PubMed · 9554480
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T W Prior. 1998. Determining gene dosage.. https://pubmed.ncbi.nlm.nih.gov/9554480/
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BACKGROUND: When generating transformed plants, a first step in their characterization is to obtain, for each new line, an estimate of how many copies of the transgene have been integrated in the plant genome because this can deeply influence the level of transgene expression and the ease of stabilizing expression in following generations. This task is normally achieved by Southern analysis, a procedure that requires relatively large amounts of plant material and is both costly and labour-intensive. Moreover, in the presence of rearranged copies the estimates are not correct. New approaches to the problem could be of great help for plant biotechnologists. RESULTS: By using a quantitative real-time PCR method that requires limited preliminary optimisation steps, we achieved statistically significant estimates of 1, 2 and 3 copies of a transgene in the primary transformants. Furthermore, by estimating the copy number of both the gene of interest and the selectable marker gene, we show that rearrangements of the T-DNA are not the exception, and probably happen more often than usually recognised. CONCLUSIONS: We have developed a rapid and reliable method to estimate the number of integrated copies following genetic transformation. Unlike other similar procedures, this method is not dependent on identical amplification efficiency between the PCR systems used and does not need preliminary information on a calibrator. Its flexibility makes it appropriate in those situations where an accurate optimisation of all reaction components is impossible or impractical. Finally, the quality of the information produced is higher than what can be obtained by Southern blot analysis.
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We describe a variation of the method to generate single-copy transgenic plants by recombinase-mediated resolution of multiple insertions. In this study, a transgene construct flanked by oppositely oriented lox sites was co-bombarded into maize cells along with a cre-expressing construct. From analysis of the regenerated plants, a high percentage of the primary transformants harbored a single copy of the introduced transgene, and among these, a majority also lacked the cre construct. We deduce that the expression of cre must have contributed to resolving concatemeric molecules either prior to or after DNA integration into the maize genome.