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Title: CODON OPTIMIZATION, GENETIC INSULATION, AND AN RTTA REPORTER IMPROVE PERFORMANCE OF THE TETRACYCLINE SWITCH.

Author
item Wells, Kevin
item Foster Frey, Juli
item Moore, Karen
item Pursel, Vernon
item Wall, Robert

Submitted to: Transgenic Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/14/1999
Publication Date: N/A
Citation: N/A

Interpretive Summary: When transgenes from one species are transferred into another species, they do not always function as expected. In addition, frequently it is useful to have a transgene function in a different tissue or at a different time than it would normally function. There are strategies based on a bacterial DNA binding protein that uses two transgenes to allow one of the transferred genes to be controlled. This system requires complicated breeding, is difficult to use in livestock, is not dependable, and is difficult to measure. It would therefore be useful to be able to easily control the transgenes that are experimentally introduced into farm animals and to be able to measure their dependability. A series of experiments were conducted in cultured cells to improve control of expression in transgenes. First, we found that all of the individual parts of a gene control strategy could be combined into a single construction. Secondly, we found that modifications to the transgene that controls the system allows more of that gene product to be produced. The modification involved changing the bacterial DNA sequences to simulate mamammalian gene. Lastly, we found that the addition of a region from a third gene results in the system that functions more often. These results are important because control of experimentally introduced genes is required before many scientific and commercial uses of this technology can be applied to improve animal agriculture.

Technical Abstract: The objective of this work was to develop a system that allows control of transgene expression. First, to circumvent the need for a binary approach, a single plasmid design was constructed and tested in tissue culture. To indirectly assay the level of rtTA expression, a dicistronic gene was built which included an internal ribosome entry site and a GFP coding region (IRES/GFP) on the same expression cassette as the coding region of rtTA (pTetGREEN). This construct did not produce fluorescent colonies when stably integrated and provided minimal expression of GFP in face of adequate expression of rtTA. The coding region of TetR was then altered by introducing 156 silent point mutations. These mutations changed the codon bias to simulate a mammalian gene. Replacement of the TetR coding region in pTetGREEN with this "mammalianized" version of TetR provided GFP expression. The adjustment of codon usage in the TetR region of rtTA nearly doubled the expression level of functional rtTA. To increase the number rtTA expressing lines, the chicken egg white lysozyme matrix attachment region (MAR) was introduced into a single plasmid design just upstream of the inducible gene. Inclusion of the MAR doubled the number of colonies that expressed rtTA (44% vs 88%). With the modifications described here, the number of lines that express rtTA and provide induction from a single plasmid design can be increased by the inclusion of a MAR and the level of rtTA expression can be further increased by adjustment of the base composition of the TetR coding region.