Proceedings of the Third World Fisheries Congress: Feeding the World with Fish in the Next Millenium—The Balance between Production and Environment

Nuclear Transfer of Somatic Cells of Transgenic Red Carp

Haobin Zhao, Shangping Chen, Zuoyan Zhu


Zhu et al. (1985, 1989) first introduced human growth hormone (hGH) gene driven by mouse metallothionein promoter into fertilized eggs of goldfish Carassius auratus and common carp Cyprinus carpio and obtained fast-growing transgenic fish. They studied the integration, expression, heredity, and growth-promoting mechanisms of novel genes and developed a model of transgenic fish.

The behavior of novel genes is sophisticated after microinjection; only a small part can ultimately reach the host genome. An integrated novel gene can insert into the host genome randomly or by recombination, and can exist as monomer or polymer. All founder transgenic animals are mosaic for the novel gene. Ordinarily, the heredity of a foreign gene does not follow Mendel’s law, and the transgenic fish with stable inheritance can be established at the F2 generation (Zhao et al. 1999). However, Wei et al. (1992) showed that the inheritance of a novel gene in F1 transgenic fish was not in Mendel’s model, like in F2 transgenic fish. The F2 transgenic fish was also mosaic; the novel gene copies were different in different tissue. Cui and Zhu (1998) detected the MThGH gene in a transgenic red carp Cyprinus carpio hematopterus (Tem. et Schl.) population from P0 to F4 by polymer chain reaction (PCR) and found that the positive ratio was increased by generation transmission. The positive ratios in P0, F1, F2, F3, and F4 were 58.3, 70.8, 77.3, 90, and 94.3%, respectively. So, it is difficult to obtain homogeneous transgenic fish with a steadily integrated novel gene by traditional breeding.

Tung et al. (1963) obtained the first nuclear transfer fish, and Chen et al. (1986) obtained the first nuclear transfer goldfish from kidney cell. Nuclear transfer may be the appropriate method for establishing homogeneous transgenic fish species and to make a transgenic fish clones.

We used nuclear transfer of F4 transgenic red carp with MThGH gene to study the stability of transgenes in F4 transgenic fish and the possibility of cloning transgenic fish.

F4 MThGH gene–transferred red carp were naturally bred from F3 red carp that were positive for MThGH gene.

Loach Misgurnus anguillicaudatus (Cantor) was purchased from a market in Dadongmen, Wuhan; Huanghe carp Cyprinus carpio (Huanghe var.) was provided by the Henan Institute of Fisheries. These fish were induced to reproduce by injection of a homogenate of carp pituitary gland, and the mature eggs were squeezed out.

The cells—kidney cells, tail fin cells, and cultured tail fin cells—were from F4 transgenic fish that were positive for MThGH as detected by PCR.

The kidney was removed from just-killed fish and digested with 0.25% trypsin or pipetted into 0.7% physiological saline; then, the suspension was centrifuged at 1,000 revolutions/min for 5 min. The supernatant was discarded, and the sediment of kidney cells was resuspended in phosphate-buffered saline (PBS) or Holtfreter’s solution.

To collect fresh tail fin cells, the tail fin of the fish was dissected and digested with 0.25% trypsin. The upper part of the sediment (tail fin cell layer) was collected and resuspended in Holtfreter’s solution for nuclear transfer after centrifugation.

The cultured tail fin cells were cultured from hGH-positive fish in M199 medium containing 25 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) plus 20% fetal bovine serum and antibiotics at 28°C. The cells were passaged from one to three. Cells at the 18th passage were dissolved with 0.25% trypsin, washed, and preserved in PBS.