Barnase gene inserted in the intron of GUS--a model for controlling transgene flow in host plants. Kuvshinov, V., Anissimov, A., & Yahya, B., M. Plant Science, 2004. abstract bibtex Public concern on possible risk of transgene flow from GM plants calls for development of reliable molecular techniques to control transgene escape from target plant into the natural and cultural environment. Reliability of the molecular systems controlling the transgene flow can however, be affected by potential rearrangements of genomic DNA. To address this problem, we introduce here an enhanced reliability Recoverable Block of Function (RBF) system where the blocking construct is inserted in a large artificial intron of a transgene of interest. This arrangement minimizes chances of transgene escape by increasing the genetic linkage between the lethal blocking gene and the gene of interest. We used barnase gene as a model of the blocking gene and GUS gene as a model of the transgene of interest. Blocking construct was inseparably linked to the transgene of interest by optimizing the coding sequence and polyadenylation signal of barnase for a high AT content and placing it in the artificial second intron of the GUS gene in such way that the transcription of GUS and barnase proceeds in opposite directions. Expression of barnase is regulated by the embryo specific cysteine endopeptidase promoter placed outside the third exon of GUS. The artificially designed splicing and polyadenylation signals were precisely recognized by the plant mRNA processing machinery. Expression levels and biological activities of both the genes matched the levels of respective native genes. Simultaneous expression of barnase and GUS led to silencing of the genes, possibly due to dsRNA formation. We suggest that the silencing effect can be avoided by placing the blocking gene and the transgene of interest in same orientation and using a mutual polyadenylation signal for the blocking gene and the transgene of interest. We have demonstrated here that it is possible to develop an artificial intron encoding another functional gene that is unknown in natural plant genome.
@article{
title = {Barnase gene inserted in the intron of GUS--a model for controlling transgene flow in host plants},
type = {article},
year = {2004},
keywords = {GUS,barnase,gene confinement},
volume = {In Press, },
websites = {http://www.sciencedirect.com/science/article/B6TBH-4C6KKHX-4/2/588fd0d5645b96e877768f3f85a16d5f},
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abstract = {Public concern on possible risk of transgene flow from GM plants calls for development of reliable molecular techniques to control transgene escape from target plant into the natural and cultural environment. Reliability of the molecular systems controlling the transgene flow can however, be affected by potential rearrangements of genomic DNA. To address this problem, we introduce here an enhanced reliability Recoverable Block of Function (RBF) system where the blocking construct is inserted in a large artificial intron of a transgene of interest. This arrangement minimizes chances of transgene escape by increasing the genetic linkage between the lethal blocking gene and the gene of interest. We used barnase gene as a model of the blocking gene and GUS gene as a model of the transgene of interest. Blocking construct was inseparably linked to the transgene of interest by optimizing the coding sequence and polyadenylation signal of barnase for a high AT content and placing it in the artificial second intron of the GUS gene in such way that the transcription of GUS and barnase proceeds in opposite directions. Expression of barnase is regulated by the embryo specific cysteine endopeptidase promoter placed outside the third exon of GUS. The artificially designed splicing and polyadenylation signals were precisely recognized by the plant mRNA processing machinery. Expression levels and biological activities of both the genes matched the levels of respective native genes. Simultaneous expression of barnase and GUS led to silencing of the genes, possibly due to dsRNA formation. We suggest that the silencing effect can be avoided by placing the blocking gene and the transgene of interest in same orientation and using a mutual polyadenylation signal for the blocking gene and the transgene of interest. We have demonstrated here that it is possible to develop an artificial intron encoding another functional gene that is unknown in natural plant genome.},
bibtype = {article},
author = {Kuvshinov, Viktor and Anissimov, Andrei and Yahya, Bukhari M},
journal = {Plant Science}
}
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Expression of barnase is regulated by the embryo specific cysteine endopeptidase promoter placed outside the third exon of GUS. The artificially designed splicing and polyadenylation signals were precisely recognized by the plant mRNA processing machinery. Expression levels and biological activities of both the genes matched the levels of respective native genes. Simultaneous expression of barnase and GUS led to silencing of the genes, possibly due to dsRNA formation. We suggest that the silencing effect can be avoided by placing the blocking gene and the transgene of interest in same orientation and using a mutual polyadenylation signal for the blocking gene and the transgene of interest. 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