Production of cloned transgenic cow expressing omega-3 fatty acids
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- Wu, X., Ouyang, H., Duan, B. et al. Transgenic Res (2012) 21: 537. doi:10.1007/s11248-011-9554-2
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n-3 Polyunsaturated fatty acids (n-3 PUFA) are important for human health. Alternative resources of n-3 PUAFs created by transgenic domestic animals would be an economic approach. In this study, we generated a mfat-1 transgenic cattle expressed a Caenorhabditis elegans gene, mfat-1, encoding an n-3 fatty acid desaturase. Fatty acids analysis of tissue and milk showed that all of the examined n-3 PUAFs were greatly increased and simultaneously the n-6 PUAFs decreased in the transgenic cow. A significantly reduction of n-6/n-3 ratios (P < 0.05) in both tissue and milk were observed.
KeywordsN-3 fatty acid desaturase genemfat-1Transgenic cattleNuclear transfer
Clinic studies have showed that long-chain omega-3 polyunsaturated fatty acids (n-3 PUFAs) have beneficial effect on human health, while high levels of n-6 PUFAs in human bodies are closely related to certain diseases such as cancer, cardiovascular diseases and various mental disorders (Riediger et al. 2009). A rational balanced level of n-6 and n-3 fatty acids is important and the ideal n-6/n-3 ratio is 1:1. The current n-6/n-3 ratio in food for human consumption is as high as 8–16 (Enser et al. 1996; Simopoulos. 2003). Because mammals has neither enzymes to synthesize n-6 and n-3 fatty acids nor convert n-6 to n-3 fatty acids (Spychalla et al. 1997; Kang et al. 2001), directly and constantly intake of n-6 and n-3 PUFAs from food is the only source of n-6 and n-3 fatty acids for mammals (Horrocks and Yeo 1999; Arterburn et al. 2006). At present, product from sea fish is main dietary source of n-3 fatty acids for human. However, the serious environment contamination, especially water pollutions, cause methyl mercury and other heavy poisonous metals flow and deposit into fish, and subsequently transfer to fish product consumers (Sidhu. 2003; Park and Johnson 2006; Foran et al. 2005). Furthermore, although higher concentration of n-3 PUFAs in livestocks can be obtained by feeding them with fishmeal and other marine products, it is time-consuming and costly, and is limited by the huge demand of marine products and it has same issue of mercury accumulation in livestocks (Marmer et al. 1984; Mitchell et al. 1991; Naylor et al. 2000). Alternatively, transgenic technology may become direct and effective approach to enrich n-3 fatty acids in livestocks.
Kang et al. reported that expression of a humanized fat-1 gene (hfat-1), encoding an n-3 fatty acid desaturase from Caenorhabditis elegans, in mice resulted in a significant increase of n-3 fatty acids as well as a sharply decreased ratio of n-6/n-3 fatty acids in the hfat-1 transgenic mice (Kang et al. 2004). n-3 fatty acids in the milk of transgenic mice were also significantly increased. When neonatal mice were fed with the transgenic milk, the docosahexaenoic acid (DHA) content in their brains increased (Kao et al. 2006; Bongiovanni et al. 2007). Lai et al. generated healthy hfat-1 transgenic pigs and reported a significantly increased level of n-3 fatty acids and reduced n-6/n-3 ratio in the transgenic pigs compared with wild-type pigs (Lai et al. 2006). Other reports have also showed that fatty acid desaturase genes isolated from higher plants such as maize (Tao et al. 2006), spinach (Saeki et al. 2004), and scarlet weeds (Indo et al. 2009) worked well in mammals after being introduced into animal cells.
In this study, a mammalianized fat-1 (mfat-1) expression vector was constructed and introduced into primary fetal bovine fibroblasts. A transgenic cow that expressed mfat-1 gene was produced by somatic cell nuclear transfer (NT) and compositions of n-3 and n-6 PUFA in the transgenic were analyzed.
Materials and methods
Construct of mfat-1 vector
Construct of the mfat-1 gene expression vector. The coding region of the fat-1 gene from C. elegans (GenBank: NM_001028389) was optimized for mammalian cell expression and named as mfat-1. The mfat-1 cDNA was synthesized and constructed into the mfat-1 expression vector, pST200, consisting of a mouse muscle creatine kinase enhancer, cytomegalovirus enhancer with a chicken β-actin promoter. A pgk-neo expression cassette flanked by loxP sites was linked to the construct as the selection marker.
Cell culture and preparation of transfected cells
Donor cell lines were established from a 50-days fetus of Holstein cow. Tissue was minced, suspended in DMEM/Ham’s F12 (1:1) supplemented with 15% FBS and antibiotics, seeded in 25 cm2 tissue culture flasks, and cultured at 39°C in a humidified atmosphere of 5% CO2 in air for several days. The mfat-1 expression vector, pST200, was linearized and transfected into early passage of the primary bovine fetal fibroblast by electroporation. The transfected cells were selected with 250 μg/ml of G418. The G418-resistant colonies were pooled and used to clone mfat-1 transgenic embryos by NT as described below.
Oocyte maturation in vitro (IVM)
Bovine ovaries were collected from a local abattoir and cumulus-enclosed oocyte complexes (COC) were aspirated from 3 to 8 mm follicles. Oocytes with evenly shaded cytoplasm and intact layers of cumulus cells were selected and cultured in maturation medium: M199 containing 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT), 0.5 μg/ml FSH (Sioux Biochemicals, Sioux City, IA), 5 μg/ml LH (Sioux Biochemicals), and 100 U/ml penicillin/100 μg/ml streptomycin (HyClone Laboratories, Logan, UT) and cultured for 20 h prior to NT.
After maturation (IVM) for 20 h, the cumulus cells were removed and oocytes with the first polar body (PB1) were enucleated as cytoplast recipients. Single transfected cells were transferred to the perivitelline space of enucleated oocytes. The reconstructed couplets were fused in mannitol fusion buffer by two DC pulses of 1.8 kV/cm for 20 μs. Then the fused clones were activated by 5 μM ionomycin for 5 min and treated with 10 mg/ml cycloheximide in CR1aa medium for 5 h in 5% CO2 in air at 38°C. Following activation, the embryos were cultured in CR1aa medium supplemented with BSA for 40 h, then the cleaved embryos were transferred and cultured in CR1aa medium + 4% FBS feeding with a single-layer cumulus cells under 5% CO2 in air at 38°C with high humidity.
Embryo transfer and pregnancy evaluation
On day 7, an average of 2 blastocysts was transferred non-surgically to cow synchronized ±1 day to the stage of the embryos. Pregnancy was detected by trans-rectal ultrasound at around 60 days, and pregnant recipients were checked by ultrasound or palpation at approximately 30-days intervals to confirm ongoing pregnancies.
Analysis of the founder with RT-PCR and Southern blotting
Ear tissues were taken from the transgenic founder calves. Total RNAs were extracted from the tissues with TriZol (Invitrogen Inc., USA) according to the manufacturer’s instructions. The RNAs were then reverse-transcribed by avian myoloblastosis virus (AMV) reverse transcriptase (Takara Bio, Japan) with 18-mer oligo-dT primers in a 25 μl reaction solution. Primers specific for the mfat-1 gene (5′-GGA CCT GGT GAA GAG CAT CCG-3′ and 5′-GCC GTC GCA GAA GCC AAA C-3′) were used for subsequent cDNA amplification. The PCR conditions for the cDNA amplification included 35 cycles of incubation at 94°C for 30 s, 65°C for 30 s, and 72°C for 60 s. The amplified products were subjected to electrophoresis on 1% agarose gel.
The genomic DNA from the ear tissue of the founders was extracted according to standard molecule cloning instructions for Southern blotting. Out and inner primers were designed according to the mfat-1 sequence: outer primer 1: 5′-tcaacgccaacaccaagc-3′; outer primer 2: 5′-taggtcacgatcaccagcat-3′; inner primer 1: 5′-cgccaacaccaagcagg-3′; inner primer 2: 5′-ccgtcgcagaagccaaa-3′. The genome DNA was used for template to amplify 727 bp probe, and the probe as template for amplification of a 534 bp product with inner primers. The Genomic DNAs were digested into smaller fragments with excess amounts of EcoRI for 18 h. The concentrated samples were hybridized as standard protocol. Expression vector pIE-fat1 was as positive control.
Tissue fatty acid analysis
Lipids were extracted as in a previous report (Lu et al. 2008). Briefly, the samples collected from ear tissue were homogenized in a mixture of methanol, chloroform, and water. After 15 min, chloroform was added and the samples were vortexed and centrifuged. The lower phase was dried under nitrogen and resuspended in boron trifluoride methanol. The samples were heated at 90°C for 30 min and extracted with 4.0 ml pentane and 1.5 ml water. The mixtures were vortexed and the upper phase was recovered. The extracts were dried, resuspended, and injected into a capillary column (SP-2380, 105 m × 53 mm ID, 0.20 μm film thickness; Sigma). The gas chromatography was done on a Perkin-Elmer Clarus 500. Identification of components was done by comparison of retention times with those of authentic standards (Sigma).
Reproduction and lactation of the transgenic cow
The signs of estrus were observed when the transgenic calves grew up to 13-month old. At about 14-month-old both transgenic cow and the control were artificially inseminated (AI) with frozen and thawed semen from a proven Holstein sire. Pregnancy was detected by trans-rectal ultrasound at around 60 days after AI and maintained pregnancy up to birth. After their delivery, the milk samples from both transgenic and the control cows were collected. The analysis protocol of milk fatty acids has been described above.
All paired comparisons were subject to a two-tailed Student t test with P < 0.05 considered statistically significant.
Development of reconstructed embryos
Total of 224 reconstructed oocytes obtained after transfer of mfat-1 transgenic cells into enucleated oocyte cytoplasts. The fusion, cleavage and blastocyst development rates were 83, 84, and 34%, respectively. Forty-eight blastocysts were transferred to 23 synchronized recipient cow. Pregnancy rates were 8.7% (2/23) at 60, 90 days and at term. Generation of transgenic calves and expression of the mfat-1 gene After pregnancy for 281 days, two female calves were naturally delivered at the same day, named ZK001 and ZK002, the birth weights were 32.2 and 32.6 kg, respectively.
RT-PCR and Southern blotting proved transgenic positive
Tissue fatty acid composition of the transgenic cow
PUFAs composition of total lipids from the transgenic calf and the controls
ZK002 (mfat-1 transgenic)
Fatty acid composition of the milk from the transgenic cow
PUFAs composition of total lipids from the transgenic and the control dairy milk
It is known that the n-3 fatty acid desaturases is the key enzyme in the synthesis of n-3 PUFAs, since they can efficiently covert n-6 PUFAs into n-3 PUFAs. Because mammals lack n-3 desaturases, the n-3 and n-6 PUFAs are not interconvertible in mammalian animals (Goodnight et al. 1981; Knutzon et al. 1998). Dietary are the only supply for mammalian animals’ n-3 PUFAs. Desaturases genes from fungus (Pereira et al. 2004; Sakuradani et al. 2005), round worms (Spychalla et al. 1997; Kang et al. 2001, 2004; Lai et al. 2006; Ge et al. 2002), spinach (Saeki et al. 2004) and scarlet weeds (Indo et al. 2009) have been isolated and used for transgenic research. A humanized n-3 desaturase gene, mfat-1, from C. elegans, has been successfully induced to mice and pigs. In the tissues and organs of the founder animals and their offspring, all n-3 PUFAs were significantly increased, while n-6 PUFAs were decreased, resulting in a greatly reduced n-6/n-3 ratio (Kang et al. 2004; Lai et al. 2006).
In the present study, a newly constructed mfat-1 expression vector was introduced to primary bovine fetal fibroblast cells and one mfat-1 transgenic founder calf, ZK002 was generated by NT. Fatty acid analyses showed that all major n-3 fatty acid peaks were elevated and all major n-6 peaks were in lower level in the calf. The ratio of n-6 and n-3 in ZK002 was several times less than its recipient mother cow 005 as well as the non-transgenic calf (ZK001) and the control (Fig. 2 and Table 1). This result indicated that the mfat-1 transgenic calf, ZK002, has efficiently converted n-6 fatty acids into n-3 fatty acids in her body. ZK001 is a cloned calf but not transgenic, which may result from a mixed non-transfected fibroblasts. The result was confirmed by her n-6/n-3 ratio, which is similar to the control cows and significantly higher than the transgenic calf ZK002.
In mouse, the fat-1 genes from both C. elegans and C. briggsae possess great value for the production of n-3 PUFAs in transgenic animals (Kang et al. 2004; Zhu et al. 2008). The proportion of n-6 PUFAs decreased and n-3 PUFAs increased considerably, particularly for DHA and docosapentaenoic acid (DPA) in transgenic mice (Zhu et al. 2008). In the transgenic pigs, Lai et al. reported that n-3 PUFAs such as eicosapentaenoic acid (EPA) and DPA showed a 15-fold and fourfold increase, respectively, and the concentration of total n-6 fatty acids in the transgenic piglets was reduced by 23%. The ratio of n-6/n-3 in hfat-1 transgenic piglets showed fivefold reduction. The major tissues (muscle, liver, kidney, heart, spleen, tongue, brain and skin) from hfat-1 transgenic piglets showed a substantially lower n-6/n-3 ratio (Lai et al. 2006). Due to the limit number of the transgenic calf in the present study, it is not possible to sacrifice the animal for analysis of tissues except ear.
The transgenic cow ZK002 grows healthy and showed signs of estrus from 13 month old. After artificial insemination the cow got pregnancy and naturally delivered a healthy calf. Her milk content of n-3 PUAFs was significantly increased and the ratio between n-6 and n-3 was fourfold reduction compared with the non-transgenic ZK001. This result suggested that the mfat-1 transgenic cow has normal reproduction and the fat-1 transgene has been expressed well in her mammary gland and the n-3 fatty acids were enriched in her milk. Thus, results from pigs and cow indicate that fat-1 transgenic domestic animals can produce meat and milk enriched in n-3 fatty acids, which probably become an efficient and economical approach to meet the increasing demand for n-3 PUAFs.
This work was supported by Hi-Tech Research and Development Program of China (863 Programs, Nos. 2007AA100505 and 2008AA10Z159) and grant from Inner Mongolia University for Chang Jiang Scholars Program to GPL.