部分 鱼类基因转移模型建立 Novel Gene Transfer into the Fertilized Eggs of Goldfish (Carassius auratus L. 1758)* Z. ZHU G. LI L. HE S. CHEN Institute of Hydrobiology， Academia Sinica， Luojiashan， Wuhan 430072 Summary Novel gene which was microinjected into fertilized eggs of the goldfish replicated during the embryogenesis. A proportion of the novel gene has been integrated into the host DNS of the 50-day-old transgenic goldfish. Zusammenfassung übertragung von Fremdgenen in befrucbtete Eier des Goldfisches (Carassius auratus L. 1758) Durch Mikroinjektion in befruchtete Eier des Goldfisches eingebrachte Fremdgene wurden w？hrend der Embryogenese repliziert. Ein Teil der Fremd-DNS ist in die DNS der 50 Tage alten Goldfische integriert worden. Résumé Transmission des gènes aliens dans des ceufs fertilisés du Cyprin doré (Carassius auratus L. 1758) De gènes nouveaux sont microinjectés dans des ceufs fertilisés des embryons de Carassius auratus. Ces gènes sont répliqués. Un part de la DNS nouveau était intégré dans la DNS des Carassius auratus originaux à ce moment agés 50 journées. In recent years， purified genes have been microinjected into fertilized eggs or embryos of mammals， amphibians， and insects species in order to assess the fate and expression of the novel gene during the development in whole animal system [for review， see (3)]. Results of experiments performed on Xenopus laevis demonstrated that the microinjected， cloned DNA sequences could be replicated and faithfully transcribed during embryogenesis (2，7). PALMITER et al. (1982) (5) introduced into the pronuclei of fertilized eggs of mice a hybrid gene containing the promoter of the mouse metallothionein-1 gene fused to the structural gene for rat growth hormone. Some transgenic mice grew significantly larger than their littermates. Thus it is hoped to use the gene transfer method to develop rapidly growing strains of domestic animals. Compared with mammals and amphibians， fish， being at a lower level in evolution among the vertebrates， are much more suitable for micromanipulation during embryogenesis. Our previous experiences in nucleo-transplantation in fish (1) indicated that the affinity of the nucleus and cytoplasm among different species or even different genera of fish and the adaptability of fish embryonic development to its environment are greater than among the other vertebrates. It is naturally imaginable that a superfish could be bred by the microinjection of a cloned and properly modified fish growth hormone gene. To this end， as a first step， we performed an experiment of novel gene transfer into the fertilized eggs of gold fish to assay its stability and existence during the host embryogenesis and in the adult fish as well. Novel Gene Preparation: The cloned DNA sequence used here for microinjection came from a recombinate plasmid pBPVMG-6 (6) (Fig. 1)， a gift of Dr. D. HAMER at NHI， U.S.A. A 9.4 kb linear DNA fragment of BPVMG was isolated from pBPVMG-6 with a procedure of restriction enzyme BamH1 digestion， 0.7% agarose gel electrophoresis separation， phenol and chloroform extraction， and alcohol precipitation. It was finally dissolved in 88 mM NaCl and 10 mM Tris HCl (pH 7.5) at an injection concentration of 27 ng/μl. The Preparation of Fertilized Eggs of Gold Fish: In April， 1-2 year old gold fish， males and females， were induced for reproduction by injection of a homogenate of carp pituitary gland (1 mg pituitary gland/1 kg body weight). About 10 hours later， the eggs were squeezed out and inseminated artifily. The chorion of the eggs was removed after 10 minute treatment in 0.25% trypsine solution， and the naked eggs were then transferred into Holtfreter’s solution (4) in an agar layered Petri dish. The Novel Gene Introduction: A glass micro-needle with a tip inner diameter of 3 μm for microinjection was drawn with a needle drawing apparatus (Leitz). A plastic tube connected the micro-needle with a 50 ml syringe. After loading the DNA solution into the microneedle， the micromanipulater was carefully adjusted to insert the tip of the micro-needle into the central position of the germinal disc of the fertilized egg. A 1-2 nl dose of DNA solution (about 7×106 copies of BPVMG molecules) was then released into each egg before the first cleavage occurred by applying a proper pressure to the syringe. The microinjected eggs were allowed to develop in Holtfreter’s solution up to blastula stage and then transferred into “aged” boiling water (boiled and then left at room temperature for more than three days). Fate of the Novel Gene in Fish Development: More than 3000 gold fish eggs received the microinjection during the spawnning season. Random samples of 20-30 embryos were collected immediately after injection and at the specified stages of embryogenesis. The totalDNA in the embryos was isolated by the procedure of RUSCONI and SCHAFFNER (1981) (7). DNA equivalents of one em
