|Authors: ||Y. Takikawa, H. Toyoda, Y. Matsuda, T. Nonomura, K. Kakutani|
|Keywords: ||RT-PCR, nested PCR, chitinase gene, retrotransposon, inducible expression gene, constitutive expression gene|
A microscopy-based needle micromanipulation technique for direct injection of foreign material into target cells was refined as a novel method of aspiration of cellular contents from a single target cell using a micropipette.
Subsequently, we performed direct PCR amplification of mRNAs transcribed in target tomato callus cells.
Friable calli were induced from leaf explants, and single cells from active small cell aggregates were used as targets for aspiration.
The aspirated cellular contents were subjected to RT-PCR and subsequent nested PCR amplification analyses.
Five tomato genes (LHA2, GAPDH, CHI3, PI2 and TLC1) were selected from the cDNA database as PCR targets.
The GAPDH and LHA2 genes were amplified from all aspirated samples and were used as indicators of successful PCR amplification.
Aspiration of only the cytosol (excluding the nucleus), facilitated amplification of mature target gene mRNA that was not contaminated with PCR products derived from the genomic DNA sequences of the target genes.
We identified novel stimulus-activated genes, such as CHI3 and TLC1, which were constitutively transcribed in tomato callus cells.
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