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.

Factors Determining the Efficiency of Porcine Somatic Cell Nuclear Transfer: Data Analysis with Over 200,000 Reconstructed Embryos

“…Also, standardization is easier, with the possibility for future automation. So far, HMC has been successfully established in cattle (Vajta et al, 2004), pig (Du et al, 2007), horse (Lagutina et al, 2007), goat (Nasr-Esfahani et al, 2011), sheep (Zhang et al, 2013), and water buffalo (Saha et al, 2013).…”

Double Transgenesis of Humanized fat1 and fat2 Genes Promotes Omega-3 Polyunsaturated Fatty Acids Synthesis in a Zebrafish Model

“…5a). In the previous study of fat1-transgenic mammal (Kang et al 2004;Lai et al 2006;Wu et al 2012;Zhang et al 2013), the 20:4n-6 was abundant in dietary and should be converted to 20:5n-3 by fat1-encoded omega-3 desaturase effectively. In our study, however, the 20:4n-6 content of wild-type zebrafish was much low, because little 20:4n-6 was provided by the routine diet of zebrafish.…”

Efecto del uso de medio secuencial humano en la producción de blastocistos de hembra ovis canadensis mexicana por clonación manual

“…La clonación manual ha permitido producir embriones de mamíferos domésticos, tales como: bovinos , porcinos (Du et al, 2008), caprinos (Akshey et al, 2008), ovinos (Zhang et al, 2013), entre otros. En cuanto a especies silvestres, existen dos estudios de producción de embriones por clonación manual interespecies, el antílope tibetano (Pantholops hodgsonii) (Yu et al, 2011) y el búfalo Arnee (Bubalus arnee) (Priya et al, 2014).…”