The Biological Analysis Of Embryonic Stem Cells From Hfad--3 Transgenic Mice

The co-3 desaturase enzyme FAD3 coded by ω-3 fatty acid desaturase gene(fad-3), is one of the membrane proteins located in the endoplasmic reticulum membrane, which convert ω-6 into co-3 PUFAs. In this study, fad-3 gene cloned from Linum usitatissimum linum was humanized and named hfad-3. After microinjection of hfad-3 into zygote pronucleus, transgenic mice were obtained. The embryonic stem cells from the fad-3 transgenic mice were isolated and named as Fad3-ESL1,2,3,4 and 5. The results showed that 1) the fad-3 embryonic stem cells had positive expressions of Sox-2, Oct-4, Nanog, SSEA-1 and alkaline phosphatase. The stem cells could formed bodies which differentiated into three germ layers.2) Identification of the transgenic stem cells by PCR showd that the hfad-3 gene integrated in the chromosome steadily. The hfad-3 gene was expressed in all of the five Fad3-ES cell lines, and Fad3-ESL2 showed the highest expression level. 3) At 24-48h, the stem cells got into a rapid growth phase, and reached a plateau after 72h. The dates of double time showed that the growth of Fad3-ES has a little slower than the control. The Fad3-ES cells had a relatively lag in contrast to the control.4) The cell apoptosis detection results showed that the portions of early apoptosis, apoptosis and death in Fad3-ES cells were significantly lower than the controls. Moreover, the cell apoptosis and death rose with increasing passages.5) Fatty acid analysis indicated that the ratio of ω-6/ω-3 of Fad3-ES cells obviously decreased when compared to the control, and Fad3-ESL2 was the minimum, only about one-third of the control. The fatty acid analysis of the cell culture medium was in consistent with the results of cells. The polyunsaturated fatty acid ratio ω-6/ω-3 of Fad3-ES cells also decreased obviously.In summary, ES cells derived from the fad3 transgenic had highly comparable developmental polypotency to the non-transgenic ES cells. The ernbryoid bodies proved that three germ layer differentiation formed. The fad3 expressed well in different passages of cells from passage 5 to passage 30. The ω-3 polyunsaturated fatty acids expressed continuely and the ratio of ω-6/ω-3 constantly decreased.