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. 2004 May;2(2):97-108.
doi: 10.1016/s1672-0229(04)02015-7.

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

Wei Tang  1 , Vanessa Samuels, Nicki Whitley, Nicole Bloom, Tinya DeLaGarza, Ronald J Newton
Affiliations

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

Wei Tang et al. Genomics Proteomics Bioinformatics. 2004 May.

Abstract

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.

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Figures

Fig. 1 A and B
Fig. 1 A and B
Linear maps of m-gfp5-ER gene indicating the localization of the m-gfp5-ER, a modified GFP protein with an endoplasmic reticulum targeting sequence. CaMV35SPro, the cauliflower mosaic virus 35S promoter; NosTer, the terminator from nopaline synthase gene. Arrows indicate gene translation orientation. The probe used in Northern blot analysis of transgenic cells is the 816-bp fragment of the m-gfp5-ER gene. The sequences of sense and antisense siRNA were indicated immediately below the m-gfp5-ER gene, and the positions of siRNAs were between nucleotides 183 and 203 (siRNA-a) and between nucleotides 557 and 577 (siRNA-b) of the m-gfp5-ER gene.
Fig. 2
Fig. 2
Northern blot analysis of total RNA from transgenic cell lines of rice (OS), cotton (GH), Fraser fir (AF), and Virginia pine (PV). RNA (10 µg) was extracted from transgenic cells bombarded with two siRNAs 48 h after bombardment, and was hybridized at 65°C with the 816-bp m-gfp5-ER probe corresponding to the m-gfp5-ER gene, which was labeled with DIG. The control panel is Virginia pine gfp transgenic cells that did not bombarded with siRNA. The integrity and the amount of RNA applied to each lane were verified by the control of 25S rRNA (lower panel).
Fig. 3
Fig. 3
Laser scanning microscopy of silenced GFP expression by two siRNAs in transgenic cells 48 h after bombardment with siRNAs in rice (OS), cotton (GH), Fraser fir (AF), and Virginia pine (PV). The control is the GFP expression in Virginia pine gfp transgenic cells not bombarded with siRNAs. GFP fluorescence was decreased more in all transgenic cells after bombardment with siRNA-b than with siRNA-a. No GFP fluorescence was observed in non-transgenic control cells (bars = 0.04 mm).
Fig. 4
Fig. 4
Northern blot analysis of total RNA from transgenic cell lines of Virginia pine (PV). RNA (10 µg) was extracted from transgenic cells bombarded with two siRNAs at 0 (transgenic control), 12, 24, 36, 48, and 60 h after bombardment, and was hybridized at 65°C with the 816-bp m-gfp5-ER probe corresponding to the m-gfp5-ER gene, which was labeled with DIG. The control panel is Virginia pine gfp transgenic cells that did not bombarded with siRNA. The integrity and the amount of RNA applied to each lane were verified by the control of 25S rRNA (lower panel).
Fig. 5
Fig. 5
Detection of silenced GFP expression by siRNA in transgenic cells and at a time course in Virginia pine (PV). The control is the GFP expression in gfp transgenic cells not bombarded with siRNA. GFP fluorescence in gfp transgenic cells was decreased at 12, 24, 36, 48, and 60 h after bombardment with siRNA. No GFP fluorescence was observed in non-transgenic control cells (bars = 0.04 mm).
Fig. 6
Fig. 6
Quantitative analysis of gfp expression silenced by siRNA in transgenic cells in rice (OS), cotton (GH), Fraser fir (AF), and Virginia pine (PV) 48 h after bombardment. GFP fluorescence was expressed as fluorescence intensity (arbitrary unit). Fluorescence of gfp transgenic cells without bombardment with siRNA was also presented as a control. Experiments were repeated three times, and each replicate consisted of 30–50 cells. Values represent the means ± S.D.
Fig. 7
Fig. 7
Quantitative analysis of gfp expression silenced by siRNA in transgenic cells in Virginia pine (PV) at 0 (transgenic control), 12, 24, 36, 48, and 60 h after bombardment. GFP fluorescence was expressed as fluorescence intensity (arbitrary unit). Fluorescence of gfp transgenic cells without bombardment with siRNA was also presented as a control. Experiments were repeated three times, and each replicate consisted of 30–50 cells. Values represent the means ± S.D.
Fig. 8
Fig. 8
Detection of small RNAs. Low molecular weight RNA fractions were isolated from transgenic cells of Virginia pine, separated on polyacrylamide gels, blotted onto Hybond N+ membranes, and hybridized with 816-bp gfp-coding sequences. The 21-nt siRNA oligomers were used as size controls (size indicated in nucleotides). Each numbered lane contains the low molecular weight RNA fraction of transgenic cells bombarded with siRNA-a and siRNA-b. No specific signal could be detected in transgenic cells without bombardment of siRNAs with the probes. Lane A: transgenic cells bombarded with siRNA-a gave rise to small gfp-specific RNAs of approximately 21 nucleotides. Lane B: transgenic cells bombarded with siRNA-b gave rise to small gfp-specific RNAs of approximately 21 nucleotides. Lane C: gfp transgenic cells without bombardment with siRNA was also presented as a control. Lane M: the 21-nt small gfp-specific RNAs were used as a marker.
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