BioTechniquesVol. 46, No. 1 CitationsOpen AccessCitationsNijsje Dorman†Nijsje Dorman††a freelance writer in Boston, MA.Search for more papers by this authorPublished Online:25 Apr 2018https://doi.org/10.2144/000113049AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Fertile GroundIsotope labeling, a popular tool for quantitative proteomics, is often performed in a tagging step after protein isolation. However, this workflow cannot correct for inconsistencies in sample handling. By contrast, in vivo metabolic labeling, exemplified by SILAC (stable isotope labeling with amino acids in cell culture), controls for uneven sample losses that occur prior to the liquid chromatography and mass spectrometry steps. SILAC has been used with cultured plant cells, but to date isotopic labeling for soil-grown plants has not been described. Using tomato plants as an example, Schaff et al. propose a new methodology they term SILIP, or stable isotope labeling in planta, which uses 15N-enriched fertilizer. The swap has no discernible effect on growth, and achieves over 98% incorporation of 15N across all plant tissues. This is a significant improvement over SILAC, for which incorporation rates of 70–80% have been reported for Arabidopsis cell culture (a consequence of autotrophic production of amino acids). Moreover, as this report in The Plant Journal explains, the ability to perform labeling of whole plants grown in soil opens up the option of studying plant-environment interactions, including symbiotic and parasitic relationships with rhizobacteria and nematodes, respectively.Tomato plant. Image reproduced with permission from Wikimedia Creative Commons, creativecommons.org/licenses/by 2.0-Schaff et al. SILIP: a novel stable isotope labeling method for in planta quantitative proteomic analysis. Plant J. [Epub ahead of print, Sep 5, 2008; doi: 10.1111/j.1365-313X.2008.03639.x].Regulatory AffairsThe ability to control alternative splicing would enable functional studies of different mRNA isoforms, and represents a therapeutic approach to diseases arising from splicing errors. Antisense RNA that masks particular splice sites or regulatory elements can alter splicing, but constitutive expression of these modulators limits their utility for functional studies and would raise safety concerns for gene therapy. Although regulating expression of protein-coding genes is routine, options for RNA-encoding cassettes are more limited. For example, because the U7 snRNA-based expression cassette directs both transcription and downstream processing, regulatory elements cannot be easily swapped in and out. Writing in Gene Therapy, Marquis et al. introduce a doxycycline-responsive gene silencing strategy that can tune expression of splicing modulators. In this system, the Tet repressor is fused to the heterochromatin-inducing KRAB protein; in the presence of dox, the Tet repressor recruits KRAB to tet operator sequences, silencing nearby genes. Marquis et al. show that this system achieves dox-dependent control of splicing in cell culture models of splicing defects involved in spinal muscular atrophy and β-thalassemia. Extent of splicing correction was 77% in the first case and near-total in the second test system, and remained equally responsive throughout several induction/repression cycles. Given the availability of tTR-KRAB transgenic mice and mouse models for human splicing defects, the authors suggest that their system should allow in vivo elucidation of the pathogenesis of splicing disorders.-Marquis et al. Doxycycline-controlled splicing modulation by regulated antisense U7 snRNA expression cassettes. Gene Ther. [Epub ahead of print, Aug 14, 2008; doi:10.1038/gt.2008.138].Delivery ChargeEfficient entry of exogenous DNA into cells is a prerequisite for gene therapy. The most efficient DNA delivery vehicles are recombinant viruses; however, worries about immunogenicity and safety persist. Recently, biofunctionalized nanoparticles have been introduced as potential nonviral gene delivery vehicles. A paper from Ghosh et al. appearing in ACS Nano explores the influence of size and charge of functionalized gold nanoparticles on transfection efficiency. Looking to histones for design clues, the authors selected gold nanoparticles 6 nm in diameter—the approximate size of a histone core protein—and functionalized them with ammonium groups to approximate the basic amino acids that stud the histone octamers and interact with the negatively charged DNA backbone. DNA complexed with nanoparticles decorated with trimethyl ammonium transfected Cos-1 cells less efficiently than DNA-polylysine complexes, and was associated with moderate cytotoxicity. Lysine-functionalized nanoparticle-DNA complexes were roughly the same size and charge as the trimethyl ammonium-conjugated nanoparticles, but showed over 5-fold enhanced transfection. However, using lysine dendrimers for functionalization resulted in the most condensed DNA-nanoparticle complexes (∼91 nm in diameter), significantly improved transfection (over 28 times better than polylysine alone), and showed no cytotoxic effects. These results reveal the potential for adjusting size and charge properties of DNA-binding nanoparticles and, given the bioinert properties of gold colloids, suggest a fruitful alternative to traditional virus-based gene transduction.-Ghosh et al. Efficient gene delivery vectors by tuning the surface charge density of amino acid-functionalized gold nanoparticles. ACS Nano [Epub ahead of print, Oct 24, 2008; doi:10.1021/nn800507t].