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Abstract
Technology of somatic cell nuclear transfer (SCNT) has been adapted worldwide to generate transgenic animals, although the traditional procedure relies largely on instrumental micromanipulation. In this study, we used the modified handmade cloning (HMC) established in cattle and pig to produce transgenic sheep with elevated levels of omega-3 (n−3) fatty acids. Codon-optimized nematode mfat-1 was inserted into a eukaryotic expression vector and was transferred into the genome of primary ovine fibroblast cells from a male Chinese merino sheep. Reverse transcriptase PCR, gas chromatography, and chromosome analyses were performed to select nuclear donor cells capable of converting omega-6 (n−6) into n−3 fatty acids. Blastocysts developed after 7 days of in vitro culture were surgically transplanted into the uterus of female ovine recipients of a local sheep breed in Xinjiang. For the HMC, approximately 8.9% (n = 925) of reconstructed embryos developed to the blastocyst stage. Four recipients became pregnant after 53 blastocysts were transplanted into 29 naturally cycling females, and a total of 3 live transgenic lambs were produced. Detailed analyses on one of the transgenic lambs revealed a single integration of the modified nematode mfat-1 gene at sheep chromosome 5. The transgenic sheep expressed functional n−3 fatty acid desaturase, accompanied by more than 2-folds reduction of n−6/n−3 ratio in the muscle (p<0.01) and other major organs/tissues (p<0.05). To our knowledge, this is the first report of transgenic sheep produced by the HMC. Compared to the traditional SCNT method, HMC showed an equivalent efficiency but proved cheaper and easier in operation.
Highlights
Sheep is one of the most important domestic animal species for human consumption of meat protein and milk
With new knowledge and understanding that a number of human diseases can be effectively prevented via improved and balanced nutrition, the nutritional value of sheep meat and milk could be further increased by elevated levels of polyunsaturated fatty acids
Recombinant cells were screened by PCR and RT-Quantitative PCR (qPCR) for the presence and level of mfat-1 mRNA expression (Figure 1C), and the cellular fatty acids composition was assessed by gas chromatography for mfat-1 function (Figure 1D)
Summary
Sheep is one of the most important domestic animal species for human consumption of meat protein and milk. Both of the introduced mfat-1 and the host gene Cep120 were functional, and three live births of the transgenic sheep were produced by the HMC. Tissue examination of one of the three new born transgenic lambs showed that the introduced mfat-1 effectively lowered the n26/n23 ratio in the muscle and other major organs/tissues.
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