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.

Figure 1. Establishment and analysis of transgenic clonal donor cells.

(A) Schematic representation of n−3 fatty acid desaturase gene with linearized expression vectors. (B) Detection of the mfat-1 gene in Geneticin-resistant cell clones by PCR and RT-qPCR. mfat-1 expression vector was used as the template for positive control (PC) and untransfected syngenic cells was used as the negative control (NC). (C) Quantitative PCR analysis of mfat-1 expression in positive cell clones. cDNA representing mfat-1 was amplified with sequence specific primers. The beta-actin was used as internal control and the expression level observed in the transgenic donor cell was normalized to the value of A-3-1. (D) Partial gas chromatograph traces showing the polyunsaturated fatty acid profiles of total cellular lipids from the H-6-6 cells and the control cells. Note the level of n-6 polyunsaturated acids are lower whereas n−3 fatty acids are abundant in the mfat-1 cells (right) as compared with the control cells (left), in which there is very little n−3 fatty acid.

Figure 2. Production of transgenic lambs by handmade cloning.

(A) The recipient #0907 and the transgenic lamb (PP-01). (B) Detection of the mfat-1 gene in umbilical cord samples of three cloned lambs by PCR and RT-qPCR. (C) Insertion site of mfat-1 vector in the sheep genome. Arrows indicate the mfat-1 transcriptional direction, which is identical with the endogenous putative sheep Cep120 gene. PCR fragment obtained in this study are shown by black bars. (D) Southern blot using the ³²P-labled mfat-1 specific sequence as a probe to hybridize the genomic DNA from the transgenic donor cells and the cloned lambs. The genomic DNA was digested with BamHI before the gel electrophoresis. (E) Northern blot analysis. Total RNAs were loaded on each lane (15 µg per sample) and the coding region of mfat-1 was used as a probe. Shown below is the gel electrophoresis of rRNA as control. (F) Quantitative PCR analysis of mfat-1 expression in major tissues from the transgenic lambs (PP-02). Compared with the mRNA expression level normalized to the donor cell, the highest level of mfat-1 expression was observed in transgenic muscle sample.

Figure 3. Partial gas chromatograph of fatty acids in muscle sample of mfat-1 transgenic (PP-02) and the control lamb.

Fatty acid methyl esters were quantified using a fully automated 6890 Network GC System with an Agilent J&W fused-silica DB-23 capillary column. The peaks were identified by comparison with the internal fatty acid standards, and area percentage for all of resolved peaks was analyzed using GC ChemStation software. Compared with the wild-type control (left), the level of n−6 polyunsaturated acids in the transgenic muscle (right) are significantly lower, whereas n−3 fatty acids are abundant.