Technical development for production of gene-modified laboratory rats

Area of Science:

• * Reproductive biology and genetic engineering.
• * Development of animal models for biomedical research.

Background:

• * Transgenic rats are crucial models for human diseases, organ transplantation, and protein production.
• * Conventional methods for creating transgenic rats involve pronuclear microinjection of exogenous DNA.
• * Improvements in survival rates have been observed with micro-vibration techniques.

Purpose of the Study:

• * To evaluate and enhance the efficiency of producing transgenic rats.
• * To explore the application of Intracytoplasmic Sperm Injection (ICSI) for creating transgenic rat models.
• * To identify optimal conditions for DNA integration and production efficiency.

Main Methods:

• * Comparison of conventional pronuclear microinjection with ICSI for creating transgenic rats.
• * Optimization of exogenous DNA concentration for both methods (5 µg/ml for microinjection, 0.1–0.5 µg/ml for ICSI).
• * Investigated sperm head membrane disruption techniques (detergent, ultrasound) to improve DNA uptake during ICSI.

Main Results:

• * Exogenous DNA concentration of 5 µg/ml was found to be optimal for conventional microinjection.
• * ICSI technique successfully produced transgenic rats, with optimal DNA concentration between 0.1–0.5 µg/ml.
• * Disrupting sperm head membranes enhanced production efficiency in the ICSI-transgenic (ICSI-tg) method.

Conclusions:

• * ICSI presents a more efficient alternative for producing transgenic rats compared to conventional pronuclear microinjection.
• * Further advancements in establishing rat embryonic stem cell lines and male germline stem cell lines, alongside somatic cell nuclear transfer, are required for modifying endogenous genes.