Efficient generation of sFat-1 transgenic rabbits rich in n-3 polyunsaturated fatty acids by intracytoplasmic sperm injection

Abstract

N-3 polyunsaturated fatty acids (n-3 PUFAs) have their first double bond at the third carbon from the methyl end of the fatty-acid chain and had been proven to be beneficial to human health. However, mammals cannot produce n-3 PUFAs by themselves because they lack the n-3 fatty-acid desaturase (Fat-1) gene. Thus, the possibility of producing sFat-1 transgenic rabbits was explored in this study. The transgenic cassette of pPGK1–sFat-1–CMV–EGFP was constructed and transgenic rabbit embryos were produced by intracytoplasmic sperm injection (ICSI). When 123 EGFP-positive embryos at the 2–8-cell stage were transplanted into the oviduct of four oestrous-synchronised recipients, two of them became pregnant and gave birth to seven pups. However, transfer of embryos into the uterus of oestrous-synchronised recipients and oviduct or uterus of oocyte donor rabbits did not result in pregnancy. The integration of the sFat-1 gene was confirmed in six of the seven live pups by PCR and Southern blot. The expression of the sFat-1 gene in the six transgenic pups was also detected by reverse transcription polymerase chain reaction (RT-PCR). Gas chromatography–mass spectrometry analysis revealed that transgenic rabbits exhibited an ~15-fold decrease in the ratio of n-6 : n-3 PUFAs in muscle compared with wild-type rabbits and non-transgenic rabbits. These results demonstrate that sFat-1 transgenic rabbits can be produced by ICSI and display a low ratio of n-6 : n-3 PUFAs.