Embryonic stem cell line and method for preparing the same
a stem cell line and embryonic technology, applied in the field of embryonic stem cell lines, can solve the problems of not being cultured, and no es cell line developed from human oocytes utilizing nuclear transfer technology
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example 1
Preparation of Oocyte and Nuclear Donor Cell
[0141]Voluntary oocyte donors were screened carefully through physical and mental examinations, and administered with follicle stimulation hormone (FSH) to induce superovulation.
[0142]About 36 hours after the administration of human chorionic gonadotropin (hCG) to the donors, cumulus-oocyte complexes (COCs) were recovered and cultured for 40 minutes in the G1.2 medium using an incubator maintained at 37° C., 5% CO2 and saturated humidity. Such COCs were treated with 0.1% (w / v) hyaluronidase (Sigma Co., St. Louis, Mo., U.S.A.) for 1 hour to disperse cumulus cells.
[0143]The oocytes were obtained by separating such cumulus cells from the COCs. The separated cumulus cells were isolated through a mouth pipette and washed with the G1.2 medium. Those cumulus cells having a modal diameter ranging from 10 to 12 mm were selected as nuclear donor cells.
example 2
Enucleation of Oocyte and Cell Fusion
[0144]One of the oocytes obtained in Example 1 was cultured in the G1.2 medium for 1 to 2 hours in order to induce the maturation of its nucleus. Thereafter, enucleation, nuclear transfer and electrofusion thereof were performed as follows.
[0145](2-1) Enucleation of Oocyte and Nuclear Transfer from Somatic Cell
[0146]The oocyte was washed once with the G1.2 medium. Such oocyte was transferred to a hyaluronidase solution prepared by mixing 1 ml of the G1.2 medium with 111 μl of a solution, wherein 0.05 g of hyaluronidase was dissolved in 5 ml of the G1.2 medium, and adjusted to a 0.1% (w / v) hyaluronidase concentration. The oocyte was stripped of any remaining cumulus cells, washed three times with the G1.2 medium and placed in the same medium. Then, the oocyte was transferred to a cytochalasin B solution prepared by mixing 1 ml of the G1.2 medium supplemented with 10% fetal bovine serum (FBS) with 1 μl of a solution wherein cytochalasin B was disso...
example 3
Reprogramming, Activation and In Vitro Culturing of Nucleus-Transferred Oocyte
[0155]Since a sperm-mediated activation, which is one of the major factors for a normal embryonic development, was absent in case of the nucleus-transferred oocyte obtained in Example 2, an artificial stimulus was needed instead. In order to determine the optimum conditions for artificial embryogenesis, therefore, nucleus-transferred oocytes were reprogrammed, activated and in vitro cultured under various conditions as shown in Tables 2 to 4.
[0156]First, to examine the effect of the reprogramming time on the rate of blastocyst formation, the reprogramming times were set at about 2, 4, 6 and 20 hours, respectively, while applying the same conditions for activation and in vitro culturing as can be seen from Table 2. As a result, the highest rate of blastocyst formation was obtained when the reprogramming time was about 2 hours.
TABLE 2No. of nucleus-transferred oocytesIn vitro culturedeveloped toReprogramming...
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