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Site-Specific Fat-1 Knock-In Enables Significant Decrease of n-6PUFAs/n-3PUFAs Ratio in Pigs.

Mengjing Li1, Hongsheng Ouyang1, Hongming Yuan1

  • 1Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun, Jilin Province, People's Republic of China.

G3 (Bethesda, Md.)
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Summary

Researchers created marker-free transgenic pigs using the CRISPR/Cas9 system to insert the fat-1 gene. These pigs efficiently convert n-6 polyunsaturated fatty acids to n-3 polyunsaturated fatty acids, improving pork

Keywords:
fat-1genome editingomega-3 polyunsaturated fatty acidspig

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Area of Science:

  • Genetics and Molecular Biology
  • Animal Science
  • Biotechnology

Background:

  • The fat-1 gene from Caenorhabditis elegans encodes a desaturase enzyme.
  • This enzyme converts n-6 polyunsaturated fatty acids (n-6PUFAs) to n-3 polyunsaturated fatty acids (n-3PUFAs).
  • Previous fat-1 transgenic animals used random integration, leading to variable expression and biosafety concerns.

Purpose of the Study:

  • To generate marker-free, site-specific fat-1 transgenic pigs.
  • To utilize the safe and ubiquitous Rosa26 locus for transgene insertion.
  • To assess the efficacy of fat-1 gene expression and its impact on fatty acid profiles in pigs.

Main Methods:

  • Site-specific insertion of the fat-1 gene into the porcine Rosa 26 (pRosa26) locus using CRISPR/Cas9.
  • Southern blot analysis to confirm single-copy gene integration.
  • Gas chromatography to analyze fatty acid composition in tissues.

Main Results:

  • Successfully generated pigs with a single copy of the fat-1 gene integrated at the pRosa26 locus.
  • Demonstrated satisfactory fat-1 gene expression across various tissues in F1 generation pigs.
  • Significantly increased n-3PUFAs and decreased the n-6PUFAs/n-3PUFAs ratio from 9.36 to 2.12.

Conclusions:

  • Site-specific knock-in of the fat-1 gene in pigs is feasible and efficient.
  • These transgenic pigs possess improved fatty acid profiles with higher n-3PUFAs.
  • Fat-1 knock-in pigs offer potential for enhanced pork nutritional value and as models for n-3PUFA-related disease research.