ABSTRACT
Transgenic fish have been routinely produced by microinjecting or electroporating foreign DNA into one-cell stage embryos or unfertilized eggs. While both techniques are effective in producing transgenic fish species from which unfertilized or newly fertilized eggs can be easily obtained, these techniques are not applicable to live-bearing fish and many crustacean species where unfertilized or newly fertilized eggs are not readily available. In this paper, we describe a new method of introducing foreign DNA into the live-bearing fish, Poeciliposis lucida, and crayfish, Procambarus clarkii, by directly transforming the immature ovary or testis of these animals with replication-defective pantropic retroviral vectors carrying a reporter gene (neo(R)). A significant fraction of the progeny derived from these treated animals contains the neo(R) reporter gene, determined by a PCR-based assay. The PCR-positive individuals were crossed with nontransgenic individuals, and about 50% of the resulting progeny carried the transgene, suggesting that the F(1) animals are germline transgenic. Integration of the transgenes was confirmed by detecting the junction fragments of the genomic DNA associated with transgene constructs. The expression of reporter genes was detected by reverse transcription (RT) PCR assay. These results showed that foreign genes could be reproducibly transferred into live-bearing fish and crustaceans by directly transforming the immature gonads with replication-defective pantropic retroviral vectors.
ABSTRACT
Transgenic animals have been routinely produced by microinjecting or electroporating naked DNA into 1-cell-stage embryos or unfertilized eggs. However, these techniques are inapplicable to live-bearing fish and many crustacean species for which unfertilized or newly fertilized eggs are not readily obtainable. In the present study, replication-defective pantropic retroviral vectors carrying a reporter gene (neo(R) or beta-gal) were used to directly transform the immature ovary or testis of a live-bearing fish (Poeciliopsis lucida) and crayfish (Procambarus clarkii). The fraction of the progeny derived from these treated individuals shown to contain the neo(R) reporter gene by an assay based on polymerase chain reaction (PCR) was significant. The PCR-positive individuals were crossed with nontransgenic individuals, and about 50% of the resulting progeny carried the transgene, suggesting that the F(1) animals are germline transgenic. Integration of the transgenes was confirmed by detecting the junction fragments of the genomic DNA associated with transgene constructs. Expression of reporter genes was detected by a reverse transcription-nested PCR assay. These results showed that transgenic live-bearing fish and crustaceans could be easily produced by directly transforming the immature gonads with replication-defective pantropic retroviral vectors.
ABSTRACT
The marine rotifer, Brachionus plicatilis, is able to digest Chlorella efficiently, suggesting that the rotifer contains a powerful cellulolytic enzyme system. A multi-component cellulolytic complex, including endoglucanase (CM-cellulase), cellobiohydrolase and beta-glucosidase, was found in Brachionus plicatilis. Endoglucanase (endo-beta-1,4 glucanase) was purified to homogeneity from rotifer homogenates using a sequential chromatographic method. The purified enzyme exhibits a strong hydrolytic activity with carboxymethyl(CM)-cellulose. The optimum temperature and pH for the endoglucanase activity were 37 degrees C and 7.0, respectively. 80% of the CM-cellulase activity was retained in salt mixture that ranged from 150 to 500 mM NaCl equivalent. The purified protein was isolated with a molecular weight of approximately 62 kDa estimated by SDS-polyacrylamide gel electrophoresis.
