Studies on Nuclear Transplantation in Mammalian Embryos

A series of experiments were conducted to study the major procedures in nuclear transplantation such as oocyte enucleation and activation, electrofusion and developnent of the nuclear transplant embryos in the mouse, rabbits and sheep. The important results are as follows:11. In the mouse, only 35% of the oocytes collected 15～16 h after hCG had a notable first polar body (FPb) and those without FPb were enucleated by removing cytoplasm from the PVS-wider side and the enucleation rate was similar to that in the oocytes with FPb, and the enucleation rate of removing 1/3 cytoplasm was remarkably higher than that of removing 1/4 cytoplasm. 2. Among the three fusion media tested, mannitol and sucrose solutions produced better results than M2 in electrofusion of mouse 2-cell embryos. Under favorable pulse conditions, the osmotic pressure of fusion medium had no motable effect on electrofusion, but as the conditions became so unfavorable that some embryos began to lyse, the fusion rates in hypertonie mannitol solution were significantly higher than those in isotonic or hypotonic solutions. A wide range of pulse strengths (0.31～2.04 by/ cm) and durations(10～1280 μs) were used and 100% of fusion were obtained in many cases. Optimal pulse durations were plotted for field strengths to obtain high fusion rates (96%～ 100%) in mouse2-cell embryos. 3. With one pulse of 0.45 by / cm, satisfactory results of mouse oocyte activation were obtained only when the duration increased to 160 μs or longer. The activation rate increased as the oocytes got older. Some of the oocytes ar. rested at metaphase Ⅲ after electrical stimulation and their proportion to the number of oocytes not activated increased with egg age. 4. 10% and 31% of the nuclear transplant embryos developed to morula or blastocyst stage in sheep and rabbits, respectively, with Chinese-made hormones and chemicals.

Factors Affecting Electro－Fusion and Electro－Activation In Serial Nuclear Transplantation In Goat（Carpa hircus） Embryo

The effects of the status of both donor and recipient cells (different maturity and different transplantation generations) on serial nuclear transplantation in goat were studied, using, as indices, electro-fusion rate, and the cleavage rate and development rate of the cultured embryos. As shown by the results,no significant difference in fusion rate was noted between donor cells of different maturity (recovered 26-28 hrs as against 32-36 hrs after LRH treatment ) and both types of donor cells responded to the given electrostimulation. With cells from ICM as donor, the fusion rate was lower than that using cells from 8-16 celled embryos and morula (G0: 0.01<p<0.05; Gl:p>0.05). The generation of transplantation produced no apparent effect on fusion rate. With the given electro-stimulation conditions, the average fusion rate was 88.58% fur GO and 89.38% for G1; average cleavage rate, 39.36%fur G0 and 47.75% for G1; and the proportion of embryos developing to morula and blastula was 15.17% for G0 and 18.2% fur G1.

Development of Inter-Family Nuclear Transplant Embryos by Transplanting the Nuclei from the Loach Blastulae into the Non-Enucleated Zebrafish Eggs

The developmental fate of the pronuclei in recombined embryos obtained by transplanting the donor nuclei into the non-enucleated eggs remains controversial in the case of fish. In the present study, the nuclei from the loach blastulae were transplanted into non-enucleated zebrafish eggs, the resulting 9 inter-family nuclear transplant embryos developed to larval stages. Although the development timing of the nuclear transplants resembled that of zebrafish, chromosome examination revealed that most of the recombined embryos were diploids with karyotype characteristic of loach, which was also proved by RAPD analysis. Moreover, 3 out of the 9 larval fish formed barb rudiments specific to loach. It was therefore concluded that the nuclear transplant larval fish were inter-family nucleo-cytoplasmic hybrids; and that only the donor nuclei were involved in the development of the nuclear transplant embryos, while the pronuclei in the non-enucleated eggs were likely automatically eliminated during the development.

Embryonic and genetic manipulation in fish

Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been thoroughly studied in China since 1960s. Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults. Most importantly, nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish. This was the first case of cloned fish with somatic cells. Based on the technique of microinjection, recombinant MThGH gene has been transferred into fish eggs and the first batch of transgenic fish were Produced in 1984. The behavior of foreign gene was characterized and the onset of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis. This eventually led to the transgenic mosaicism. The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults. The transgenic common carp were more efficient in utilizing dietary protein than the controls. An 'all-fish' gene construct CAgcGH has been made by splicing the common carp β-actin gene (CA) promoter onto the grass carp growth hormone gene (goGH) coding sequence. The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait. Combination of techniques of fish cell culture, gene transformation with cultured cells and nuclear transplantation should be able to generate ho- mogeneous strain of valuable transgenic fish to fulfil human requirement in 21st century

Medaka fish stem cells and their applications

Stem cells are present in developing embryos and adult tissues of multicellular organisms. Owing to their unique features, stem cells provide excellent opportunities for experimental analyses of basic developmental processes such as pluripotency control and cell fate decision and for regenerative medicine by stem cell-based therapy. Stem cell cultures have been best studied in 3 vertebrate organisms. These are the mouse, human and a small laboratory fish called medaka. Specifically, medaka has given rise to the first embryonic stem (ES) cells besides the mouse, the first adult testis-derived male stem cells spermatogonia capable of test-tube sperm production, and most recently, even haploid ES cells capable of producing Holly, a semi-cloned fertile female medaka from a mosaic oocyte created by microinjecting a haploid ES cell nucleus directly into a normal oocyte. These breakthroughs make medaka a favoring vertebrate model for stem cell research, the topic of this review.