Development of an effective method for production of cattle that contain enhanced healthy polyunsaturated fatty acids.

本研究的目的是生产含有哺乳动物不能生物合成的健康多不饱和脂肪酸的牛。为了实现这一目的，我们开发了一种方法来生产转基因牛，该转基因牛携带从能够新生物合成多不饱和脂肪酸的植物中提取的脂肪酸去饱和酶基因。我们从陆地植物中α-亚麻酸（18：3 n-3）含量最高的亚麻种子中获得了编码ω3脂肪酸去饱和酶的FAD 3 cDNA。当将cDNA导入酵母并检查其脂肪酸组成时，观察到18：3 n-3的新合成。此外，建立了外源基因稳定转染哺乳动物原代培养细胞的方法。利用此方法，将pβ-act/FAD 3/IRES/EGFP（neor）转染牛成纤维细胞和小鼠3 T3-L1细胞，通过测定18：3 n-3的组成率检测其功能表达。FAD 3基因来源于亚麻种子， ...更多信息 在哺乳动物培养的细胞中，由于与野生型细胞相比，FAD 3基因转染的细胞中18：3 n-3的比例增加，因此在功能上受到压制。然而，转染细胞中的比率较低，仅为0.4%（野生型：0.2%）。因此，根据哺乳动物密码子使用，将植物型的FAD 3的DNA序列改变为拟南芥型DNA序列。在此基础上，我们获得了在哺乳动物细胞中优化的FAD 3基因（FAD 3 opt），并构建了表达载体（pCAG/FAD 3 opt/IRES/EGFP（neor））。转染pCAG/FAD 3 opt/IRES/EGFP（neor）的小鼠3 T3-L1细胞中观察到EGFP的强表达。此外，我们还获得了一株携带pCAG/FAD 3 opt/IRES/EGFP（neor）的牛原代成纤维细胞系。本研究对转基因细胞的脂肪酸组成进行了分析，并对牛克隆胚胎的基因表达与其进一步发育的关系进行了研究，以期建立一种以转基因细胞为供体细胞生产克隆牛的有效方法。我们检测了用携带荧光素酶表达载体（pβ-act/luc+/IRES/EGFP（neor））的牛成纤维细胞重建的胚胎中的基因表达。结果，当在融合后60小时（hpf）的转基因克隆胚胎中检查LUC+表达强度时，强表达胚胎以高速率发育。此外，当胚胎然后被分类为Luc-positive，马赛克，或Luc-negative取决于是否所有，一些或没有卵裂球发光，分别，发育率的囊胚期的阳性胚胎高于马赛克胚胎。所有阴性胚胎均未发育成囊胚。然后，我们用5-甲基胞嘧啶抗体进行免疫染色，检测胚胎中的DNA甲基化水平。Lucs阳性胚胎的甲基化水平低于Lucs阴性胚胎。这些结果表明，核移植胚胎的发育能力与基因表达能力呈正相关，与4- 8-细胞期DNA甲基化水平呈负相关。少

The purpose of this study is to produce cattle containing healthy polyunsaturated fatty acids that are not biosynthesized by mammals. To achieve this purpose, we developed a method to produce transgenic cattle carrying a gene for a fatty acid desaturase taken from plants that are able to newly biosynthesize the polyunsaturated fatty acids.We obtained an FAD3 cDNA which encoded an ω3 fatty acid desaturase from flax seeds that contained the highest rate of α-linolenic acid (18:3n-3) contents in land plants. New synthesis of 18:3n-3 was observed when the cDNA was introduced into yeast and examined their fatty acid composition. Furthermore, a transfection method of a foreign gene stably into mammalian primary cultured cell was established. Using this method, pβ-act/FAD3/IRES/EGFP(neor) was transfected into bovine fibroblast cells and mouse 3T3-L1 cells, and then the functional expression was examined by determination of composition rates of 18:3n-3. The FAD3 gene derived from flax seeds ex … More pressed functionally in mammalian cultured cells, because the ratio of the 18:3n-3 increased in the transfected cells with the FAD3 gene compared with that in wild-type cells. However, the ratio in the transfected cells were low and only 0.4% (wild type : 0.2%). Therefore, the DNA sequence of the FAD3 that was a plant type was changed to the mammalian-type DNA sequence according to the mammalian codon usage. Then we obtained a new FAD3 gene optimized in mammalian cells (FAD3opt), and expression vector (pCAG/FAD3opt/IRES/EGFP(neor)) was produced. Strong expression of EGFP was observed in the mouse 3T3-L1 cells transfected with pCAG/FAD3opt/IRES/EGFP(neor). In addition, we have obtained a bovine primary fibroblast cell lines carrying pCAG/FAD3opt/IRES/EGFP(neor). We now examine the fatty acid composition in the transfected cells.We also examined the relation of gene expression in bovine cloned embryos to their further development, to establish an effective method for production of cloned calves with the transgenic cells as donor cells. We examined gene expression in embryos reconstructed with bovine fibroblasts carrying a luciferase expression vector (pβ-act/luc+/IRES/EGFP(neor)). As a result, when intensity of LUC+ expression was examined in transgenic cloned embryos 60 hours post fusion (hpf), strongly expressed-embryos developed at a high rate. Moreover, when The embryos were then classified as being Luc-positive, mosaic, or Luc-negative depending on whether all, some or no blastomeres were luminescent, respectively, the developmental rates to the blastocyst stage of positive embryos were higher than those of mosaic embryos. Any negative embryos did not developed to blastocysts. Then, we examined DNA methylation levels in the embryos by immunostaining with a 5-methyl cytosine antibody. The methylation level of the Luc-positive embryos was lower than that of the Luc-negative embryos. These results indicated that the developmental capacity of NT embryos could be correlated positively to the gene expression ability and negatively to the DNA methylation level at 4- to 8-cell stage. Less