2024 Society of Chemical Industry.

Advances and new horizons in metabolic engineering of heterotrophic bacteria and cyanobacteria for enhanced lactic acid production.

Bacteria species such as E.Coli, Lactobacilli, and pediococci play an important role as starter strains in fermentation food or polysaccharides into lactic acid. These bacteria were metabolically engineered using multiple proven genome editing methods to enhance relevant phenotypes. The efficacy of these procedures varies depending on the editing tool used and researchers' ability to pick suitable recombinants, which significantly increased genome engineering throughput. Cyanobacteria produce oxygenic photosynthesis and play an important role in carbon dioxide fixing. The fixed carbon dioxide is then retained as polysaccharides in cells and metabolised into various low carbon molecules such as lactate, succinate, and ethanol. Lactate is used as a building ingredient in various bioplastics, food additives, and medicines. This review covers the recent advances in lactic acid production through metabolic and genetic engineering in bacteria and cyanobacteria.

Is Lactococcus lactis a Suitable Candidate for Use as a Vaccine Delivery System Against Helicobacter pylori?

Helicobacter pylori was described in 1979. This bacterium, which thrives in the harsh conditions of the stomach, is typically acquired during childhood and can remain colonized for life. Approximately, 90% of the global population is affected, and H. pylori is linked to various conditions, including gastritis, peptic ulcers, lymphoproliferative gastric lymphoma, and even gastric cancer. Currently, antibiotics are the primary treatment method, but the associated challenges of antibiotic use have led to the consideration of oral vaccination as a viable preventive measure against this infection. However, the stomach's harsh environment characterized by its acidic conditions and numerous proteolytic enzymes poses significant obstacles to the development and effectiveness of oral vaccines. To address these challenges, researchers have proposed and evaluated several delivery systems. One of the most promising options is the use of probiotics. Among the various probiotics, Lactococcus lactis stands out as a suitable candidate for oral vaccine delivery against H. pylori due to the advancements in genetic engineering that have been applied to it. This review article discusses the limitations of current treatment strategies and rationalizes the shift toward vaccination, particularly oral vaccination for this infection. It also explores the advantages and challenges of using probiotic bacteria, with a focus on L. lactis as a delivery system. Ultimately, despite the existing challenges, L. lactis continues to be recognized as a promising delivery system. Nonetheless, further research is essential to fully assess its effectiveness and address the challenges associated with this approach.

Genetic Engineering Pharmaceutical Technology: A Brief Review

Genetic engineering has emerged as a transformative technology in the pharmaceutical industry, offering unprecedented control over the manipulation of genetic material. This technology enables the precise editing, combining, and expression of target genes in various organisms, leading to the production of specific proteins, enzymes, and biologics essential for therapeutic applications. This review provides an overview of the basic principles and strategies of gene therapy, the development of genetic engineering pharmaceuticals, and their applications in producing physiologically active substances, antibodies, and vaccines. The review also highlights the significant advancements in the field, and explores the future potential of genetic engineering in advancing medical science and improving human health.

Ninety-Day Feeding Test of Stacked DBN9936 × DBN9501 Maize on Sprague Dawley Rats.

The transgenic maize DBN9936 × DBN9501, which confers resistance to insects and tolerance to herbicides, was developed via conventional cross breeding of transgenic maize DBN9936 and DBN9501. In our present study, a 90-day feeding toxicity study was conducted on Sprague Dawley rats to evaluate the safety of the maize. A total of 140 rats were randomly assigned to seven groups (n = 10/sex/group): one control group, three genetically modified (GM) groups with 17.5%, 35%, and 70% (wt/wt) GM maize, respectively, and three non-GM groups with corresponding incorporation rate of parental maize DBN318. The rats of control group were fed with AIN93G diet. The parameters including body weights, food consumption, hematology, serum biochemistry, organ weights, and histopathology were examined during the course of the study. Compared with the non-GM group or AIN93G control group, minor statistical differences were observed for some parameters in some groups, yet none of them was considered a GM-related adverse effect. In conclusion, the results demonstrated that no adverse effect was observed on rats following 90 days feeding with diet containing up to 70% GM maize. The results indicated that stacked maize DBN9936 × DBN9501 was as safe as its parental DBN318 maize.

Efficient genome editing of two-cell mouse embryos via modified CRISPR/Cas electroporation

Creating genetically modified (GM) animals using CRISPR/Cas mediated through the electroporation of two-cell stage embryos, rather than fertilized eggs, holds considerable potential. The full potential of genome editing using two-cell stage embryos is only beginning to be explored. We developed an improved electroporation method to prevent blastomere fusion in two-cell-stage embryos, enabling efficient genome editing. Using this method, we demonstrated that the indel mutation rates and ssODN knock-in (KI) efficiencies in two-cell-stage embryos are comparable to those in fertilized eggs, with a tendency for higher efficiency in long DNA KI. This study highlights the potential value of two-cell-stage embryos and provides enhanced animal model production opportunities. Furthermore, realizing genome editing in two-cell-stage embryos extends the editing timeframe from fertilized egg to two-cell-stage embryo, offering promising avenues for future research in embryo genome editing techniques.

Genome-wide association study of metabolic traits in the giant duckweed Spirodela polyrhiza.

The exceptionally high growth rate and high flavonoid content make the giant duckweed Spirodela polyrhiza (L.) Schleid. (Arales: Lemnaceae Martinov) an ideal organism for food production and metabolic engineering. To facilitate this, identification of the genetic basis underlying growth and metabolic traits is essential. Here, we analysed growth and content of 42 metabolites in 137 S. polyrhiza genotypes and characterized the genetics underpinning these traits using a genome-wide association (GWA) approach. We found that biomass positively correlated with the content of many free amino acids, including L-glutamine, L-tryptophan, and L-serine, but negatively correlated with specialized metabolites, such as flavonoids. GWA analysis showed that several candidate genes involved in processes such as photosynthesis, protein degradation, and organ development were jointly associated with multiple metabolic traits. The results suggest the above genes are suitable targets for simultaneous optimization of duckweed growth and metabolite levels. This study provides insights into the metabolic diversity of S. polyrhiza and its underlying genetic architecture, paving the way for industrial applications of this plant via targeted breeding or genetic engineering.

Honing CAR T cells to tackle acute myeloid leukemia.

Acute myeloid leukemia (AML) remains a dismal disease with poor prognosis, particularly in the relapsed/refractory (r/r) setting. Chimeric antigen receptor (CAR) therapy has yielded remarkable clinical results in other leukemias and thus has, in principle, the potential to achieve similar outcomes in r/r AML. Re-directing the approved CD19-specific CAR designs against the myeloid antigens CD33, CD123 or CLEC12A has occasionally yielded morphological leukemia-free states (MLFS) but has so far been marred by threatening myeloablation and early relapses. These safety and efficacy limitations owe in large part to the challenge of identifying suitable target antigens and designing adequate receptors for effective recognition and safe elimination of AML. Building on lessons learned from the initial clinical attempts, a new wave of CAR strategies relying on alternative target antigens and innovative CAR designs is about to enter clinical evaluation. Adapted multi-antigen targeting, logic-gating and emerging cell engineering solutions offer new possibilities to better direct T cell specificity and sensitivity towards AML. Pharmacologic modulation and genetic epitope engineering may extend these approaches by augmenting target expression in AML cells or minimizing target expression in normal hematopoietic cells. On/off switches or CAR T cell depletion may curb excessive or deleterious CAR activity. Investigation of AML-intrinsic resistance and leukemic microenvironmental factors is poised to reveal additional targetable AML vulnerabilities. We summarize here the findings, challenges and new developments of CAR therapy for AML. These illustrate the need to specifically adapt CAR strategies to the complex biology of AML to achieve better therapeutic outcomes.

Genome-Wide Identification and Characterization of OSC Gene Family in Gynostemma pentaphyllum (Cucurbitaceae)

Gynostemma pentaphyllum is a traditional Chinese medicinal plant of considerable application value and commercial potential, primarily due to its production of various bioactive compounds, particularly dammarane-type triterpenoid saponins that are structurally analogous to ginsenosides. Oxidosqualene cyclase (OSC), a pivotal enzyme in the biosynthesis of triterpenoid metabolites in plants, catalyzes the conversion of oxidosqualene into triterpenoid precursors, which are essential components of the secondary metabolites found in G. pentaphyllum. To elucidate the role of OSC gene family members in the synthesis of gypenosides within G. pentaphyllum, this study undertook a comprehensive genome-wide identification and characterization of OSC genes within G. pentaphyllum and compared their expression levels across populations distributed over different geographical regions by both transcriptome sequencing and qRT-PCR experimental validation. The results identified a total of 11 members of the OSC gene family within the genome of G. pentaphyllum. These genes encode proteins ranging from 356 to 767 amino acids, exhibiting minor variations in their physicochemical properties, and are localized in peroxisomes, cytoplasm, plasma membranes, and lysosomes. All GpOSCs contain highly conserved DCTAE and QW sequences that are characteristic of the OSC gene family. A phylogenetic analysis categorized the GpOSCs into four distinct subfamilies. A cis-element analysis of the GpOSC promoters revealed a substantial number of abiotic stress-related elements, indicating that these genes may respond to drought conditions, low temperatures, and anaerobic environments, thus potentially contributing to the stress resistance observed in G. pentaphyllum. Expression analyses across different G. pentaphyllum populations demonstrated significant variability in OSC gene expression among geographically diverse samples of G. pentaphyllum, likely attributable to genetic variation or external factors such as environmental conditions and soil composition. These differences may lead to the synthesis of various types of gypenosides within geographically distinct G. pentaphyllum populations. The findings from this study enhance our understanding of both the evolutionary history of the OSC gene family in G. pentaphyllum and the biosynthetic mechanisms underlying triterpenoid compounds. This knowledge is essential for investigating molecular mechanisms involved in forming dammarane-type triterpenoid saponins as well as comprehending geographical variations within G. pentaphyllum populations. Furthermore, this research lays a foundation for employing plant genetic engineering techniques aimed at increasing gypenoside content.

Using Biotechnology to Create Transgenic Crops that Resist Climate Change

Climate change is a major threat to food security, especially for farmers in vulnerable areas facing the challenges of climate change. This study aimed to modify the plant genome to increase tolerance to extreme climatic conditions such as drought and high temperatures using genetic engineering methods based on CRISPR-Cas9 technology. Transgenic and control plants were grown in a laboratory experimental design with a quantitative approach. The results showed that the transgenic plants consistently outperformed the control plants under drought, high temperature, and low nutrient conditions. The transgenic plant showed a growth rate of 15.2 and average productivity of 25.7, higher than the control (12.3 and 19.1). Under high temperature conditions, the increase in productivity was even greater, reaching 33.2%, indicating that the inserted HSP gene successfully protects important proteins from heat stress damage. Genetic modification through the expression of genes such as DREB1A, HSP70, and PHR1 was shown to increase plant resistance to environmental stress. With these findings, biotechnology provides a real opportunity to build more adaptive and sustainable agricultural systems, supporting global food needs amid the intensifying challenges of global warming. This research is very relevant in answering the big challenge in the future, which is how to ensure biotechnology can be utilised sustainably to meet the world's growing food needs, without compromising ecosystems and biodiversity.

Strategies and Protocols for Optimized Genome Editing in Potato.

The potato family includes a highly diverse cultivar repertoire and has a high potential for nutritional yield improvement and refinement but must in line with other crops be adapted to biotic and abiotic stresses, for example, accelerated by climate change and environmental demands. The combination of pluripotency, high ploidy, and relative ease of protoplast isolation, transformation, and regeneration together with clonal propagation through tubers makes potato highly suitable for precise genetic engineering. Most potato varieties are tetraploid having a very high prevalence of length polymorphisms and small nucleotide polymorphisms between alleles, often complicating CRISPR-Cas editing designs and strategies. CRISPR-Cas editing in potato can be divided into (i) characterization of target area and in silico-aided editing design, (ii) isolation and editing of protoplast cells, and (iii) the subsequent explant regeneration from single protoplast cells. Implementation of efficient CRISPR-Cas editing relies on efficient editing at the protoplast (cell pool) level and on robust high-throughput editing scoring methods at the cell pool and explant level. Gene and chromatin structure are additional features to optionally consider. Strategies and solutions for addressing key steps in genome editing of potato, including light conditions and schemes for reduced exposure to hormones during explant regeneration, which is often linked to somaclonal variation, are highlighted.

Insights into the functional mechanisms of three terpene synthases from Lavandula angustifolia (Lavender)

Lavender species are of significant economic value being cultivated extensively worldwide for their essential oils (EOs), which include terpenes that play crucial roles in the cosmetic, personal care, and pharmaceutical industries. The terpene synthases in lavender, such as Lavandula angustifolia linalool synthase (LaLINS), limonene synthase (LaLIMS), and bergamotene synthase (LaBERS), are key enzymes in terpene biosynthesis. However, the functional mechanisms underlying these enzymes remain poorly understood. Here, we used AlphaFold2 to predict the three-dimensional structures of LaLINS, LaLIMS, and LaBERS. The hydrodynamic radii of LaLINS, LaLIMS, and LaBERS were 5.7 ± 0.2, 6.2 ± 0.3, and 5.4 ± 0.2 nm, respectively. Mutations D320A or D324A led to a complete loss of activity in LaLINS compared to the wild-type (WT) enzyme; similarly, mutations D356A or D360A abolished activity in LaLIMS, and D291A or D295A eliminated activity in LaBERS. Furthermore, the genes LaLINS, LaLIMS, and LaBERS exhibited significantly higher expression levels in leaves compared to stems and flowers, with peak expression occurring at 8:00 a.m. Our findings contribute to a deeper understanding of terpene biosynthesis in lavender and offer insights for improving essential oil production through genetic engineering.

Utilizing Plant Growth-Promoting Rhizobacteria (PGPR) to Advance Sustainable Agriculture

Plant growth-promoting rhizobacteria (PGPR) are beneficial bacteria that play a crucial role in sustainable agriculture by enhancing plant growth through various mechanisms. This review examines the contributions of PGPR in improving nutrient availability, producing phytohormones, providing biocontrol against pathogens, and enhancing abiotic stress tolerance. By reducing the necessity for chemical fertilizers and pesticides, PGPR mitigate environmental impacts, enhance soil health, and support long-term agricultural productivity. However, challenges such as inconsistent performance across various soils, regulatory barriers, and limited farmer awareness, hinder their widespread adoption. Recent advancements in nano-encapsulation technology, genetic engineering, and bioinformatics, present promising solutions for overcoming these obstacles and enhancing PGPR efficacy. The incorporation of PGPR into biofertilizers, biopesticides, and integrated plant management (IPM) offers a sustainable resolution to global agricultural challenges. This review addresses the current state of PGPR research, applications, and future directions for optimizing their use in promoting sustainable agriculture.

Harnessing the Power of Plants: Innovative Approaches to Pollution Prevention and Mitigation

Innovative and sustainable environmental management strategies are urgently required to address the escalating global pollution crisis. Phytoremediation, which involves using plants to mitigate, remediate, or contain environmental contaminants, is a promising, cost-effective, and environmentally friendly alternative to conventional remediation methods. This review summarizes current research to elucidate the multifaceted roles of plants in pollution mitigation, detailing mechanisms such as phytoextraction, phytostabilization, phytodegradation, and rhizofiltration; we highlight successful case studies that demonstrate practical applications across diverse environments, such as the use of hyperaccumulator plants for heavy metal removal and genetically engineered species for organic pollutant degradation. Furthermore, this review explores recent technological advancements that have enhanced the effectiveness of phytoremediation, such as the integration of nanotechnology and genetic engineering. It also analyzes the economic and social implications of adopting plant-based pollution control strategies, emphasizing their potential for community involvement and socioeconomic benefits. Despite the promising outlook, we acknowledge the inherent challenges and limitations of phytoremediation, including public acceptance and scalability issues. Finally, we identify key opportunities for future research and innovative approaches that could expand the scope and impact of phytotechnologies in pollution mitigation. This comprehensive review underscores the potential of plants as both agents of environmental restoration and essential components of sustainable pollution management systems.

Introducing CCD1 into isolated Rhodotorula strain enhances flavor production and improves cigar fermentation

IntroductionThe fermentation process plays an important role in enhancing the quality of cigar tobacco leaves. Through fermentation, microbial metabolism can degrade aromatic precursors and macromolecules, which increases the content of aroma compounds and reduces irritancy of tobacco leaves.MethodsTo further enhance the fermentation effect of cigar tobacco leaves, a Rhodotorula strain (Rh3), capable of producing carotenoids and improving fermentation quality, was isolated from cigar tobacco leaves. Subsequent genetic engineering techniques introduced the carotenoid cleavage dioxygenase 1 (CCD1) gene into the isolated Rh3.ResultsThe modified Rh3 exhibits a significant increase in carotenoid degradation products compared with the original Rh3 in culture medium (from 0.29 μg/mg to 15 μg/mg). Subsequent cigar tobacco leaf fermentation experiments revealed that the modified Rh3 produced 65.9% more carotenoid degradation products compared to the control group, outperforming the original strain, which achieved a 41.4% increase. Furthermore, the modified strain preserves its ability to improve the intrinsic chemical composition of cigar tobacco leaves.DiscussionWe show here that modified Rh3 can increase the content of carotenoid degradation products, thereby enhancing the fermentation effect of cigar tobacco leaves. This study presents a beneficial exploration to improve the quality of cigar tobacco leaves for future use and offers a promising strategy for producing flavor compounds from discarded tobacco leaves.

Boosting the efficacy of fungal biocontrol: miRNA339-5p-mediated mosquito immunity regulation.

Aedes mosquitoes are vectors for numerous viral diseases, including dengue, zika, chikungunya, and yellow fever. Therefore, underscoring the urgent need for eco-friendly alternatives to combat insecticide resistance and the scarcity of effective vaccines. Entomopathogenic fungi present a sustainable alternative to chemical insecticides; however, their widespread application is limited by their relatively low virulence. Here, we investigated the immunological interactions between Metarhizium anisopliae and Aedes albopictus, demonstrating that fungal infection significantly up-regulated immune-related genes in the Toll and melanization pathways, thereby enhancing antifungal and antibacterial defenses at 48 h post-infection (hpi). Small RNA sequencing identified miR339-5p as a crucial modulator, targeting the immune genes Gram-Negative Binding Protein 1 (GNBP1) and CLIP-domain Serine Protease B15 (CLIPB15), which are critical for Toll and phenoloxidase (PO) pathway activation. The administration of a synthetic miR339-5p mimic increased fungal virulence, resulting in a higher mortality rate among adult mosquitoes and a significant increase in the mortality rate of mosquito larvae within 24 hpi. GNBP1 was found to regulate both Toll and PO pathways, while CLIPB15 specifically modulated the PO system by cleaving prophenoloxidase (PPO). This research highlights the potential of leveraging Ae. albopictus-encoded miR339-5p through advanced genetic engineering techniques to bolster the efficacy of existing fungal-based mosquito control strategies, providing a promising approach in the fight against mosquito-borne diseases. © 2024 Society of Chemical Industry.

Evaluation of Vaccinia Virus Infection in Mice Using Two-Reporter Recombinant Virus.

The family Poxviridae comprises multiple viruses with large double-stranded (ds) DNA genomes that can infect numerous vertebrate and invertebrate hosts, including humans. The development of genetic engineering methods for Vaccinia virus (VACV), the prototypic member in the family, have allowed the manipulation of the genomes of poxviruses for the generation of recombinant (r)VACV expressing easily traceable luciferase and/or fluorescent reporter genes. These recombinant viruses have significantly contributed to progress in the field of poxvirus research and accelerated the development of novel prophylactic vaccines and therapeutic antiviral treatments. Recently, we described two reporter rVACV expressing luciferase (Nluc) and fluorescent (GFP or Scarlet) proteins to easily track viral infections in different systems, overcoming the limitations associated with the use of rVACV expressing a single luciferase or fluorescent reporter gene. Here, we describe the experimental procedures to carry out in vitro, in vivo and ex vivo studies using these novel bireporter-expressing rVACV, which also represent an excellent option to study the biology of VACV, including the use of these reporter viruses for testing new antivirals and vaccines, using cultured cells and/or well-characterized animal models of infection.