With the help of somatic cell transplantation technology, goats, cloned cattle and goats have been successfully obtained; this method has also been used in special reports on cloned pigs, namely undifferentiated early embryonic cells (4 cell oocyte embryos). As a donor cell fusion with enucleated oocytes. Pig clonal propagation after phenotypic selection has potential significance in the production of internal products. In addition, the combination of genetic modification technology and cloning technology can create a potential donor for exogenous transplant organs in humans. The first step from cloned cells to pigs is to investigate various factors that may affect pig embryo development in vitro. Mature oocytes harvested from the sow white sows are stimulated by parthenogenetic reproduction after being stimulated by either of two electrical activation methods; this embryo is then placed on microfibrillation inhibitors (cell division inhibition In B), to avoid loss of chromosomes due to cell division, these embryos are finally incubated in any of 3 different culture media (other environmental conditions remain unchanged) (Table 1). Under a certain intensity of electrical shock, the number of embryos stimulated by multiple pulses was less than that of embryos that used a single pulse of electrical stimulation; in addition, the type of media also affected the development of embryos, and the development of embryos in NCSU23 medium. optimal. Subsequently, embryos in NCSU23 medium were stimulated with a single pulse current of 1.5 kv/cml OO[mu]s and the developmental potential of in vitro matured oocytes was determined under these conditions (using mature oocytes in vivo as a control). Of the 167 in vitro-matured oocytes adopting this electrokinetic mode, 116 split within 48 h, but only 4 develop to blastocysts, which is significantly lower than the corresponding in vivo development of mature oocytes. Rate (p = 0.001, X2 = test). Therefore, mature oocytes that develop in vivo are used in later experiments. In many reports on the cloning of livestock, fetal fibroblasts are used as donor cells. The reason for this is that these cells can be genetically modified before cloning. Therefore, we evaluated the ability of porcine fetal fibroblasts to continue to develop after nuclear transfer. The "Meishan Meishan (Black Hair)" pigs were slaughtered on the 24th day of gestation to take their fetuses, and primary cell cultures were established after trypsinization. The cells were further subjected to high-density plate culture for 2 to 6 passages and finally fused; the cells were continuously cultured for 16 days without the supplemented medium, and the resulting cells were in the G0 phase. Finally, the sex of each embryo was identified by the PCR method. Due to their fibroblast antigenicity, cultured cells were negative for cytokeratin and stage-specific type I embryonic antigens but strongly positive for the marker vimentin of the mesoderm. The mouse system cloned from adult somatic cells adopts a special non-fusion method in which donor nuclei are selectively introduced into enucleated oocytes by voltage-driven microinjection. We applied this method to nuclear transfer in pigs. In a NCSU23 medium containing cytochalasin, the metaphase type II chromosomes and the first polar body taken from the oocytes of Landrace gilts or Holstein sorghum (Dow White Duroc) were removed by voltage-driven microinjection The fetal fibroblast nuclei of Meishan Meishan (Black Hair) hybrid pigs were individually introduced into each enucleated oocyte by voltage-driven microinjection. The regenerated embryonic precursors were incubated at 38°C for 3 to 4 hours, then pulsed and activated, and finally the obtained nuclear transfer embryos were cultured. Although porcine embryos develop well into the blastocyst stage in vitro, their growth and development are poor after transplantation into the uterine horns of surrogate sows. Since it is necessary to ensure that the sows can normally gestate at least 4 fetuses, it is inferred that if there is an assisted embryo within the uterus that undergoes sperm production, it will contribute to the full development of the cloned embryo. To study the ability of assisted embryos to promote the development of cloned embryos in the uterus, two experiments were designed. The cloned embryos were cultured in vitro for 20 h in test I (single cell embryos) or in vitro for 40 h in test II (2-4 cells After embryo transfer), all transplanted piglets (9 pigs in trial I and 24 piglets in series II) were all white, and therefore these piglets can be proved to be non-clonal pigs. The fact that the cloned embryo failed to complete the entire 4 pregnancy process suggests that the ratio between the nuclear transfer embryo and the helper embryo is quite important. The percentage of the 2 embryos in this experiment is obviously not at the best level. Test III 11O cloned embryos that have undergone nuclear transfer between 2-8 cell stages were passaged in fibroblast culture medium for 2 to 6 passages and transplanted into 4 surrogate uteri of vaccinated sows without assisted insemination embryos, of which 3 were Surrogate sows began estrus at 27d, 35d, and 6ld after embryo transfer. The estrus cycle was 2ld. Delayed estrus indicates that each surrogate sow has become pregnant. Since porcine embryos are implanted in the uterine wall from 13 days to 14 days of embryonic development, it is likely that two of the pregnant sows will terminate embryonic development after the placenta is formed. The reasons for embryonic development termination are unknown. In the third experiment, the fourth surrogate sow continued her pregnancy after the tubal transplantation of 36 cloned embryos, which were taken out and transplanted in the fibroblast medium to the second passage. One of the cloned embryos developed into full-term embryos and was naturally born on July 2, 2000. The piglet produced was named Xena, the birth weight was 1.2 kg, and the placental weight was 0.3 kg, both of which were within the normal range of non-clone piglets. The anatomy of the piglet's placenta showed normality, while in some of the cloned cattle, the placenta was abnormal. The placenta of the cloned mice using the nuclear microinjection technique was always much larger than that of the non-cloned mouse. Xena cloned pigs were healthy, female, and black-colored, consistent with the predicted results of the "Meishan Meishan" nuclear donor mother. In order to confirm this result, DNA was extracted from the 3 correlates associated with Xena cloned pigs (Xena, Changbai Dairy sows, and Baji's fibroblasts) and 23 markers were set up for specific microsatellite analysis. The analysis was carried out in another laboratory and therefore did not have any subjective color. The analysis results (Figure 1) confirmed that the genome of the Xena cloned pig was the same species as the genome of the Meishan pig for fibroblasts, and that it was the same as that of the Landrace. There are significant differences in the sow’s genome.The results of the above studies show that the use of microinjection to transplant somatic cell nuclei into enucleated oocytes can successfully clone pigs. Although the mechanism has not been fully understood, it is speculated that this is due in part to micropipettes. The activation of voltage is caused by the rapid operation; In addition, the success or failure of porcine cloning may be related to the cytoplasmic chromosome contamination sensitivity of the donor cells. Relative to fusion (cell transplantation, microinjection method is more favorable for nuclear transplantation. Differently, microinjection can selectively remove most of the cytoplasm of the donor cells, and thus the cytoplasm content is relatively thin in the early embryo. In order to induce germ line mutations in cattle and sheep, genetic random integration techniques and gene target techniques for in vitro cells have been used, and later-related technologies have been used; these findings have shown people the prospect that these technologies are also It can be used in pigs.Since porcine embryonic stem cells can not be cultured, this technique has broad application prospects.It is also possible to accelerate the application of pig chromosome manipulation techniques in conventional breeding or transgenes with the help of the ideal genotype individual recultivation technology. The results together with the recently reported technique of intracytoplasmic sperm injection in pigs have shown that microinjection technology can promote the development of pig cloning technology.