title: Handmade cloned transgenic sheep rich in omega-3 Fatty acids.
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.
authors: State Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.
authors: Peng
authors: P
authors: Zhang
authors: Peng
authors: P
authors: Liu
authors: Hongwei
authors: H
authors: Dou
authors: Lei
authors: L
authors: Chen
authors: Longxin
authors: L
authors: Chen
authors: Lin
authors: L
authors: Lin
authors: Pingping
authors: P
authors: Tan
authors: Gabor
authors: G
authors: Vajta
authors: Jianfeng
authors: J
authors: Gao
authors: Yutao
authors: Y
authors: Du
authors: Runlin Z
authors: RZ
authors: Ma
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002849:Chromatography, Gas; D002999:Clone Cells; D019976:Cloning, Organism / Q000379:methods*; D015525:Fatty Acids, Omega-3 / Q000378:metabolism*; D005260:Female; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D018895:Microsatellite Repeats / Q000235:genetics; D053652:Nuclear Transfer Techniques; D012756:Sheep / Q000235:genetics*
chemical_list: D015525:Fatty Acids, Omega-3; D015801:Helminth Proteins