Biological Procedures Online最新文献

Gene delivery is a complex process with several challenges when attempting to incorporate genetic material efficiently and safely into target cells. Some of the key challenges include not only efficient cellular uptake and endosomal escape to ensure that the genetic material can exert its effect but also minimizing the toxicity of the delivery system, which is vital for safe gene delivery. Of importance, if gene delivery systems are intended for biomedical applications or clinical use, they must be scalable and easy and affordable to manufacture to meet the demand. Here, we show an efficient gene delivery method using a combination of carbon dots coated by PEI through electrostatic binding to easily generate cationic carbon dots. We show a biofunctional approach to generate optimal cationic carbon dots (CCDs) that can be scaled up to meet specific transfection demands. CCDs improve cell viability and increase transfection efficiency four times over the standard of PEI polyplexes. Generated CCDs enabled the challenging transfection protocol to produce retroviral vectors via cell cotransfection of three different plasmids into packing cells, showing not only high efficiency but also functionality of the gene delivery, tested as the capacity to produce infective retroviral particles.

Background: Generating targeted mutant mice is a crucial technology in biomedical research. This study focuses on optimizing the CRISPR/Cas9 system uptake into sperm cells using the methyl β-cyclodextrin-sperm-mediated gene transfer (MBCD-SMGT) technique to generate targeted mutant blastocysts and mice efficiently. Additionally, the present study elucidates the roles of cholesterol and reactive oxygen species (ROS) in the exogenous DNA uptake by sperm.

Results: In this study, B6D2F1 mouse sperm were incubated in the c-TYH medium with different concentrations of MBCD (0, 0.75, 1, and 2 mM) in the presence of 20 ng/µl pCAG-eCas9-GFP-U6-gRNA (pgRNA-Cas9) for 30 min. Functional parameters, extracellular ROS, and the copy numbers of internalized plasmid per sperm cell were evaluated. Subsequently, in vitro fertilization (IVF) was performed and fertilization rate, early embryonic development, and transfection rate were assessed. Finally, our study investigated the potential of the MBCD-SMGT technique in combination with the CRISPR-Cas9 system, referred to as MBCD-SMGE (MBCD-sperm-mediated gene editing), for generating targeted mutant blastocysts and mice. Results indicated that cholesterol removal from the sperm membrane using MBCD resulted in a premature acrosomal reaction, an increase in extracellular ROS levels, and a dose-dependent influence on the copy numbers of the internalized plasmids per sperm cell. Moreover, the MBCD-SMGT technique led to a larger population of transfected motile sperm and a higher production rate of GFP-positive blastocysts. Additionally, the current study validated the targeted indel in blastocyst and mouse derived from MBCD-SMGE technique.

Conclusion: Overall, this study highlights the significant potential of the MBCD-SMGE technique for generating targeted mutant mice. It holds enormous promise for modeling human diseases and improving desirable traits in animals.

Metal-organic frameworks (MOFs) are porous materials with unique characteristics that make them well-suited for drug delivery and gene therapy applications. Among the MOFs, zeolitic imidazolate framework-8 (ZIF-8) has emerged as a promising candidate for delivering exogenous DNA into cells. However, the potential of ZIF-8 as a vector for sperm-mediated gene transfer (SMGT) has not yet been thoroughly explored.This investigation aimed to explore the potential of ZIF-8 as a vector for enhancing genetic transfer and transgenesis rates by delivering exogenous DNA into sperm cells. To test this hypothesis, we employed ZIF-8 to deliver a plasmid expressing green fluorescent protein (GFP) into mouse sperm cells and evaluated the efficiency of DNA uptake. Our findings demonstrate that ZIF-8 can efficiently load and deliver exogenous DNA into mouse sperm cells, increasing GFP expression in vitro. These results suggest that ZIF-8 is a valuable tool for enhancing genetic transfer in SMGT, with important implications for developing genetically modified animals for research and commercial purposes. Additionally, our study highlights the potential of ZIF-8 as a novel class of vectors for gene delivery in reproductive biology.Overall, our study provides a foundation for further research into using ZIF-8 and other MOFs as gene delivery systems in reproductive biology and underscores the potential of these materials as promising vectors for gene therapy and drug delivery.

Background: Some of the most complex surgical interventions to treat trauma and cancer include the use of locoregional pedicled and free autologous tissue transfer flaps. While the techniques used for these reconstructive surgery procedures have improved over time, flap complications and even failure remain a significant clinical challenge. Animal models are useful in studying the pathophysiology of ischemic flaps, but when repeatability is a primary focus of a study, conventional in-vivo designs, where one randomized subset of animals serves as a treatment group while a second subset serves as a control, are at a disadvantage instigated by greater subject-to-subject variability. Our goal was to provide a step-by-step methodological protocol for creating an alternative standardized, more economical, and transferable pre-clinical animal research model of excisional full-thickness wound healing following a simulated autologous tissue transfer which includes the primary ischemia, reperfusion, and secondary ischemia events with the latter mimicking flap salvage procedure.

Results: Unlike in the most frequently used classical unilateral McFarlane's caudally based dorsal random pattern skin flap model, in the herein described bilateral epigastric fasciocutaneous advancement flap (BEFAF) model, one flap heals under normal and a contralateral flap-under perturbed conditions or both flaps heal under conditions that vary by one within-subjects factor. We discuss the advantages and limitations of the proposed experimental approach and, as a part of model validation, provide the examples of its use in laboratory rat (Rattus norvegicus) axial pattern flap healing studies.

Conclusions: This technically challenging but feasible reconstructive surgery model eliminates inter-subject variability, while concomitantly minimizing the number of animals needed to achieve adequate statistical power. BEFAFs may be used to investigate the spatiotemporal cellular and molecular responses to complex tissue injury, interventions simulating clinically relevant flap complications (e.g., vascular thrombosis) as well as prophylactic, therapeutic or surgical treatment (e.g., flap delay) strategies in the presence or absence of confounding risk factors (e.g., substance abuse, irradiation, diabetes) or favorable wound-healing promoting activities (e.g., exercise). Detailed visual instructions in BEFAF protocol may serve as an aid for teaching medical or academic researchers basic vascular microsurgery techniques that focus on precision, tremor management and magnification.

Background: Gastric cancer (GC) is a common malignancy and a leading cause of cancer-related death with high morbidity and mortality. Methyl-CpG binding domain protein 3 (MBD3), a key epigenetic regulator, is abnormally expressed in several cancers, participating in progression and metastasis. However, the role of MBD3 in GC remains unknown.

Methods: MBD3 expression was assessed via public databases and validated by western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). The prognosis of MBD3 was analysed via bioinformatics based on the TCGA dataset. The migration, invasion and proliferation of GC cells were examined by transwell, wound healing, cell counting kit (CCK)-8, colony-formation and xenograft mouse models. Epithelial-mesenchymal transition (EMT) and phosphatidylinositide 3-kinases/ protein Kinase B (PI3K/AKT) pathway markers were evaluated by Western blotting. RNA sequencing was used to identify the target of MBD3.

Results: MBD3 expression was higher in GC tissues and cells than in normal tissues and cells. Additionally, high MBD3 levels were associated with poor prognosis in GC patients. Subsequently, we proved that MBD3 enhanced the migration, invasion and proliferation abilities of GC cells. Moreover, western blot results showed that MBD3 promoted EMT and activated the PI3K/AKT pathway. RNA sequencing analysis showed that MBD3 may increase actin γ1 (ACTG1) expression to promote migration and proliferation in GC cells.

Conclusion: MBD3 promoted migration, invasion, proliferation and EMT by upregulating ACTG1 via PI3K/AKT signaling activation in GC cells and may be a potential diagnostic and prognostic target.