Developmental and Evaluation of Advanced Expression Vectors with Both Enhanced Integration and Stable Expression for Transgenic Farmed Fish

The objectives of the project were to develop expression vectors using the Sleeping Beauty transposon technology and the genetic border elements to provide both enhanced integration rate and stable transgene expression, and to evaluate the application of such vectors in farmed fish such as catfish and carp. The panel recommended adding the objective of evaluating the endogenous transposable elements, particularly in catfish, in order to evaluate the applicability of the expression vectors while reduc1ng efforts in real production of transgenic fish considering the focus of the project was to develop the vector and evaluation of its applicability, not producing transgenic fish. Efficient production of transgenic farmed fish is hindered by two major problems: mosaicism due to delayed integration after single-cell stage, and silencing of transgene expression. In this project, we proposed to combat these problems by coupling the Sleeping Beauty transposon technology that can enhance integration rate and the border elements that can insulate transgene from position effect. Our major objective was to develop a new generation of expression vector that contains both of these elements. We have developed expression vectors containing both the Sleeping Beauty transposon signals, inverted repeats and direct repeats (IR and DR, respectively), and the border elements, scs and scs'. Growth hormone minigene has been cloned into this vector for applications of such vectors in growth enhancement. Luc reporter gene has been also cloned into this vector cascades for relative easy evaluation of transgene expression. Transgenic fish have been produced using these expression vectors in both catfish (US) and carp (Israel). Much effort was also devoted to evaluation of the endogenous transposable elements in catfish as recommended by the BARD grant panel. Multiple families of Tcl-like transposons were identified from catfish. Surprisingly, many Tc I-related transcripts were identified. Among these transcripts, both the sense and antisense transcripts were present. Some of the transcripts may be useful for development of novel transposase-based technology for aquaculture applications in the future. This project has both scientific and aquaculture implications. First, to develop expression vectors containing both IR/DR and scs/scs' repeated elements have been reported being extremely technically difficult due to excision of the repeated sequences by the E. coli host during cloning processes. We have successfully constructed this advanced vector that contained very complex cascades for both gene integration and gene regulation. We have produced transgenic fish using such vectors. This advanced expression vector should be useful for production of transgenic fish. By simply replacing the growth hormone gene, any gene of interest can be readily inserted in this vector. Thus this vector should provide technological possibility for early integration and stable expression of any economically important genes in aquaculture. We have also evaluated the applications of the Sleeping Beauty-based vectors in terms of the impact of gene size and found that the size of trans gene drastically affects transposition. The system will be only useful for transferring genes smaller than 5.6 kb. We have also identified novel transposase-related transcripts that may be useful for the development of novel transposase-based technologies for general scientific research and for aquaculture applications.