Gene 578 (2016) 1–6 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene Review Pros and cons of pDNA and mRNA transfection to study mRNA translation in mammalian cells Dmitry E. Andreev a,⁎,IlyaM.Terenina,b, Sergey E. Dmitriev a,b, Ivan N. Shatsky a,⁎ a Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia b Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia article info abstract Article history: Protein synthesis in eukaryotes is subject to stringent control. The misregulation of translation of certain mRNAs Received 25 October 2015 is often a hallmark of many diseases, including malignancies and autoimmune disorders. To understand why and Accepted 7 December 2015 how it happens, it is important to investigate the translational control of specificmRNAs.Inthiscase,onecould Available online 8 December 2015 use reporter mRNAs in order to identify cis-acting elements responsible for regulation. Here we overview plasmid DNA (pDNA) and mRNA transfections, their pitfalls and limitations, as well as some emerging applications for Keywords: Plasmid DNA mRNA transfection. Reporter gene © 2015 Elsevier B.V. All rights reserved. mRNA Transfection Translation Gene therapy Contents 1. Introduction............................................................... 1 2. DNAreporters.............................................................. 2 3. mRNAtransfection............................................................ 2 4. Conclusions............................................................... 5 Acknowledgements.............................................................. 5 References.................................................................. 5 1. Introduction how translation is regulated in living cells, but the main limitation of this technique is that it cannot explain how particular mRNA species During the last decade, we rapidly entered the “omics” era. It are regulated under specific conditions. In other words, we now know changed many areas of life sciences and, for instance, studies related (or could know if we wish) what is regulated and when it is regulated, to regulation of gene expression. In particular, a breakthrough in next but we do not understand how this is achieved. generation sequencing technology allowed scientists to develop the In order to investigate mechanisms of translational control of specif- ribosome profiling technique (Ingolia et al., 2009), which for the first ic mRNAs in detail, the initial step is to address the translation behavior time addressed translation at the whole transcriptome scale. Without of a particular mRNA and then to find and dissect cis-acting elements doubt, this powerful approach has already changed our understanding responsible for such behavior. For this, the use of reporter constructs is essential. It is thought that most of the features that affect the efficiency of Abbreviations: pDNA, plasmid DNA; UTR, untranslated region; DRB, 5,6-dichloro-1-β- translation of mammalian mRNAs are located in 5′ and 3′ untranslated D-ribobenzimidazole; TLR, Toll-like receptor; ARCA, anti-reverse cap analogue; MATra, regions. The 5′-UTR or 5′ leader needs to be traversed by the 43S magnet-assisted transfection; MEF, mouse embryonic fibroblasts; ES, embryonic stem preinitiation complex on its way to the translation initiation site, and cells; Ψ, pseudouridine; IRES, internal ribosome entry site. hence different features within the 5′ leader may facilitate or inhibit ⁎ Corresponding authors. ′ E-mail addresses: [email protected] (D.E. Andreev), [email protected] translation initiation. 3 -UTRs are believed to serve as regulatory plat- (I.N. Shatsky). forms for various effectors such as RNA binding proteins and miRNAs, http://dx.doi.org/10.1016/j.gene.2015.12.008 0378-1119/© 2015 Elsevier B.V. All rights reserved. 2 D.E. Andreev et al. / Gene 578 (2016) 1–6 which in turn may affect mRNA stability, localization, and translation Another important issue for pDNA transfection is that multiple aber- efficiency (Sonenberg and Hinnebusch, 2009; Jackson et al., 2010; rant mRNA isoforms may be produced from the expression cassette. Hinnebusch, 2014). To address the relative impact of either 5′-UTR, or This might be either because of alternative promoters located in the 3′-UTR, or both on translation, reporter constructs are usually generated 5′-UTR, alternative or cryptic splicing, or a combination of both (for a re- where UTRs of interest flank a particular reporter gene. A huge selection view, see Shatsky et al., 2010). Importantly, cryptic promoters may be of reporter genes are available, including those expressing fluorescent present not only in the inserted fragment but also in reporter gene itself reporter proteins, which can be directly measured in living cells, secret- (Vopalensky et al., 2008), or in the vector backbone (Lemp et al., 2012). ed reporters for measurement in the culture media, and enzymatic re- A good example of the latter is the presence of a cryptic mammalian porters, which can be measured after cell lysis. Important properties promoter in the pGEM-4Z cloning vector that was not designed for ex- of reporter proteins that should be considered are their half-life and pression in mammalian cells but nevertheless is able to efficiently drive time required for maturation. The question addressed in this review is reporter expression (Chauhan et al., 2009). Importantly, cryptic pro- whether to use a pDNA transfection or mRNA transfection in cultured moters and/or splice sites may result in the production of reporter cells? We will share our experience regarding these techniques and mRNAs with unexpected 5′-UTRs, which either lack regulatory features highlight artifacts and caveats as well as advantages for each of them. of the original 5′-UTR or acquire some from vector backbone. Even low amounts of aberrant transcripts which are hard to detect by convention- al methods, such as Northern-blot, can significantly affect the results of 2. DNA reporters very sensitive reporter assays (Van Eden et al., 2004; Kozak, 2005, 2006; Gendra et al., 2007; Belancio, 2011). Searching for such aberrant tran- DNA reporters are plasmids (pDNA) where the expression of a scripts significantly increases the number of control experiments re- reporter gene, flanked by UTRs of interest, is driven by either a constitu- quired to perform. tively active (such as SV40, CMV etc), cell/tissue specific, or inducible pro- Moreover, various vector backbones can produce multiple cryptic moter. It is generally accepted that pDNA are translocated via endosomal non-coding transcripts that can directly influence reporter activity. uptake, cytosolic release, and nuclear entry. Because of the latter, a major Nejepinska et al. (2012) analyzed transcriptomes derived from various drawback of pDNA transfection is that it is usually ineffective for non- transiently transfected plasmids by means of high-throughput sequenc- dividing cells. ing. They detected unexpectedly complex spurious transcripts derived A combination of pDNAs bearing various reporters may be delivered from many parts of defined vector backbones, for example, a unique into cultured cells, and after a defined period of post-transfection, the population of edited sense and antisense small RNAs derived from the reporter activities may be estimated. Importantly, using single-cell Kan/Neo resistance cassette. Importantly, some vectors, when co- expression measurements from a pDNA expressing EGFP, the onset transfected, can inhibit the expression of luciferase reporters in a of fluorescence is detected 2 hours to 20 hours post-transfection dose-dependent manner. One possible explanation is that spurious (Leonhardt et al., 2014).This dispersion is probably a result of pDNA transcripts generated from the vector backbone may form dsRNAs and entry into the nucleus during cell division in unsynchronized cells then activate PKR, which in turn phosphorylates eIF2, thus leading to (Wilke et al., 1996; Mortimer et al., 1999; James and Giorgio, 2000; translation repression (Garcia et al., 2007; Sadler and Williams, 2007). Akita et al., 2007). Another explanation for such kinetics is stochasticity Finally, it should be considered that pDNA may be immunogenic be- of mRNA synthesis in mammalian cells, also known as intrinsically ran- cause the presence of DNA in the cytoplasm is not normal for eukaryotic dom “transcription bursts” (Raj et al., 2006). cells. Indeed, it was noticed some time ago that bacterial DNA with non- There is a number of disadvantages of pDNA transfection, and methylated CpG motifs induce a strong immune response through Toll- because of that, the results of experiments must be considered with like receptor 9 (TLR9) (Hemmi et al., 2000). However, there are certain some caution. First, since pDNA transfection depends on mitotic activity lines of evidence indicating that the methylation status of CpG is not a of cells, conditions that affect cell cycle progression can also affect primary determinant of an immune response, as self DNA also has the reporter production. Indeed, cells arrested in the G1 phase of the cell ability to induce TLR9 activation if it enters the endosomal compartment cycle by treatment with aphidicolin exhibited 20-fold lower reporter in which the recognition takes place (Brencicova and Diebold, 2013). gene activity than asynchronous control cells upon pDNA transfection This immune response leads to the activation