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Tzfira, T. et al. pSAT vectors: a modular series of plasmids for fluorescent protein tagging and expression of multiple genes in plants. Plant Mol. Biol. 57, 503-516

Hebrew University of Jerusalem, Yerushalayim, Jerusalem, Israel
Plant Molecular Biology (Impact Factor: 4.26). 04/2005; 57(4):503-16. DOI: 10.1007/s11103-005-0340-5
Source: PubMed

ABSTRACT

Autofluorescent protein tags represent one of the major and, perhaps, most powerful tools in modern cell biology for visualization of various cellular processes in vivo. In addition, advances in confocal microscopy and the development of autofluorescent proteins with different excitation and emission spectra allowed their simultaneous use for detection of multiple events in the same cell. Nevertheless, while autofluorescent tags are widely used in plant research, the need for a versatile and comprehensive set of vectors specifically designed for fluorescent tagging and transient and stable expression of multiple proteins in plant cells from a single plasmid has not been met by either the industrial or the academic communities. Here, we describe a new modular satellite (SAT) vector system that supports N- and C-terminal fusions to five different autofluorescent tags, EGFP, EYFP, Citrine-YFP, ECFP, and DsRed2. These vectors carry an expanded multiple cloning site that allows easy exchange of the target genes between different autofluorescence tags, and expression of the tagged proteins is controlled by constitutive promoters, which can be easily replaced with virtually any other promoter of interest. In addition, a series of SAT vectors has been adapted for high throughput Gateway recombination cloning. Furthermore, individual expression cassettes can be assembled into Agrobacterium binary plasmids, allowing efficient transient and stable expression of multiple autofluorescently tagged proteins from a single vector following its biolistic delivery or Agrobacterium-mediated genetic transformation.

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    • "Plant vacuoles from tpc1 knock-out plants appeared ideal to express animal TPC2; since plant vacuoles and animal lysosomes have several physiological functions in common, the working hypothesis was that the mechanisms of vacuolar/lysosomal targeting may be similar and conserved during evolution. Alex Costa, at that time in the group of Fiorella Lo Schiavo, cloned the human TPC2 fused with an EGFP to its C-terminus, into the plant expression vector pSAT6 [43]. Transient transformation of isolated mesophyll protoplasts [44] showed, with great satisfaction, that hTPC2 at least partially localized to the vacuolar membrane [42]. "
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    • "was subcloned into the commercial pMOS vector (GE Healthcare) by PCR using the following pair of primers, both containing BamHI restriction sites (underlined): 59-GTAACTGGATCCACAGGAAGATGG-39 and 59-AGCCAAGGATCCAGTATCTATGAG-39. The complementary DNA sequence was PCR amplified and cloned into pGEMHE (Liman et al., 1992) for oocyte expression and into pSAT6-EGFP-N1 (Tzfira et al., 2005) for transientPlant Physiol. Vol. "
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    • "We employed a simple visual assay to assess the transfection efficiency using fluorescence microscopy. We transfected protoplasts with the pSAT plasmid, expressing the nuclear-targeted redshifted GFP (EGFP) driven by two cauliflower mosaic virus 35S promoters, oriented in tandem (Tzfira et al., 2005). Protoplasts were transfected using a poly(ethylene glycol) (PEG)-calcium-mediated transfection procedure as previously described (Yoo et al., 2007), except that we used 10 times more protoplasts than detailed in (Yoo et al., 2007). "
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