Non-destructive analysis of the nuclei of transgenic living cells using laser tweezers and near-infrared raman spectroscopic technique.

Transgenic cell lines of loblolly pine (Pinus taeda L.) were analyzed by a compact laser-tweezers-Raman-spectroscopy (LTRS) system in this investigation. A low power diode laser at 785 nm was used for both laser optical trapping of single transgenic cells and excitation for near-infrared Raman spectroscopy of the nuclei of synchronized cells, which were treated as single organic particles, at the S-phase of the cell cycle. Transgenic living cells with gfp and uidA genes were used as biological samples to test this LTRS technique. As expected, different Raman spectra were observed from the tested biological samples. This technique provides a high sensitivity and enables real-time spectroscopic measurements of transgenic cell lines. It could be a valuable tool for the study of the fundamental cell and molecular biological process by trapping single nucleus and by providing a wealth of molecular information about the nuclei of cells.

Post-transcriptional gene silencing induced by short interfering RNAs in cultured transgenic plant cells.

Short interfering RNA (siRNA) is widely used for studying post-transcriptional gene silencing and holds great promise as a tool for both identifying function of novel genes and validating drug targets. Two siRNA fragments (siRNA-a and -b), which were designed against different specific areas of coding region of the same target green fluorescent protein (GFP) gene, were used to silence GFP expression in cultured gfp transgenic cells of rice (Oryza sativa L.; OS), cotton (Gossypium hirsutum L.; GH), Fraser fir [Abies fraseri (Pursh) Poir; AF], and Virginia pine (Pinus virginiana Mill.; PV). Differential gene silencing was observed in the bombarded transgenic cells between two siRNAs, and these results were consistent with the inactivation of GFP confirmed by laser scanning microscopy, Northern blot, and siRNA analysis in tested transgenic cell cultures. These data suggest that siRNA-mediated gene inactivation can be the siRNA specific in different plant species. These results indicate that siRNA is a highly specific tool for targeted gene knockdown and for establishing siRNA-mediated gene silencing, which could be a reliable approach for large-scale screening of gene function and drug target validation.