Name: induction and analysis of oxidative stress in sleeping beauty transposon-transfected human retinal pigment epithelial cells
Uploaded: 2020-12-11T13:00:00
Description: Watch this Scientific Journal Video about Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells at JoVE.com

The authors would like to thank Gregg Sealy and Alain Conti for excellent technical assistance and Prof. Zsuzsanna Izsv&#225;k from the Max-Delbr&#252;ck Center in Berlin for kindly providing the pSB100X&#160;and pT2-CAGGS-Venus&#160;plasmids. This work was supported by the Swiss National Sciences Foundation and the European Commission in the context of the Seventh Framework Programme. Z.I was funded by European Research Council, ERC Advanced [ERC-2011-ADG 294742].

Procedures for the collection and use of human eyes were approved by the Cantonal Ethical Commission for Research (no. 2016-01726).
1. Cell isolation and culture conditions
<ol>
	<li>Human ARPE-19 cell line
	<ol>
		<li>Culture 5 x 105 ARPE-19 cells, a human RPE cell line, in Dulbecco&#39;s Modified Eagle&#39;s Medium/Nutrient Mixture F-12 Ham (DMEM/Ham&#180;s F-12) supplemented with 10% fetal bovine serum (FBS), 80 U/mL penicillin, 80 &#181;g/mL streptomycin, and 2.5 &#181;g/mL am...

<ol>
	<li>Zareba, M., Raciti, M. W., Henry, M. M., Sarna, T., Burke, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+in+ARPE-19+cultures:+Do+melanosomes+confer+cytoprotection.">Oxidative stress in ARPE-19 cultures: Do melanosomes confer cytoprotection.</a> Free Radical Biology and Medicine. 40 (1), 87-100 (2006).</li><li>Gong, X., Draper, C. S., Allison, G. S., Marisiddaiah, R., Rubin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+macular+carotenoid+lutein+in+human+retinal+pigment+epithelial+cells.">Effects of the macular carotenoid lutein in human retinal pigment epithelial cells.</a> Antioxidants. 6 (4), (2017).</li><li>Sacconi, R., Corbelli, E., Querques, L., Bandello, F., Querques, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Review+of+current+and+future+management+of+geographic+atrophy.">A Review of current and future management of geographic atrophy.</a> Ophthalmology and Therapy. 6, 69-77 (2017).</li><li>Al-Zamil, W. M., Yassin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+age-related+macular+degeneration:+a+review.">Recent developments in age-related macular degeneration: a review.</a> Clinical Interventions in Aging. 12, 1313-1330 (2017).</li><li>Kumar-Singh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+complement+membrane+attack+complex+in+dry+and+wet+AMD+-+From+hypothesis+to+clinical+trials.">The role of complement membrane attack complex in dry and wet AMD - From hypothesis to clinical trials.</a> Experimental Eye Research. 184, 266-277 (2019).</li><li>Ung, L., Pattamatta, U., Carnt, N., Wilkinson-Berka, J. L., Liew, G., White, A. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+reactive+oxygen+species.">Oxidative stress and reactive oxygen species.</a> Clinical Science. 131, 2865-2883 (2017).</li><li>Beatty, S., Koh, H. H., Phil, M., Henson, D., Boulton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+oxidative+stress+in+the+pathogenesis+of+age-related+macular+degeneration.">The role of oxidative stress in the pathogenesis of age-related macular degeneration.</a> Survey of Ophthalmology. 45 (2), 115-134 (2000).</li><li>Alhasani, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gypenosides+protect+retinal+pigment+epithelium+cells+from+oxidative+stress.">Gypenosides protect retinal pigment epithelium cells from oxidative stress.</a> Food and Chemical Toxicology. 112, 76-85 (2018).</li><li>. National Institute of Health Available from: <a target="_blank" href="https://nei.nih.gov/learn-about-eye-health/resources-for-health-educators/eye-health-data-and-statistics/age-related-macular-degeneration-amd-data-and-statistics">https://nei.nih.gov/learn-about-eye-health/resources-for-health-educators/eye-health-data-and-statistics/age-related-macular-degeneration-amd-data-and-statistics</a> (2020)</li><li>Mitchell, P., Liew, G., Gopinath, B., Wong, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+macular+degeneration.">Age-related macular degeneration.</a> The Lancet. 392, 1147-1159 (2018).</li><li>He, Y., Leung, K. W., Ren, Y., Jinzhi, P., Jian, G., Tombran-Tink, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEDF+improves+mitochondrial+function+in+RPE+cells+during+oxidative+stress.">PEDF improves mitochondrial function in RPE cells during oxidative stress.</a> Investigative Ophthalmology &amp; Visual Science. 55, 6742-6755 (2014).</li><li>Cao, S., Walker, G. B., Wang, X., Cui, J. Z., Matsubara, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+cytokine+profiles+of+human+retinal+pigment+epithelium:+Oxidant+injury+and+replicative+senescence.">Altered cytokine profiles of human retinal pigment epithelium: Oxidant injury and replicative senescence.</a> Molecular Vision. 19, 718-728 (2013).</li><li>Farnoodian, M., Sorenson, C. M., Sheibani, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEDF+expression+affects+the+oxidative+and+inflammatory+state+of+choroidal+endothelial+cells.">PEDF expression affects the oxidative and inflammatory state of choroidal endothelial cells.</a> Amercian Journal of Physiology and Cell Physiology. 314 (4), 456-472 (2018).</li><li>Polato, F., Becerra, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+Degenerative+Diseases:+Mechanisms+and+Experimental+Therapies.">Retinal Degenerative Diseases: Mechanisms and Experimental Therapies.</a> Retinal Degenerative Diseases. , 699-706 (2016).</li><li>Schallenberg, M., Charalambous, P., Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM-CSF+regulates+the+ERK1/2+pathways+and+protects+injured+retinal+ganglion+cells+from+induced+death.">GM-CSF regulates the ERK1/2 pathways and protects injured retinal ganglion cells from induced death.</a> Experimental Eye Research. 89, 665-677 (2009).</li><li>Schallenberg, M., Charalambous, P., Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM-CSF+protects+rat+photoreceptors+from+death+by+activating+the+SRC-dependent+signalling+and+elevating+anti-apoptotic+factors+and+neurotrophins.">GM-CSF protects rat photoreceptors from death by activating the SRC-dependent signalling and elevating anti-apoptotic factors and neurotrophins.</a> Graefes Archives for Clinical and Experimental Ophthalmology. 250, 699-712 (2012).</li><li>Thumann, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+PEDF-expressing+primary+pigment+epithelial+cells+by+the+SB+transposon+system+delivered+by+pFAR4+plasmids.">Engineering of PEDF-expressing primary pigment epithelial cells by the SB transposon system delivered by pFAR4 plasmids.</a> Molecular Therapy - Nucleic Acids. 6, 302-314 (2017).</li><li>Garcia-Garcia, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+PEDF+release+in+rat+iris+and+retinal+epithelial+cells+after+Sleeping+Beauty+transposon-mediated+gene+delivery.">Long-term PEDF release in rat iris and retinal epithelial cells after Sleeping Beauty transposon-mediated gene delivery.</a> Molecular Therapy - Nucleic Acids. 9, 1-11 (2017).</li><li>Johnen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiangiogenic+and+neurogenic+activities+of+Sleeping+Beauty-mediated+PEDF-transfected+RPE+cells+in+vitro+and+in+vivo.">Antiangiogenic and neurogenic activities of Sleeping Beauty-mediated PEDF-transfected RPE cells in vitro and in vivo.</a> BioMed Research International. 2015, (2015).</li><li>Weigel, A. L., Handa, J. T., Hjelmeland, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+of+H2O2-,+HNE-,+or+tBH-treated+ARPE-19+cells.">Microarray analysis of H2O2-, HNE-, or tBH-treated ARPE-19 cells.</a> Free Radical Biology &amp; Medicine. 33 (10), 1419-1432 (2002).</li><li>Allen, R. G., Tresini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+gene+regulation.">Oxidative stress and gene regulation.</a> Free Radical Biology and Medicine. 28 (3), 463-499 (2000).</li><li>Tate, D. J., Miceli, M. V., Newsome, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+and+H2O2+induce+catalase+and+metallothionein+gene+expression+in+human+retinal+pigment+epithelial+cells.">Phagocytosis and H2O2 induce catalase and metallothionein gene expression in human retinal pigment epithelial cells.</a> Investigative Ophthalmology and Visual Science. 36 (7), 1271-1279 (1995).</li><li>Halliwell, B., Clement, M. V., Long, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+in+the+human+body.">Hydrogen peroxide in the human body.</a> FEBS Letters. 486 (1), 10-13 (2000).</li><li>Giblin, F. J., McCready, J. P., Kodama, T., Reddy, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+direct+correlation+between+the+levels+of+ascorbic+acid+and+H2O2+in+aqueous+humor.">A direct correlation between the levels of ascorbic acid and H2O2 in aqueous humor.</a> Experimental Eye Research. 38, 87-93 (1984).</li><li>Geiger, R. C., Waters, C. M., Kamp, D. W., Glucksberg, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KGF+prevents+oxygen-mediated+damage+in+ARPE-19+cells.">KGF prevents oxygen-mediated damage in ARPE-19 cells.</a> Investigative Ophthalmology and Visual Science. 46, 3435-3442 (2005).</li><li>Campochiaro, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vector+gene+transfer+of+endostatin/angiostatin+for+macular+degeneration+(GEM)+study.">Lentiviral vector gene transfer of endostatin/angiostatin for macular degeneration (GEM) study.</a> Human Gene Therapy. 28, 99-111 (2017).</li><li>Chen, X. -. D., Su, M. -. Y., Chen, T. -. T., Hong, H. -. Y., Han, A. -. D., Li, W. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+affects+retinal+pigment+epithelial+cell+survival+through+epidermal+growth+factor+receptor/AKT+signaling+pathway.">Oxidative stress affects retinal pigment epithelial cell survival through epidermal growth factor receptor/AKT signaling pathway.</a> International Journal of Ophthalmology. 10 (4), 507-514 (2017).</li><li>Tu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allicin+attenuates+H2O2+-+induced+cytotoxicity+in+retinal+pigmented+epithelial+cells+by+regulating+the+levels+of+reactive+oxygen+species.">Allicin attenuates H2O2 - induced cytotoxicity in retinal pigmented epithelial cells by regulating the levels of reactive oxygen species.</a> Molecular Medicine Reports. 13, 2320-2326 (2016).</li><li>Hao, Y., Liu, J., Wang, Z., Yu, L., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Piceatannol+protects+human+retinal+pigment+epithelial+cells+against+hydrogen+peroxide+induced+oxidative+stress+and+apoptosis+through+modulating.">Piceatannol protects human retinal pigment epithelial cells against hydrogen peroxide induced oxidative stress and apoptosis through modulating.</a> Nutrients. 11, 1-13 (2019).</li><li>Ballinger, S. W., Van Houten, B., Conklin, C. A., Jin, G. F., Godley, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+causes+significant+mitochondrial+DNA+damage+in+human+RPE+cells.">Hydrogen peroxide causes significant mitochondrial DNA damage in human RPE cells.</a> Experimental Eye Research. 68 (6), 765-772 (1999).</li><li>Ma, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+overexpression+of+uncoupling+protein+2+attenuates+salt-induced+vascular+dysfunction+by+inhibition+of+oxidative+stress.">Transgenic overexpression of uncoupling protein 2 attenuates salt-induced vascular dysfunction by inhibition of oxidative stress.</a> American Journal of Hypertension. 27 (3), 345-354 (2014).</li><li>Johnen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleeping+Beauty+transposon-mediated+transfection+of+retinal+and+iris+pigment+epithelial+cells.">Sleeping Beauty transposon-mediated transfection of retinal and iris pigment epithelial cells.</a> Investigative Ophthalmology and Visual Science. 53 (8), 4787-4796 (2012).</li><li>M&#225;t&#233;s, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+evolution+of+a+novel+hyperactive+Sleeping+Beauty+transposase+enables+robust+stable+gene+transfer+in+vertebrates.">Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates.</a> Nature Genetics. 41 (6), 753-761 (2009).</li><li>. Marienfeld Technical information Neubauer-improved Available from: <a target="_blank" href="https://www.marienfeld-superior.com/information-about-our-counting-chambers.html">https://www.marienfeld-superior.com/information-about-our-counting-chambers.html</a> (2020)</li><li>. Electron Microscopy Sciences. Neubauer Haemocytometry Available from: <a target="_blank" href="https://www.emsdiasum.com/microscopy/technical/datasheet/68052-14.aspx">https://www.emsdiasum.com/microscopy/technical/datasheet/68052-14.aspx</a> (2020)</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2^(-&#916;&#916;CT)+method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2^(-&#916;&#916;CT) method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Bascuas, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-virally+transfected+primary+human+pigment+epithelium+cells+overexpressing+the+oxidative+stress+reduction+factors+PEDF+and+GM-CSF+to+treat+retinal+neurodegeneration+neurodegenerationl.">Non-virally transfected primary human pigment epithelium cells overexpressing the oxidative stress reduction factors PEDF and GM-CSF to treat retinal neurodegeneration neurodegenerationl.</a> Human Gene Therapy. 30 (11), (2019).</li><li>Zhuge, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fullerenol+protects+retinal+pigment+epithelial+cells+from+oxidative+stress-induced+premature+senescence+via+activating+SIRT1.">Fullerenol protects retinal pigment epithelial cells from oxidative stress-induced premature senescence via activating SIRT1.</a> Investigative Ophthalmology &amp; Visual Science. 55 (7), 4628-4638 (2014).</li><li>Kaczara, P., Sarna, T., Burke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+H2O2+Availability+to+ARPE-19+cultures+in+models+of+oxidative+stress.">Dynamics of H2O2 Availability to ARPE-19 cultures in models of oxidative stress.</a> Free Radical Biology and Medicine. 48 (8), 1068-1070 (2010).</li><li>Gorrini, C., Harris, I. S., Mak, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+oxidative+stress+as+an+anticancer+strategy.">Modulation of oxidative stress as an anticancer strategy.</a> Nature Reviews Drug Discovery. 12, 931-947 (2013).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEDF+protects+human+retinal+pigment+epithelial+cells+against+oxidative+stress+via+upregulation+of+UCP2+expression.">PEDF protects human retinal pigment epithelial cells against oxidative stress via upregulation of UCP2 expression.</a> Molecular Medicine Reports. 19 (1), 59-74 (2019).</li><li>Donadelli, M., Dando, I., Fiorini, C., Palmieri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UCP2,+a+mitochondrial+protein+regulated+at+multiple+levels.">UCP2, a mitochondrial protein regulated at multiple levels.</a> Cellular and Molecular Life Sciences. 71, 1171-1190 (2014).</li></ol>

The protocol presented here offers an approach to analyze the anti-oxidative and protective function of PEDF and GM-CSF produced by transfected cells, which can be applied to cells transfected with any putative beneficial gene. In gene therapeutic strategies that have the objective to deliver proteins to tissue by transplanting genetically modified cells, it is critical to obtain information as to the level of protein expression, the longevity of expression, and the effectiveness of the expressed protein in a model of th...

The model described here, offers a useful approach to evaluate the efficiency ofbiopharmaceutical agents for reducing oxidative stress in cells. We have used the model to investigate the protective effects of PEDF and GM-CSF on the H2O2-mediated oxidative stress on retinal pigment epithelial cells, which are exposed to high levels of O2, and visible light, and the phagocytosis of photoreceptor outer segment membranes, generating significant levels of reactive oxygen species (ROS)1,2. They are considered a major contributor to the pathogenesis of avascular age-related macular degeneration (aAMD)3,4,5,6,7,8. Besides, there is a decrease in RPE-synthesized neuroprotective factors, specifically the pigment epithelium-derived factor (PEDF), insulin-like growth factors (IGFs), and granulocyte macrophage-colony-stimulating factor (GM-CSF) leading to the dysfunction and loss of RPE cells, followed by photoreceptor and retinal ganglion cell (RGC) death3,4,5. AMD is a complex disease that results from the interaction between metabolic, functional, genetic, and environmental factors4. The lack of treatments for aAMD is the major cause of blindness in patients older than 60 years of age in industrialized countries9,10. The reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of genetically modified RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation and supporting cell survival11,12,13,14,15,16. Even though there are several methodologies to deliver genes to cells, we have chosen the non-viral hyperactive Sleeping Beauty transposon system to deliver the PEDF and GM-CSF genes to RPE cells because of its safety profile, the integration of the genes into the host cells&#39; genome, and its propensity to integrate the delivered genes in non-transcriptionally active sites as we have shown previously17,18,19.
Cellular oxidative stress can be induced in cells cultured in vitro by several oxidative agents, including hydrogen peroxide (H2O2), 4-hydroynonenal (HNE), tertbutylhydroperoxide (tBH), high oxygen tensions, and visible light (full spectrum or UV irradiation)20,21. High oxygen tensions and light require special equipment and conditions, which limits transferability to other systems. Agents such as H2O2, HNE, and tBH induce overlapping oxidative stress molecular and cellular changes. We chose H2O2 to test the antioxidant activity of PEDF and GM-CSF because it is convenient and biologically relevant since it is produced by RPE cells as a reactive oxygen intermediate during photoreceptor outer segment phagocytosis22 and it is found in ocular tissues in vivo23. Since the oxidation of glutathione may be partially responsible for the production of H2O2 in the eye, we have analyzed the levels of GSH/glutathione in our studies, which are linked to H2O2-induced oxidative stress and the regenerative capacity of cells21,22. The analysis of glutathione levels is especially relevant since it participates in the anti-oxidative protective mechanisms in the eye24. Exposure to H2O2 is used frequently as a model to examine the oxidative stress susceptibility and antioxidant activity of RPE cells1,25,26,27,28,29,30, and, additionally, it shows similarities to light-induced oxidative stress damage, a &#34;physiological&#34; source of oxidative stress21.
To evaluate the functionality and the effectiveness of neuroprotective factors, we have developed an in vitro model that allows for the analysis to quantify the anti-oxidative effect of growth factors expressed by cells genetically modified to overexpress PEDF and GM-CSF. Here, we show that RPE cells transfected with the genes for PEDF and GM-CSF are more resistant to the harmful effects of H2O2 than are non-transfected control cells, as evidenced by cell morphology, density, viability, intracellular level of glutathione, and expression of UCP2 gene, which codes for the mitochondrial uncoupling protein 2 that has been shown to reduce reactive oxygen species (ROS)31.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well plates</td>
			<td>Corning</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Greiner</td>
			<td>7657160</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well culture plate white with clear flat bottom&#160;</td>
			<td>Costar</td>
			<td>3610</td>
			<td>Allows to check the cells before measuring the luminescence (GSH-Glo Assay)</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Corning&#160;</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamid 40%</td>
			<td>Biorad</td>
			<td>161-0144</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>AMIMED</td>
			<td>4-05F00-H</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-GMCSF</td>
			<td>ThermoFisher Scientific</td>
			<td>PA5-24184</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-mouse IgG/IgA/IgM&#160;</td>
			<td>Agilent</td>
			<td>P0260</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-PEDF&#160;</td>
			<td>Santa Cruz Biotechnology Inc</td>
			<td>sc-390172</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-penta-His&#160;</td>
			<td>Qiagen</td>
			<td>34660</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-phospho-Akt</td>
			<td>Cell Signaling Technology</td>
			<td>9271</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-rabbit IgG H&#38;L-HRP</td>
			<td>Abcam</td>
			<td>ab6721</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody donkey anti-rabbit Alexa Fluor 594&#160;</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>A11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody goat anti-mouse Alexa 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11029</td>
			<td></td>
		</tr>
		<tr>
			<td>ARPE-19 cell line</td>
			<td>ATCC</td>
			<td>CRL-2302</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9418-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>chamber culture glass slides</td>
			<td>Corning</td>
			<td>354118</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoTox-Glo Cytotoxicity Assay&#160;</td>
			<td>Promega</td>
			<td>G9291</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/Ham`s F12&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D8062</td>
			<td></td>
		</tr>
		<tr>
			<td>Duo Set ELISA kit</td>
			<td>R&#38;D Systems&#160;</td>
			<td>DY215-05</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>78440</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISAquant kit</td>
			<td>BioProducts MD</td>
			<td>PED613-10-Human</td>
			<td></td>
		</tr>
		<tr>
			<td>Eyes (human)</td>
			<td>Lions Gift of Sight Eye Bank (Saint Paul, MN)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FBS&#160;</td>
			<td>Brunschwig</td>
			<td>P40-37500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount Aqueous Mounting Medium</td>
			<td>Sigma-Aldrich</td>
			<td>F4680-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>FLUOstar Omega plate reader&#160;</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism software (version 8.0)</td>
			<td>GraphPad Software, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GSH-Glo Glutathione Assay</td>
			<td>Promega</td>
			<td>V6912</td>
			<td></td>
		</tr>
		<tr>
			<td>hydrogen peroxide (H2O2)</td>
			<td>Merck</td>
			<td>107209</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software (image processing program)</td>
			<td>W.S. Rasband, NIH, Bethesda, MD, USA; https://imagej.nih.gov/ij/; 1997&#8211;2014</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazol&#160;</td>
			<td>Axonlab</td>
			<td>A1378.0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DMI4000B microscope&#160;</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 480 Instrument II&#160;</td>
			<td>Roche Molecular Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 480 SW1.5.1 software</td>
			<td>Roche Molecular Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71376-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Axonlab</td>
			<td>3468.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Neon Transfection System&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Neon Transfection System 10 &#181;L Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK1096</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Marienfeld-superior</td>
			<td>640010</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA superflow&#160;</td>
			<td>Qiagen</td>
			<td>30410</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose&#160;</td>
			<td>VWR</td>
			<td>732-3197</td>
			<td></td>
		</tr>
		<tr>
			<td>Omega Lum G Gel Imaging System</td>
			<td>Aplegen Life Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1X</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P0781-100</td>
			<td></td>
		</tr>
		<tr>
			<td>PerfeCTa SYBR Green FastMix</td>
			<td>Quantabio</td>
			<td>95072-012</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>158127-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers&#160;</td>
			<td>Invitrogen&#160;</td>
			<td></td>
			<td>See Table 1 in Supplementary Materials</td>
		</tr>
		<tr>
			<td>pSB100X (250 ng/&#181;L)</td>
			<td></td>
			<td></td>
			<td>M&#225;t&#233;s et al., 2009. Provide by Prof. Zsuzsanna Izsvak</td>
		</tr>
		<tr>
			<td>pT2-CMV-GMCSF-His plasmid DNA (250 ng/&#181;L)</td>
			<td></td>
			<td></td>
			<td>Constructed using the existing pT2-CMV-PEDF-EGFP plasmid reported in Johnen, S. et al. (2012) IOVS, 53 (8), 4787-4796.</td>
		</tr>
		<tr>
			<td>pT2-CMV-PEDF-His plasmid DNA (250 ng/&#181;L)</td>
			<td></td>
			<td></td>
			<td>Constructed using the existing pT2-CMV-PEDF-EGFP plasmid reported in Johnen, S. et al. (2012) IOVS, 53 (8), 4787-4796.</td>
		</tr>
		<tr>
			<td>QIAamp DNA Mini Kit</td>
			<td>QIAGEN</td>
			<td>51304</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant hGM-CSF&#160;</td>
			<td>Peprotech</td>
			<td>100-11</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant hPEDF&#160;&#160;</td>
			<td>BioProductsMD</td>
			<td>004-096</td>
			<td></td>
		</tr>
		<tr>
			<td>ReliaPrep RNA Cell Miniprep System</td>
			<td>Promega</td>
			<td>Z6011</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>89901</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free DNase Set</td>
			<td>QIAGEN</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>QIAGEN</td>
			<td>74204</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Applichem</td>
			<td>A2572</td>
			<td></td>
		</tr>
		<tr>
			<td>Semi-dry transfer system for WB&#160;</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SuperMix qScript</td>
			<td>Quantabio</td>
			<td>95048-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-buffered saline (TBS)&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>15504020</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>AppliChem</td>
			<td>A4975</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>T4174</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween</td>
			<td>AppliChem&#160;</td>
			<td>A1390</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>ThermoFisher Scientific</td>
			<td>29700</td>
			<td></td>
		</tr>
		<tr>
			<td>WesternBright ECL HRP substrate</td>
			<td>Advansta</td>
			<td>K-12045-D50</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman nitrocellulose membrane</td>
			<td>Chemie Brunschwig</td>
			<td>MNSC04530301</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction and analysis of oxidative stress in sleeping beauty transposon-transfected human retinal pigment epithelial cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction of oxidative stress in human Retinal Pigment Epithelial cells 
ARPE-19 and primary hRPE cells were treated with varying concentrations of H2O2 for 24 h and the intracellular level of the antioxidant glutathione was quantified (Figure 2A,B). H2O2 at 50 &#181;M and 100 &#181;M did not affect glutathione production, whereas at 350 &#181;M there was a significant decrease...

Watch this Scientific Journal Video about Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells at JoVE.com

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Research

JoVE Journal

Medicine

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...