Mammalian cell transfection: the present and the future

Transfection is a powerful analytical tool enabling study of the function of genes and gene products in cells. The transfection methods are broadly classified into three groups; biological, chemical, and physical. These methods have advanced to make it possible to deliver nucleic acids to specific subcellular regions of cells by use of a precisely controlled laser-microcope system. The combination of point-directed transfection and mRNA transfection is a new way of studying the function of genes and gene products. However, each method has its own advantages and disadvantages so the optimum method depends on experimental design and objective

In situ/Subcellular localization of arabinogalactan protein expression by fluorescent in situ hybridization, FISH

31 p. Methods Mol Biol 2149:403-427The arabinogalactan proteins are highly glycosylated and ubiquitous in plants. They are involved in several aspects of plant development and reproduction; however, the mechanics behind their function remains for the most part unclear, as the carbohydrate moiety, covering the most part of the protein core, is poorly characterized at the individual protein level. Traditional immunolocalization using antibodies that recognize the glycosidic moiety of the protein cannot be used to elucidate individual proteins' distribution, function, or interactors. Indirect approaches are typically used to study these proteins, relying on reverse genetic analysis of null mutants or using a reporter fusion system. In the method presented here, we propose the use of RNA probes to assist in the localization of individual AGPs expression/mRNAs in tissues of Arabidopsis by fluorescent in situ hybridization, FISH. An extensive description of all aspects of this technique is provided, from RNA probe synthesis to the hybridization, trying to overcome the lack of specific antibodies for the protein core of AGPs.This work was financed by FEDER through the COMPETE program, and by Portuguese National funds through FCT, Fundação para a Ciência eTecnologia (Project PTDC/AGR-GPL/115358/2009 and FCT - 02-SAICT-2017 – POCI-01-0145-FEDER-027839) and PhD grant SFRH/BD/111781/2015), and received support from Spanish–Portuguese Joint Project Nº E 30/12. EU project 690946 ‘SexSeed’ (Sexual Plant Reproduction – Seed Formation) funded by H2020-MSCA-RISE-2015.Peer reviewe