Transfection is a pivotal technique in small animal research, enabling the introduction of exogenous nucleic acids into cells for gene expression modulation, functional genomics, and therapeutic applications. The ability to control gene expression in vivo has significantly advanced our understanding of disease mechanisms, drug responses, and regenerative processes. Various transfection strategies, including chemical, physical, and viral-mediated methods, facilitate gene expression in different tissues while presenting unique advantages and limitations. This review explores the mechanisms governing gene expression in transfected cells, highlights the most commonly used transfection techniques for small animal research, and discusses the challenges and innovations aimed at enhancing transfection efficiency, tissue specificity, and long-term gene expression.
Gene expression modulation through transfection has revolutionized biomedical research by enabling precise control over protein production, genetic modification, and cellular responses. In small animal models, transfection serves as a fundamental tool for investigating gene function, modeling human diseases, and testing therapeutic strategies. The success of gene expression studies depends on efficient delivery methods, stability of the transfected nucleic acids, and the ability of host cells to process and express the introduced genetic material.
Small animal models, particularly rodents, provide a physiologically relevant platform for in vivo transfection studies, allowing researchers to evaluate gene expression dynamics in living organisms. However, achieving robust and reproducible gene expression in transfected tissues remains challenging due to variable transfection efficiency, immune responses, and degradation of introduced nucleic acids. Advances in transfection technologies, including nanoparticle-based delivery, electroporation, and viral vectors, have improved the precision and efficacy of gene expression studies in small animals. This review discusses the principles of gene expression following transfection, the various delivery approaches employed in small animal research, and the emerging innovations addressing current limitations in the field.
Mechanisms of Gene Expression in Transfected Cells
Gene expression following transfection involves multiple cellular processes, from nucleic acid uptake to protein translation. The efficiency of gene expression depends on the stability, localization, and processing of transfected DNA or RNA within the cell.
Plasmid DNA Transfection and Transgene Expression
For plasmid DNA transfection, nucleic acids must enter the nucleus to be transcribed into mRNA before translation occurs in the cytoplasm. The efficiency of nuclear entry is a critical determinant of transgene expression, as only a fraction of transfected plasmids successfully reach the nucleus. Promoter selection plays a crucial role in transcriptional activity, with strong viral promoters (such as CMV or SV40) driving high expression levels in many cell types. However, these promoters may exhibit variable activity across tissues, necessitating the use of tissue-specific or inducible promoters to achieve controlled gene expression in small animal models.
mRNA Transfection and Direct Protein Translation
mRNA transfection bypasses the nuclear entry requirement, leading to more rapid and transient gene expression. Once delivered into the cytoplasm, transfected mRNA is immediately translated into protein, making this method highly suitable for short-term studies of gene function and protein expression. The stability of transfected mRNA depends on modifications such as the addition of 5’ caps and poly(A) tails, which enhance translation efficiency and reduce degradation by cellular exonucleases.
RNA Interference and Gene Silencing
RNA interference (RNAi)-based transfection enables post-transcriptional gene silencing through small interfering RNA (siRNA) or short hairpin RNA (shRNA). Following transfection, siRNA associates with the RNA-induced silencing complex (RISC), guiding the degradation of complementary mRNA molecules and effectively reducing gene expression. While siRNA-mediated silencing is transient, shRNA-expressing plasmids or viral vectors enable stable and long-term knockdown by integrating into the host genome.
Transfection Strategies for Gene Expression in Small Animals
Chemical Transfection Methods
Chemical transfection agents, including lipid-based and polymer-based carriers, facilitate the delivery of nucleic acids by forming complexes that protect genetic material and enhance cellular uptake. Lipid nanoparticles (LNPs) have emerged as a leading technology for in vivo gene delivery, particularly for mRNA-based vaccines and therapeutic applications. Polyethyleneimine (PEI)-based transfection is another widely used method that enhances plasmid DNA delivery by promoting endosomal escape and nuclear localization. While chemical transfection methods offer ease of use and scalability, their efficiency in vivo can be affected by tissue penetration barriers and serum stability.
Physical Transfection Methods
Electroporation is a highly effective physical transfection technique that applies electrical pulses to permeabilize cell membranes, facilitating nucleic acid uptake. In vivo electroporation has been successfully used in muscle, liver, and skin tissues to achieve transient or stable gene expression. Hydrodynamic injection, which involves the rapid administration of DNA in large fluid volumes, has been employed for gene delivery to the liver in rodent models. Biolistic transfection, or particle bombardment, utilizes high-velocity gold or tungsten particles coated with nucleic acids to introduce genes into target tissues, offering a localized gene delivery approach. While physical methods enhance transfection efficiency, their application is often limited by potential tissue damage and procedural complexity.
Viral-Mediated Gene Delivery
Viral vectors remain one of the most efficient means of achieving sustained gene expression in small animals. Adeno-associated virus (AAV) vectors are widely used due to their ability to achieve long-term expression with minimal immunogenicity. Lentiviral vectors, which integrate into the host genome, enable stable expression of transgenes for functional genomics and gene therapy research. However, concerns regarding immune responses, insertional mutagenesis, and biosafety regulations necessitate careful selection of viral vectors for specific applications.
Applications of Gene Expression Studies in Small Animal Research
Neuroscience and Neurodegenerative Disease Models
Transfection-based gene expression studies have significantly advanced neuroscience research by enabling targeted genetic modifications in small animal models. In vivo gene delivery to the brain via lipid nanoparticles, electroporation, or viral vectors has facilitated the study of neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. CRISPR-based transfection allows precise genome editing to investigate neural circuit function, synaptic plasticity, and neuroprotection strategies.
Cancer Research and Gene Therapy
Transfection-mediated gene expression studies are critical for oncology research, allowing the investigation of oncogene activation, tumor suppressor functions, and therapeutic interventions. Transient and stable transfection of cancer cells in xenograft models facilitates tumor profiling and drug screening. In vivo gene expression strategies, including CRISPR-mediated knockout studies and RNAi-based silencing, have provided insights into tumor progression and resistance mechanisms.
Immunology and Vaccine Development
Gene expression studies in immunology have driven advances in vaccine development and immune response characterization. In vivo transfection of dendritic cells with antigen-encoding mRNA has been used to enhance antigen presentation and immune activation. The development of mRNA-based vaccines, facilitated by optimized transfection technologies, has led to breakthroughs in infectious disease control.
Regenerative Medicine and Stem Cell Research
In regenerative medicine, transfection is used to reprogram stem cells and modulate gene expression in tissue engineering applications. The transfection of transcription factors into induced pluripotent stem cells (iPSCs) has enabled the generation of patient-specific cell lines for disease modeling and regenerative therapies. Gene expression studies in mesenchymal stem cells have provided insights into their differentiation potential and therapeutic efficacy.
Challenges and Future Perspectives
Despite the progress in transfection technologies, achieving high-efficiency gene expression in small animals remains challenging due to tissue penetration barriers, immune responses, and variability in transgene expression. Future advancements in non-viral gene delivery, including synthetic vectors, biodegradable nanoparticles, and microfluidic-assisted transfection, aim to enhance in vivo transfection efficiency and minimize off-target effects. The integration of transfection with high-throughput sequencing, CRISPR genome engineering, and artificial intelligence-driven gene delivery optimization is expected to further refine gene expression studies in small animal research.
Conclusion
Gene expression studies enabled by transfection have become essential in small animal research, providing critical insights into disease mechanisms, drug development, and therapeutic strategies. Advances in chemical, physical, and viral-mediated transfection techniques have expanded the scope of in vivo gene expression studies, improving efficiency, specificity, and reproducibility. Continued innovations in gene delivery technologies will further enhance transfection-based research, bridging the gap between experimental models and clinical applications.