Cloning and in vitro function analysis of codon-optimized FatI gene

Area of Science:

• Biotechnology
• Genetics
• Nutritional Science

Background:

• n-3 polyunsaturated fatty acids (n-3 PUFAs) are essential nutrients not synthesized by mammals.
• Dietary supplementation is the primary source of n-3 PUFAs for humans and animals.
• The fatty acid dehydrogenase I (FatI) gene can convert n-6 PUFAs to n-3 PUFAs.

Purpose of the Study:

• To develop a method for endogenous synthesis of n-3 PUFAs in mammals.
• To investigate the efficacy of the FatI gene in Boer goat cells.
• To establish a foundation for producing transgenic animals rich in n-3 PUFAs.

Main Methods:

• Codon-optimized FatI gene synthesis and construction of a eukaryotic expression vector.
• Transfection of Boer goat fetal fibroblasts using electroporation and selection of stable cell lines.
• Verification of gene integration (PCR, Southern blot) and expression (RT-PCR), followed by fatty acid profile analysis.

Main Results:

• Successful integration and expression of the FatI gene in Boer goat fibroblasts.
• Significant increase in total n-3 PUFAs (0.61% to 0.95%) and decrease in n-6 PUFAs (10.34% to 9.85%).
• Remarkable 63.49% increase in the n-3:n-6 PUFA ratio, indicating FatI gene activity.

Conclusions:

• The FatI gene can be effectively introduced into mammalian cells to enhance n-3 PUFA synthesis.
• This study provides a viable strategy for creating transgenic animals capable of producing n-3 PUFAs.
• The findings support the potential for improving human nutrition and wellness through animal-derived n-3 PUFAs.