recombinant DNA technology

Gene knock-out (KO) mouse models for DNA polymerase beta (Polβ) revealed that loss of Polβ leads to neonatal lethality, highlighting the critical organismic role for this DNA polymerase. While biochemical analysis and gene KO cell lines have confirmed its biochemical role in base excision repair and in TET-mediated demethylation, more long-lived mouse models continue to be developed to further define its organismic role. The Polb-KO mouse was the first of the Cre-mediated tissue-specific KO mouse models...

Sperm-associated antigen 5 (SPAG5), also known as Astrin, was previously demonstrated as a biomarker for cellular resistance to major breast cancer therapies, including chemo-, endocrine- and targeted therapy. However, the contribution of SPAG5 to anthracycline- and taxane-based chemotherapy in triple-negative breast cancer (TNBC) remains controversial. In the present study, the SPAG5 knockout cell model was established by using clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system in MDA-MB-231 and BT549 TNBC cell lines...

In the post-genome era, great progress has been made in metabolic engineering using recombinant DNA technology to enhance the production of high-value products by Streptomyces. With the development of microbial genome sequencing techniques and bioinformatic tools, a growing number of secondary metabolite (SM) biosynthetic gene clusters in Streptomyces and their biosynthetic logics have been uncovered and elucidated. In order to increase our knowledge about transcriptional regulators in SM of Streptomyces, this review firstly makes a comprehensive summary of the characterized factors involved in enhancing SM production and awakening SM biosynthesis...

Advancements in recombinant DNA technology have made proteins and peptides available for diagnostic and therapeutic applications, but their effectiveness when taken orally leads to poor patient compliance, requiring clinical administration. Among the alternative routes, transmucosal delivery has the advantage of being noninvasive and bypassing hepato-gastrointestinal clearance. Various mucosal routes-buccal, nasal, pulmonary, rectal, and vaginal-have been explored for delivering these macromolecules. Nanofibers, due to their unique properties like high surface-area-to-volume ratio, mechanical strength, and improved encapsulation efficiency, serve as promising carriers for proteins and peptides...

Expanding the genetic alphabet enhances DNA recombinant technologies by introducing unnatural base pairs (UBPs) beyond the standard A-T and G-C pairs, leading to biomaterials with novel and increased functionalities. Recent developments include UBPs that effectively function as a third base pair in replication, transcription, and/or translation processes. One such UBP, Ds-Px, demonstrates extremely high specificity in replication. Chemically synthesized DNA fragments containing Ds bases are amplified by PCR with the 5'-triphosphates of Ds and Px deoxyribonucleosides (dDsTP and dPxTP)...

Alpha amylase belonging to starch hydrolyzing enzymes has significant contributions to different industrial processes. The enzyme production through recombinant DNA technology faces certain challenges related to their expression, solubility and purification, which can be overcome through fusion tags. This study explored the influence of SUMO, a protein tag reported to enhance the solubility and stability of target proteins when fused to the N-terminal of the catalytic domain of amylase from Pyrococcus abyssi (PaAD)...

Abies koreana E.H.Wilson is an endangered evergreen coniferous tree that is native to high altitudes in South Korea and susceptible to the effects of climate change. Hybridization and reticulate evolution have been reported in the genus; therefore, multigene datasets from nuclear and cytoplasmic genomes are needed to better understand its evolutionary history. Using the Illumina NovaSeq 6000 and Oxford Nanopore Technologies (ONT) PromethION platforms, we generated complete mitochondrial (1,174,803 bp) and plastid (121,341 bp) genomes from A...

BACKGROUND: SARS-CoV-2, a highly dynamic beta-coronavirus, can afflict all age groups. Notably, over 16100 mortalities have been recorded among children as yet. In this regard, many vaccine projects are operational to assess immuno-potency among young cohorts. A bulk of reports have evidenced the efficacy of these immunization technologies in the elderly population, though the impact is yet to be determined among children. OBJECTIVES: This review is envisioned to outline the current efficacy of contributing vaccine technologies and examine the dose-dependent impact of immunization regimens in lowering the risks of SARS-CoV-2 infections among children and adolescents...

Monoclonal antibody-based therapeutics have achieved remarkable success in treating a wide range of human diseases. However, conventional systemic delivery methods have limitations in insufficient target tissue permeability, high costs, repeated administrations, etc. Novel technologies have been developed to address these limitations and further enhance antibody therapy. Local antibody delivery via respiratory tract, gastrointestinal tract, eye and blood-brain barrier have shown promising results in increasing local concentrations and overcoming barriers...

Investigating the function of target proteins for functional prospection or therapeutic applications typically requires the production and purification of recombinant proteins. The fusion of these proteins with tag peptides and fluorescently derived proteins allows the monitoring of candidate proteins using SDS-PAGE coupled with western blotting and fluorescent microscopy, respectively. However, protein engineering poses a significant challenge for many researchers. In this protocol, we describe step-by-step the engineering of a recombinant protein with various tags: TAT-HA (trans-activator of transduction-hemagglutinin), 6×His and EGFP (enhanced green fluorescent protein) or mCherry...

BACKGROUND: Follitropin delta is a novel recombinant follicle stimulating hormone preparation uniquely expressed in a human fetal retinal cell line by recombinant DNA technology. To date, no systematic review was available about the safety and the efficacy of the follitropin delta. The objective of this study was systematically reviewing the available literature and to provide updated evidence regarding the efficacy-safety profile of follitropin delta when compared to other gonadotropin formulations for ovarian stimulation in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles...

BACKGROUND: One of the leading current trends in technology is the miniaturization of devices to the microscale and nanoscale. The highly advanced approaches are based on biological systems, subjected to bioengineering using chemical, enzymatic and recombinant methods. Here we have utilised the biological affinity towards cellulose of the cellulose binding domain (CBD) fused with recombinant proteins. RESULTS: Here we focused on fusions with 'artificial', concatemeric proteins with preprogrammed functions, constructed using DNA FACE™ technology...

E. coli plays a substantial role in recombinant protein production. Its importance increased with the discovery of recombinant DNA technology and the subsequent production of the first recombinant insulin in E. coli. E. coli is a widely used and cost-effective host to produce recombinant proteins. It is also noteworthy that a significant portion of the approved therapeutic proteins have been produced in this organism. Despite these advantages, it has some disadvantages, such as toxicity and lack of eukaryotic post-translational modifications that can lead to the production of misfolded, insoluble, or dysfunctional proteins...

Inorganic biomaterials are used in various orthopedic and dental implants. Nevertheless, they cause clinical issues such as loosening of implants and patient morbidity. Therefore, inspired by mussel adhesive proteins, we aimed to design an adhesive and dimer-forming highly active bone morphogenetic protein-2 (BMP-2) using bioorthogonal chemistry, in which recombinant DNA technology was combined with enzymatic modifications, to achieve long-term osseointegration with titanium. The prepared BMP-2 exhibited substantially higher binding activity than wild-type BMP-2, while the adhered BMP-2 was more active than soluble BMP-2...

Recombinant adeno-associated viral vectors (rAAVs) are one of the most used vehicles for gene therapy, with five rAAV therapeutics commercially approved by the FDA. To improve product yield, we optimized the suspension production process of rAAV8 vectors carrying a proprietary transgene using a commercially available transfection reagent, FectoVIR-AAV. Using a miniaturized automated 250 mL scale bioreactor system, we generated models of vector genome (vg) titer, capsid (cp) titer, and Vg:Cp percentage from two multivariate design of experiment studies, one centered around bioreactor operating parameters, and another based on the transfection conditions...

SARS-CoV-2, the causative agent of COVID-19, has imposed a major public health threat, which needs effective therapeutics and vaccination strategies. Several potential candidate vaccines being rapidly developed are in clinical evaluation and recombinant vaccine has gained much attention thanks to its potential for greater response predictability, improved efficacy, rapid development and reduced side effects. Recombinant vaccines are designed and manufactured using bacterial, yeast cells or mammalian cells. A small piece of DNA is taken from the virus or bacterium against which we want to protect and inserted into the manufacturing cells...

Gene synthesis efficiency has greatly improved in recent years but is limited when it comes to repetitive sequences, which results in synthesis failure or delays by DNA synthesis vendors. This represents a major obstacle for the development of synthetic biology since repetitive elements are increasingly being used in the design of genetic circuits and design of biomolecular nanostructures. Here, we describe a method for the assembly of small synthetic genes with repetitive elements: First, a gene of interest is split in silico into small synthons of up to 80 base pairs flanked by Golden-Gate-compatible overhangs...

Bronchopulmonary dysplasia (BPD) is a serious chronic lung disease affecting extremely preterm infants. While mitochondrial dysfunction has been investigated in various medical conditions, limited research has explored mitochondrial DNA (mtDNA) gene mutations, specifically in BPD. This study aimed to evaluate mitochondrial mtDNA gene mutations in extremely preterm infants with BPD. In this prospective observational study, we enrolled a cohort of extremely preterm infants diagnosed with BPD. Clinical data were collected to provide comprehensive patient profiles...

An integrated and projected-based laboratory course was described, integrating interconnected knowledge points and biochemistry and molecular biology techniques on a research project-based system. The program, which served as an essential extension of theoretical courses to practice, was conducted with a sophomore of basic medical science who had completed the course in medical biochemistry and molecular biology. This course engaged students in learning "genetic manipulation" and "recombinant DNA technology" to understand the target gene's role in disease mechanics, thus altering evaluation and treatment for clinical disease...

The neuroscience and neurobiology of gene editing to enhance learning and memory is of paramount interest to the scientific community. The advancements of CRISPR system have created avenues to treat neurological disorders by means of versatile modalities varying from expression to suppression of genes and proteins. Neurodegenerative disorders have also been attributed to non-canonical DNA secondary structures by affecting neuron activity through controlling gene expression, nucleosome shape, transcription, translation, replication, and recombination...