Small animal models, particularly rodents, have long been instrumental in biomedical research, providing valuable insights into human disease pathophysiology and treatment development. The ability to manipulate gene expression in these models through transfection has significantly advanced studies in functional genomics, disease modeling, and therapeutic interventions. Transfection, the process of introducing exogenous nucleic acids into cells, serves as a non-viral alternative to viral-mediated gene delivery, offering a safer and more versatile approach to genetic modification.
The application of transfection in small animals extends beyond basic research to translational medicine, including drug discovery, vaccine development, and regenerative therapies. However, achieving efficient and reproducible transfection in vivo presents unique challenges, including low delivery efficiency, immune activation, and variable transgene expression. Advances in delivery technologies, including lipid-based nanoparticles, electroporation, hydrodynamic injection, and magnetofection, have improved the success of transfection strategies in small animal models. This review provides an overview of the major transfection methods employed in small animal research, their applications in various biomedical fields, and future perspectives for enhancing in vivo gene delivery.
Transfection Methods in Small Animal Research
Chemical Transfection
Chemical transfection remains one of the most widely used non-viral methods for delivering nucleic acids into small animal models. Cationic lipid-based transfection reagents, such as liposomes and lipid nanoparticles, enable efficient delivery of DNA, mRNA, and siRNA by facilitating endocytosis and intracellular release. These formulations have been optimized for in vivo applications to improve stability, tissue penetration, and biocompatibility. Polymeric transfection agents, including polyethyleneimine (PEI) and dendrimers, have also been developed for enhanced nucleic acid condensation and efficient delivery to target tissues. Despite their advantages, chemical transfection methods often require repeated administrations due to transient gene expression and are limited by variable uptake and biodistribution in vivo.
Physical Transfection Techniques
Physical methods of transfection, such as electroporation, hydrodynamic injection, and biolistic particle bombardment, have been increasingly utilized for gene delivery in small animal models. Electroporation applies controlled electrical pulses to create transient pores in the cell membrane, allowing nucleic acids to enter cells efficiently. This technique has been particularly effective for in vivo applications in muscle and liver tissues, where it facilitates high gene transfer efficiency with minimal systemic effects. Hydrodynamic injection, a technique involving the rapid intravenous administration of DNA in large volumes, has been widely employed for gene delivery to the liver in rodent models. Biolistic transfection, also known as particle bombardment, employs high-velocity gold or tungsten particles coated with nucleic acids to penetrate cell membranes, enabling localized gene transfer in tissues such as skin and muscle. These physical techniques offer advantages in precision and reproducibility, but they often require specialized equipment and may induce tissue damage or inflammation.
siRNA Electroporation Buffer (30 ml)
Efficient gene silencing via RNA interference (RNAi) relies on the successful transfection of small interfering RNA (siRNA), which binds to the RNA-induced silencing complex (RISC) to degrade target mRNA molecules. However, many conventional DNA transfection reagents are incompatible with siRNA due to serum sensitivity, degradation, or cytotoxicity, which can compromise gene expression studies. To overcome these challenges, specialized siRNA transfection reagents have been developed to enhance cellular uptake, protect siRNA from enzymatic degradation, and facilitate efficient gene knockdown without reducing cell viability. These optimized formulations allow transfection in the presence of serum and eliminate the need for extensive siRNA optimization, streamlining RNAi experiments. Additionally, these transfection reagents are compatible with negatively charged biomolecules, including DNA, RNA, and proteins, broadening their applications in gene silencing, functional genomics, and therapeutic research… Continue Reading
Viral-Mediated Transfection (Transduction)
Viral vectors, including adenoviruses, lentiviruses, and adeno-associated viruses (AAVs), have been extensively used for stable gene expression in small animal research. AAV-based gene transfer has gained prominence due to its ability to achieve long-term transgene expression with low immunogenicity, making it a preferred choice for neurological and systemic gene therapy applications. Lentiviral vectors, with their ability to integrate transgenes into the host genome, provide sustained gene expression and are commonly used in disease modeling and regenerative medicine. However, viral transfection methods present challenges, including potential insertional mutagenesis, immunogenic responses, and the need for biosafety containment, which limit their widespread application compared to non-viral approaches.
Applications of Transfection in Small Animal Research
Neuroscience and Neurodegenerative Disease Modeling
Transfection has been instrumental in advancing neurobiology research by enabling precise genetic modifications in small animal models. In vivo transfection of neural tissues using lipid nanoparticles and AAV vectors has facilitated the study of neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. The introduction of CRISPR-Cas9 components via transfection has allowed targeted gene editing in neural circuits, enhancing our understanding of synaptic plasticity, memory formation, and neuroprotection strategies. Transfection technologies such as In Vivo BrainFect have been optimized for stereotaxic delivery into specific brain regions, enabling localized gene expression while minimizing off-target effects.
Cancer Research and Gene Therapy
Transfection-based approaches have been widely applied in oncology research, particularly for gene silencing and gene therapy strategies. RNA interference (RNAi)-mediated transfection allows for targeted knockdown of oncogenes, providing insights into tumor progression and resistance mechanisms. CRISPR-based transfection enables precise gene editing to create genetically modified cancer models in rodents, facilitating drug screening and immunotherapy development. In vivo transfection using lipid-based nanoparticles has also been explored for mRNA-based cancer vaccines, offering promising advances in personalized medicine.
Immunology and Infectious Disease Research
The role of transfection in immunology research has expanded with the development of RNA-based vaccines and gene-modified immune cell therapies. Lipid nanoparticle-mediated mRNA transfection has been pivotal in vaccine development, as demonstrated by the success of mRNA COVID-19 vaccines. In small animal models, transfection is utilized to study immune responses, cytokine signaling, and host-pathogen interactions. Electroporation-based transfection has also been employed to enhance the uptake of DNA vaccines, improving their immunogenicity and efficacy.
Regenerative Medicine and Stem Cell Research
Transfection plays a critical role in regenerative medicine by enabling genetic modification of stem cells and progenitor cells for tissue repair and disease modeling. Transfection of induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) using non-viral delivery systems has facilitated advancements in cell-based therapies for cardiovascular, musculoskeletal, and neurodegenerative disorders. CRISPR-mediated genome editing through transfection further enhances the therapeutic potential of stem cells by allowing precise correction of genetic mutations.
Xenograft Animal Service: Immunocompromised NOD/SCID Mice
Xenografting is a preclinical technique in which tumor tissue from one species is transplanted into another, enabling the study of tumor biology and therapeutic response in a physiologically relevant in vivo system. The use of immunocompromised NOD/SCID mice, which lack functional T, B, and NK cells, allows for the establishment of patient-derived and cell line-derived xenografts without immune rejection, providing a more accurate model of human tumor progression and drug efficacy than in vitro studies. These models are essential for evaluating tumor growth kinetics, metastasis, drug sensitivity, and resistance mechanisms. Altogen Labs offers comprehensive xenograft services, including tumor implantation, pharmacokinetic and pharmacodynamic analysis, histopathology, and biomarker validation, supporting translational oncology research and the development of novel cancer therapies… Continue Reading
Challenges and Future Directions
Despite significant advancements, in vivo transfection in small animal models presents several challenges. Delivery efficiency, tissue specificity, and immune responses remain major obstacles, particularly for systemic gene transfer. Non-viral transfection methods, while safer, often exhibit lower transfection efficiency compared to viral vectors. Conversely, viral-based approaches require careful risk assessment due to their potential immunogenicity and genomic integration.
Recent innovations in nanotechnology, microfluidics, and synthetic biology are driving improvements in transfection methods. The development of targeted lipid nanoparticles, polymeric carriers, and biodegradable scaffolds has enhanced the specificity and biocompatibility of non-viral transfection systems. The integration of transfection with single-cell RNA sequencing and AI-driven gene delivery optimization is expected to further refine transfection strategies for small animal research.
Conclusion
Transfection has become a vital tool in small animal research, enabling precise genetic modifications for disease modeling, therapeutic discovery, and regenerative medicine. Advances in chemical, physical, and viral-mediated transfection techniques have expanded the scope of in vivo gene delivery, improving efficiency and specificity. While challenges remain, continued innovations in nanotechnology and genome editing are poised to enhance transfection-based applications, bridging the gap between basic research and translational medicine.
What are xenograft models?
The term xenograft refers to a transplant of an organ or tissue from a donor that comes from a different species than the recipient. It is possible to engraft human and other species’ cells and tissues into immune-deficient models, especially from cancer cells.
A wide range of medically relevant research has been conducted using the laboratory rat, one of the most extensively studied mammals. The popularity of this species is attributed to such characteristics as size, fecundity, behavior, ease of surgical techniques, tissue sampling, and general laboratory management.
therapeutic development… Continue Reading
Successful examples of xenotransplantation:
Non-human heart to a human:
In 1984, Baby Fae, an American infant with hypoplastic left heart syndrome, became the first infant to receive a baboon heart transplant. Loma Linda University Medical Center in Loma Linda, California, performed the procedure under the direction of Leonard Lee Bailey. He died 21 days later owing to a humoral-based graft rejection mainly caused by ABO blood type mismatch, considered unavoidable due to the rarity of type O baboons.
It was intended to be a temporary graft, but a suitable allograft replacement was not found in time. Despite not advancing the field of Xenotransplantation, the procedure revealed the lack of organs for infants. There was a dramatic improvement in the crisis of infant organ shortages due to the story.
A genetically modified pig’s heart was transplanted in January 2022 to a terminally ill patient, David Bennett Sr., who was ineligible for a standard human heart transplant. The pig was edited to remove enzymes that produce the sugar antigens associated with human hyperacute organ rejection. Using compassionate use criteria, the US medical regulator approved the procedure. After two months of transplantation, the recipient died.
Skin xenotransplantation:
Animal and human skin grafts became relatively popular in the 19th century. A pedicle skin graft or a free skin graft was used for the xenograft. Various types of skin grafts were used for the graft, including pedicle and free skin grafts.
It was difficult to perform pedicle grafts because the donor, for example, a sheep, had to be strapped immobile to the recipient for several days until it became vascularized. The graft could become disconnected from the donor if this occurs. Although some “successes” were reported, it is almost certain that none of these grafts were successful.
Although many species used as donors had hair, feathers, or fur growing from the skin, the surgeons did not appear concerned. The trend was to use animals with no hair, feathers, or fur. In some cases, grafts made from frogs, sometimes “skinned alive,” may have proved successful by protecting for several days while the wound healed. Most likely, none of the grafts were permanent.
Altogen Labs offers extensive preclinical research services utilizing over 90 in-house validated xenograft models, including a diverse range of tumor types for oncology research and drug development. These models encompass brain carcinoma, pancreatic cancer, breast cancer, epidermoid carcinoma, and nasopharyngeal carcinoma, along with xenograft studies for melanoma, colorectal, lung, prostate, and additional solid tumors. The laboratory provides both patient-derived xenograft (PDX) models, which preserve the genetic heterogeneity of patient tumors for personalized medicine research, and cell line-derived xenograft (CDX) models, which offer reproducible tumor growth kinetics and molecular characterization in immune-compromised mouse and rat models. These xenograft platforms enable the evaluation of tumor progression, therapeutic efficacy, drug resistance mechanisms, and biomarker discovery under controlled experimental conditions. Through rigorous validation and optimization, Altogen Labs supports translational oncology studies by providing high-fidelity in vivo models that closely mimic human tumor pathophysiology, advancing the development of targeted therapies and immuno-oncology approaches… Continue Reading
Xenotransplantation and Ethics
Researchers studying Xenotransplantation at the beginning of the 20th century viewed animals as a “natural” alternative to allografts, and few questioned their morality. Although some plays mocked Xenografters like Serge Voronoff and some images showed emotionally distraught primates – whose testicles Voronoff had removed – no genuine attempts had been made to question the science. The first half of the 20th century saw little interest in Xenotransplantation, at least in France.
In 1984, animal rights activists began protesting after the Baby Fae incident, gaining media attention and demonstrating that some people believed it was unethical to use an animal’s organs to prolong a sick human’s life. By treating animals like mere tools for slaughter on demand, humans would create a world they do not want.
Many supporters of the transplant argued that saving a human life justified the sacrifice of an animal. Animal rights activists consider Primate organs more reprehensible than pigs’ organs. Several primates exhibit stronger social structures, communication skills, and affection than mentally deficient humans and human infants, according to Peter Singer et al. Despite this, Xenotransplantation is unlikely to be prohibited by regulators due to animal suffering.
Read more: Mouse transfection