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Comparison of Various Transfection Methods in Human and Bovine Cultured Cells

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Comparison of Various Transfection Methods in Human and Bovine Cultured Cells International Journal of Oral Biology, Vol. 39, No. 4 December 31 2014, p. 177-185 Copyright ⓒ 2014, The Korean Academy of Oral Biology http://dx.doi.org/10.11620/IJOB.2014.39.4.177 Comparison of Various Transfection Methods in Human and Bovine Cultured Cells Longxun Jin, Daehwan Kim, and Sangho Roh Cellular Reprogramming & Embryo Biotechnology Laboratory, Dental Research Institute and School of Dentistry, Seoul National University, Korea (received September 15, 2014; revised November 20, 2014; accepted November 24, 2014) Transfection is a gene delivery tool that is a popular means NeonTM electroporation can be used to deliver foreign genes of manipulating cellular properties, such as induced efficiently in human and bovine somatic cells. pluripotent stem cell (iPSC) generation by reprogramming factors (Yamanaka factors). However, the efficiency of Key words: transfection, magnetofection, non-liposomal transfection needs to be improved. In the present study, three transfection, electroporation transfection protocols - non-liposomal transfection (NLT), magnetofection and electroporation - were compared by analysis of their transfection efficiencies and cell viabilities Introduction using human dental pulp cells (hDPC) and bovine fetal fibroblasts (bFF) as cell sources. Enhanced green fluorescent Viral vectors are commonly used to generate induced protein gene was used as the delivery indicator. For pluripotent stem cells (iPSCs) because of their high efficiency magnetofection, Polymag reagent was administrated. NLT, to introduce foreign DNA into mammalian cells [1-3]. FuGENE-HD and X-treme GENE 9 DNA transfection reagents were used for NLT. For electroporation, the NeonTM However, virus-free and/or integration-free plasmids in and NEPA21TM electroporators were tested. NeonTM combination with non-viral transfection methods are often electroporation showed highest transfection efficiency when recommended due to the concerns of clinical application [4,5]. compared with NLT, magnetofection, and NEPA21TM So far, many scientific research groups have introduced electroporation, with transfection efficiency of about 33% in numerous devices or materials for non-viral transfection hDPC and 50% in bFF, based on viable cell population in methods including diethylaminoethyl-dextran (DEAE-dextran), each cell type. These results suggest that transfection by calcium phosphate, liposome (lipofection), non-liposomal based transfection (NLT) reagent, polymer, magnetic bead and *Correspondence to: Sangho Roh, Cellular Reprogramming and electroporator. Embryo Biotechnology Laboratory, Seoul National University DEAE-dextran and calcium phosphate are the chemicals School of Dentistry 101 Daehak-Ro Jongno-gu Seoul 110-744 that are traditionally used to increase transfection efficiency of Republic of Korea Tel: +82-2-740-8681; Fax: +82-2-745-1907 viral RNA and DNA into mammalian cells [6,7]. Due to their E-mail: [email protected] positively charged property, they can easily bind to DNA, form co-precipitates and ultimately deliver DNA either into This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creati- nucleus or cytoplasm of the cells [8,9]. Liposome is one of vecommons.org/licenses/by-nc/3.0) which permits unrestricted non- the widely used transfection methods, taking advantage of the commercial use, distribution, and reproduction in any medium, pro- vided the original work is properly cited. ability to fuse with lipid bilayers of the cells, and this 177 178 Longxun Jin, Daehwan Kim, and Sangho Roh endocytosis-like action can easily uptake foreign DNA and the final vector product is more than twofold longer than the induce the expression of introduced gene [10]. Other than monocistronic vector, often larger than 10 kb, and this liposomal based transfection, recently developed non- reversely decreases transfection efficiency [33]. Therefore the liposomal based transfection reagent also became a potent optimal transfection protocol needs be investigated to elevate transfection tool [11]. Electroporation makes electric field transfection efficiency when delivering longer plasmid DNA which drives negatively charged DNA into targeting cells into cells. Despite a series of successful reports on [12,13]. Among polymers developed, polyethyleneimine (PEI) establishment of iPSCs, low efficiency is still observed in and poly-beta-amino ester are effective “proton sponge” that human or large animal iPSCs when compared with a murine can facilitate the transfection efficiently [14,15]. Using the model. Therefore, an optimal transfection protocol is required magnetic bead is in the limelight among recent transfection to the cells of these species. methods because of its relatively higher transfection efficiency In the present study, the episome-derived enhanced green and less toxic effects when compared with lipofection and fluorescent protein (eGFP) vector was used to transfect bovine NLT methods [16]. For magnetofection, PEI which binds to fetal fibroblasts (bFF) and human dental pulp cells (hDPC) to DNA is coated over magnetic bead [17]. Then magnetic force compare three different transfection protocols, NLT, exerted by magnetic plate attracts magnetic bead-PEI-DNA magnetofection and electroporation by examining their complex into cells [18]. transfection efficiency and cell viability. This may elucidate In general, primary cell cultures are more difficult to be transfection efficacy of larger plasmid when applying to transfected than immortalized cell lines. Recent reports human and bovine somatic cells. suggest that transfection of a specific factor could ameliorate some injured tissues when subsequently transplant the transfected primary cells [19]. Hence, improving transfection Materials and Methods efficiency for cultured primary cells is at the forefront of gene-, cell-based therapy, tissue engineering and regenerative Chemicals and plastic wares medicine [20]. Moreover, there is less information about All inorganic and organic compounds were obtained optimal transfection protocol for bovine primary cultured cells from Life Technologies (Grand Island, NY, USA) unless [21,22]. To increase the transfection efficiency other than indicated in the text. All plastic ware were purchased from viral infection in primary cell cultures, various transfection Corning (One Riverfront Plaza, NY, USA). methods has been developed [23-25]. Primary culture of human and bovine cells Recent advancement of transfection technologies can To isolate human dental pulp tissue, mesiodens (maxillary achieve iPSC generation using a couple of non-viral central supernumerary teeth) were extracted from children at transfection methods above mentioned [4,26]. Due to its high the Department of Pediatric Dentistry in Dental Hospital of efficiency for non-viral plasmid transfection, electroporation Seoul National University according to the guidelines has been applied to iPSC generation from the cells in various provided by ethics committee (S-D20100005). Dental pulp mammalian species [27-29]. Other than electroporation, tissues were dissociated using 1% (w/v) collagenase type I, polymer-based transfection and magnetofection were also 2.4 mg/ml dispase and 0.25% (v/v) trypsin, and then successfully applied to generate iPSCs in mouse model proceeded to whole-tissue culture. After culturing for several [30,31]. In spite of the iPSC's successful application, its days, hDPC can be observed propagating from center of the comparably lower efficiency of virus-free transfection than tissue to the peripheral area. The hDPC were isolated from viral transfection is remained as the biggest hurdle to be two individual children were named to hDPC-1 and hDPC-2, overcome [32]. When generating iPSCs, poor efficiency is respectively. mainly caused by individual co-transfection of several vectors. The bFF were isolated from femoral skins of 60 – 90 days To overcome this problem, a polycistronic vector is cloned old fetuses. Bovine femoral skins were dissociated with and applied by many groups [29]. In this case, however, as 0.25% trypsin for 30 min and dissected using blades. Cells several factors need to be cloned in one polycistronic vector, are well propagated after culturing in dishes. To eliminate Comparison of Various Transfection Methods in Human and Bovine Cultured Cells 179 heterogeneous cell populations, the cells were cultured at least OptiMEM and subsequently mixed with triple volume of for five passages before transfection. Polymag (Chemicell GmbH, Berlin, Germany) for 20 min in room temperature. After replacing culture medium, Plasmid DNA preparation Polymag-DNA complex dissolved in OptiMEM was added to pCXLE episomal DNA (plasmid 27082; Addgene, the cells in culture, then incubated in a humidified 37℃ Cambridge, MA, USA) was obtained from Addgene. Midi incubator. prep was carried out either by using HiSpeed Midi Kit Electroporation was conducted using either NEPATM or (Qiagen, Valencia, CA, USA) or Purelink Maxi Kit. Final NeonTM transfection system. In NEPATM electroporation plasmid DNA concentration was used between 1.0 μg/μl to system, for poring pulse, voltage, length, interval, number, 1.5 μg/μl. decay rate, polarity were set respectively as 150 V, 5 ms, 50 ms, 2, 10%, ‘+’ for hDPC, and 275 V, 1.5 ms, 50 ms, 2, 10% Cell culture and ‘+’ for bFF. For transfer pulse, voltage, length, interval, The bFF were cultured in Dulbecco’s modified Eagle’s number, decay rate, polarity were set respectively as 20 V, 50 medium
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