Introduction

Gene therapy involves transferring genetic material into target cells to correct mutations or introduce new biological functions. Among delivery systems, lentiviral vectors are considered efficient and reliable tools due to their ability to integrate stably into the host genome and transduce both dividing and non-dividing cells. This property provides long-term gene expression, which is highly valuable for therapeutic and experimental applications.

The PDX1 (Pancreatic and Duodenal Homeobox 1) gene plays a central role in pancreatic organogenesis and regulation of insulin-producing beta cells. It acts as a transcription factor controlling genes critical for endocrine differentiation and insulin secretion. Chick embryos are a useful experimental model due to their accessibility, rapid development, and the responsiveness of their fibroblast and germ cells to gene transfer systems. These features make them suitable for studying gene delivery efficiency and expression stability.

Aim

The aim of this study was to construct and produce recombinant lentiviral vectors carrying the PDX1 gene in HEK293T-LentiX cells and evaluate their transfer efficiency in chick embryonic fibroblast and germ cells. This work was conducted to assess the potential of lentiviral systems for stable gene delivery in avian cells.

Materials and Methods

HEK293T-LentiX cells were selected as producer cells due to their high transfection efficiency and viral packaging capability. They were cultured in DMEM supplemented with 10% fetal bovine serum, penicillin, and streptomycin under standard incubation conditions (37°C, 5% CO₂).

Lentiviral particles were generated using a three-plasmid packaging system, including a transfer vector containing the PDX1 gene and two helper plasmids. Transfection was carried out using the calcium phosphate method. After 48–72 hours, the viral-containing supernatant was collected, filtered, and concentrated.

Viral titers were determined by evaluating GFP expression in target cells through fluorescence microscopy and flow cytometry. Chick embryonic fibroblast and primordial germ cells were isolated and infected with various viral concentrations in the presence of 8 µg/mL polybrene to enhance infection. After incubation, cells were examined for GFP signal as evidence of successful gene transfer.

Results

High-titer recombinant lentiviral particles were successfully produced in HEK293T cells. Fluorescence microscopy revealed strong GFP expression, confirming the presence of functional viral particles. Flow cytometry analysis provided quantitative confirmation of high viral titers.

Following transduction, chick embryonic fibroblast and germ cells exhibited clear GFP expression, indicating efficient infection and gene transfer. The PDX1 gene was successfully delivered and expressed within target cells. Although transduction efficiency varied slightly between cell types, the overall results demonstrated that the lentiviral system provided stable and effective gene delivery to chick embryo-derived cells.

Discussion

The study confirmed that lentiviral vectors carrying the PDX1 gene could be efficiently produced and used to achieve stable gene transfer in chick embryonic cells. This system’s ability to integrate permanently into the host genome ensures consistent gene expression over time without repeated transfection. For functional genes like PDX1, this stability is crucial for maintaining insulin-related pathways and pancreatic cell differentiation.

Chick embryos serve as an advantageous model because their cells are easily accessible, grow rapidly, and respond well to viral vectors. Such characteristics make them ideal for investigating genetic regulation during early development.

Evaluation of viral titers using fluorescence microscopy and flow cytometry provided reliable data confirming efficient vector production. The integration of new tools such as CRISPR/Cas9 can further enhance lentiviral design precision, allowing targeted modification of specific genes. Combining these technologies may open promising avenues for studying metabolic disorders and for gene-based therapies.

Conclusion

Recombinant lentiviral vectors carrying the PDX1 gene were successfully generated and used to transduce chick embryonic fibroblast and germ cells. The system exhibited high production efficiency, stable gene expression, and suitability for in vitro studies. These findings demonstrate that lentiviral vectors represent a powerful and versatile platform for gene transfer and experimental modeling in avian systems. Moreover, coupling lentiviral vectors with genome editing technologies could expand future applications in regenerative medicine and genetic engineering.