Name: electroporation-based genetic modification of primary human pigment epithelial cells using the sleeping beauty transposon system
Uploaded: 2021-02-04T13:00:00
Description: Watch this Scientific Journal Video about Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System at JoVE.com

This work was supported by the European Union&#39;s Seventh Framework Programme for research, technological development and demonstration, grant agreement no. 305134. Zsuzsanna&#160;Izsv&#225;k was funded by the European Research Council, ERC Advanced (ERC-2011-ADG 294742). The authors would like to thank Anna Dobias and Antje Schiefer (Department of Ophthalmology, University Hospital RWTH Aachen) for excellent technical support, and the Aachen Cornea Bank (Department of Ophthalmology, University Hospital RWTH Aachen) for providing the human donor eyes.

Human donor eyes were obtained from the Aachen Cornea Bank of the Department of Ophthalmology (University Hospital RWTH Aachen) after obtaining informed consent in accordance with the Declaration of Helsinki protocols. Procedures for the collection and use of human samples have been approved by the institutional ethics committee.
1. Isolation of primary human RPE cells
<ol>
	<li>Lay out sterile protective clothing and gloves. Place a sterile drape under a laminar flow.</li>
	<li>Place steril...

In our project, we aim for the non-viral production of genetically modified primary human RPE cells that continuously overexpress and secrete an effective factor in order to use the transfected cells as long-term therapeutic for the establishment and maintenance of a protective environment. We have established the introduction of the gene encoding PEDF, an ubiquitously expressed multi-functional protein with anti-angiogenic and neuroprotective functions. The protocol described here can be used to stably and reproducibly ...

Advanced age is described to be the main risk for neurodegenerative diseases. Age-related macular degeneration (AMD), a polygenic disease leading to severe vision loss in patients older than 60 years of age, belongs to the four most common causes of blindness and vision impairment1 and is expected to increase to 288 million people in 20402. Dysfunctions of the retinal pigment epithelium (RPE), a single layer of tightly packed cells located between the choriocapillaris and the retinal photoreceptors, contribute to the pathogenesis of AMD. The RPE fulfills multiple tasks that are essential for a normal retinal function3 and secretes a variety of growth factors and factors essential to maintain the structural integrity of the retina and the choriocapillaris, thereby supporting photoreceptor survival and providing a basis for the circulation and the supply of nutrients.
In healthy eyes, pigment epithelium-derived factor (PEDF) is responsible for balancing the effects of vascular endothelial growth factor (VEGF) and protects neurons against apoptosis, prevents endothelial cell proliferation, and stabilizes the capillary endothelium. A shifted VEGF-to-PEDF ratio is related to ocular neovascularization, which was observed in animal models4,5 as well as in samples of patients with choroidal neovascularization (CNV) due to AMD and proliferative diabetic retinopathy6,7,8,9,10. The enhanced VEGF concentration is the target for the current standard treatment. The anti-VEGF pharmaceuticals bevacizumab, ranibizumab, aflibercept and, most recently, brolucizumab improve visual acuity in about one third of CNV patients or rather stabilize vision in 90% of cases11,12,13. However, the frequent, often monthly, intravitreal injections bear the risk of adverse events14, impair patient compliance, and represent a significant economic burden to healthcare systems15. Moreover, a certain percentage of patients (2%-20%) do not respond or only poorly react to the anti-VEGF therapy16,17,18,19. These negative concomitants necessitate the development of alternative treatments, e.g., intraocular implants, cell and/or gene therapeutic approaches.
Gene therapy has evolved as promising treatment for hereditary and non-hereditary diseases and intends to restore non-functional gene sequences or suppress malfunctioned ones. For polygenic diseases, where identification and replacement of the causative factors is hardly possible, strategies aim for the continuous delivery of a protective factor. In the case of AMD, various additive therapies have been developed, such as the stable expression of endostatin and angiostatin20, the VEGF antagonist soluble fms-like tyrosine kinase-1 (sFLT-1)21,22, the complement regulatory protein cluster of differentiation 59 (CD59)23 or PEDF24,25. The eye, and especially the retina, is an excellent target for a gene-based medication due to the enclosed structure, good accessibility, small size, and immune privilege, thus allowing for a localized delivery of low therapeutic doses and making transplants less susceptible to rejection. Moreover, the eye enables non-invasive monitoring, and the retina can be examined by different imaging techniques.
Viral vectors are, because of their high transduction efficiency, the main vehicle to deliver therapeutic genes into target cells. However, depending on the viral vector used, different adverse reactions have been described, such as immune and inflammatory responses26, mutagenic and oncogenic effects27,28, or dissemination in other tissues29. Practical limitations include a restricted packaging size30 as well as difficulties and costs associated with the production of clinical grade lots31,32. These drawbacks have promoted the further development of non-viral, plasmid-based vectors that are transferred via lipo-/polyplexes, ultrasound or electroporation. However, genomic integration of the transgene into the host genome is usually not promoted with plasmid vectors, thus resulting in a transient expression.
Transposons are naturally occurring DNA fragments that change their position within the genome, a characteristic that has been adopted for gene therapy. Due to an active integration mechanism, transposon-based vector systems allow for a continuous and constant expression of the inserted transgene. The Sleeping Beauty (SB) transposon, reconstituted from an ancient Tc1/mariner-type transposon found in fish33 and further improved by molecular evolution resulting in the hyperactive variant SB100X34, enabled efficient transposition in various primary cells and was used for the phenotypic correction in different disease models35. At present, 13 clinical trials have been initiated using the SB transposon system. The SB100X transposon system consists of two components: the transposon, which comprises the gene of interest flanked by terminal inverted repeats (TIRs), and the transposase, which mobilizes the transposon. Following plasmid DNA delivery to the cells, the transposase binds the TIRs and catalyzes the excision and integration of the transposon into the cell&#39;s genome.
We have developed a non-viral cell-based additive therapy for the treatment of neovascular AMD. The approach comprises the electroporation-based insertion of the PEDF gene into primary pigment epithelial (PE) cells by means of the SB100X transposon system36,37,38. The genetic information of the transposase and PEDF are provided on separate plasmids, thereby enabling the adjustment of the ideal SB100X-to-PEDF transposon ratio. Electroporation is performed using a pipette-based capillary transfection system that is characterized by a maximized gap size between the electrodes while minimizing their surface area. The device was shown to achieve excellent transfection rates in a wide range of mammalian cells39,40,41. The small electrode surface area provides a uniform electric field and reduces the various side effects of electrolysis42.
The anti-angiogenic functionality of PEDF secreted by transfected pigment epithelial cells was shown in various in vitro experiments analyzing the sprouting, migration, and apoptosis of human umbilical vein endothelial cells43. In addition, transplantation of PEDF-transfected cells in a rabbit model of corneal neovascularization44 as well as a rat model of CNV43,45,46 showed decline of neovascularization.
Here, we describe a detailed protocol for the stable insertion of the PEDF gene into primary human RPE cells via the SB100X transposon system using a capillary transfection system. The transfected cells were kept in culture for 21 days and subsequently analyzed in terms of PEDF gene expression by quantitative polymerase chain reaction (qPCR) and in terms of PEDF protein secretion by immunoblotting and enzyme-linked immunosorbent assay (ELISA, Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Isolation of primary human RPE cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Cell Culture Plate</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>0030722019</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B [250 &#181;g/mL] (AmphoB)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Colibri Forceps</td>
			<td>Geuder, Heidelberg, Germany</td>
			<td>G-18950</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Iris Forceps&#160;</td>
			<td>Geuder, Heidelberg, Germany</td>
			<td>G-18856</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpel (No. 11)</td>
			<td>Feather, Osaka, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium/Ham&#8217;s F-12 Nutrient Mixture (DMEM/F12)</td>
			<td>PAN-Biotech, Aidenbach, Germany</td>
			<td>P04-41150</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra Fine Pointed Eye Scissor&#160;</td>
			<td>Geuder, Heidelberg, Germany</td>
			<td>G-19405</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum [0.2 &#181;m Sterile Filtered] (FBS)</td>
			<td>PAN-Biotech, Aidenbach, Germany</td>
			<td>P40-37500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur Pipettes</td>
			<td>Brand, Wertheim, Germany</td>
			<td>747715</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin [10,000 units/mL] and Streptomycin [10 mg/mL] (Pen/Strep)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips (1000 &#181;L)</td>
			<td>Starlab, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel Pipette (100-1000 &#181;L)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Drape</td>
			<td>Lohmann &#38; Rauscher, Rengsdorf, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Gauze Compress&#160;</td>
			<td>Fink-Walter, Merchweiler, Germany</td>
			<td>321063</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Gloves</td>
			<td>Sempermed, Wien, Austria</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Petri Dish (Falcon 60 mm x 15 mm)</td>
			<td>Corning, Corning, NY</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Surgical Gown</td>
			<td>Halyard Health, Alpharetta, GA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Iris Forceps&#160;</td>
			<td>Geuder, Heidelberg, Germany</td>
			<td>G-18855</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporation of primary human RPE cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM Tris-HCl (pH 8.5)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12-Well Cell Culture Plate</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>150628</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Cell Culture Plate</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>0030722019</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B [250 &#181;g/mL] (AmphoB)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium/Ham&#8217;s F-12 Nutrient Mixture (DMEM/F12)</td>
			<td>PAN-Biotech, Aidenbach, Germany</td>
			<td>P04-41150</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock Microcentrifuge Tubes (1.5 mL)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum [0.2 &#181;m Sterile Filtered] (FBS)</td>
			<td>PAN-Biotech, Aidenbach, Germany</td>
			<td>P40-37500</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Leica Mikrosysteme, Wetzlar, Germany</td>
			<td>Leica DMi8</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvolume Spectrophotometer (NanoDrop Spectrophotometer)</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary Transfection System (Neon Transfection System)</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>MPK5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Neon Transfection System 10 &#181;L Kit</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>MPK1096</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer (Neubauer Chamber)</td>
			<td>Paul Marienfeld, Lauda-K&#246;nigshofen, Germany</td>
			<td>0640110</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Dulbecco w/o Ca2+ w/o Mg2+</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>L182-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin [10,000 units/mL] and Streptomycin [10 mg/mL] (Pen/Strep)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips (10 &#181;L)</td>
			<td>Starlab, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips (1000 &#181;L)</td>
			<td>Starlab, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips (200 &#181;L)</td>
			<td>Starlab, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Maxi Kit</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>12163</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel Pipette (0.1-10 &#181;L)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel Pipette (100-1000 &#181;L)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel Pipette (10-200 &#181;L)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0,05&#160;%)</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Analyses of transfected primary human RPE cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% SDS-Polyacrylamide Gel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1x Incubation Buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2x SDS Sample Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4x Incubation Buffer (200 mM NaH2PO4, 1.2 M NaCl, 40 mM imidazole, pH 8.0)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham Protran Supported 0.2 &#181;m&#160;Nitrocellulose Blotting Membrane</td>
			<td>Cytiva, Marlborough, MA</td>
			<td>10600015</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B [250 &#181;g/mL] (AmphoB)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PEDF Antibodies (Rabbit Polyclonal)</td>
			<td>BioProducts, Middletown, MD</td>
			<td>AB-PEDF1</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Penta-His Antibodies (Mouse Monoclonal)</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>34660</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium/Ham&#8217;s F-12 Nutrient Mixture (DMEM/F12)</td>
			<td>PAN-Biotech, Aidenbach, Germany</td>
			<td>P04-41150</td>
			<td></td>
		</tr>
		<tr>
			<td>Elution Buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0)&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum [0.2 &#181;m Sterile Filtered] (FBS)</td>
			<td>PAN-Biotech, Aidenbach, Germany</td>
			<td>P40-37500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer (Neubauer Chamber)</td>
			<td>Paul Marienfeld, Lauda-K&#246;nigshofen, Germany</td>
			<td>0640110</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish Peroxidase-Conjugated Anti-Mouse Antibodies (Rabbit Polyclonal)</td>
			<td>Agilent Dako, Santa Clara, CA</td>
			<td>P0260</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish Peroxidase-Conjugated Anti-Rabbit Antibodies (Goat Polyclonal)</td>
			<td>Abcam, Cambridge, United Kingdom</td>
			<td>ab6721</td>
			<td></td>
		</tr>
		<tr>
			<td>Human PEDF ELISA Kit&#160;</td>
			<td>BioProducts, Middletown, MD</td>
			<td>PED613</td>
			<td></td>
		</tr>
		<tr>
			<td>LAS-3000 Imaging System</td>
			<td>Fujifilm, Minato, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 1.2 Instrument</td>
			<td>Roche Life Science, Penzberg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler FastStart DNA Master SYBR Green I</td>
			<td>Roche Life Science, Penzberg, Germany</td>
			<td>12239264001</td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler&#160;Capillaries (20 &#956;l)</td>
			<td>Roche Life Science, Penzberg, Germany</td>
			<td>4929292001</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvolume Spectrophotometer (NanoDrop Spectrophotometer)</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN&#160;Tetra Cell Casting Module</td>
			<td>Bio-Rad Laboratories, Feldkirchen, Germany</td>
			<td>1658015</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN&#160;Tetra Vertical Electrophoresis Cell for Mini Precast Gels, 4-gel</td>
			<td>Bio-Rad Laboratories, Feldkirchen, Germany</td>
			<td>1658004</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Superflow</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>30410</td>
			<td></td>
		</tr>
		<tr>
			<td>PageRuler Prestained Protein Ladder</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin [10,000 units/mL] and Streptomycin [10 mg/mL] (Pen/Strep)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips (10 &#181;L)</td>
			<td>Starlab, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips (1000 &#181;L)</td>
			<td>Starlab, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips (200 &#181;L)</td>
			<td>Starlab, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PowerPac Basic Power Supply</td>
			<td>Bio-Rad Laboratories, Feldkirchen, Germany</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp DNA Mini Kit</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>51304</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse Transcription System&#160;</td>
			<td>Promega, Madison, WI</td>
			<td>A3500</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-Free DNase Set</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit&#160;</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocking Shaker</td>
			<td>Cole-Parmer, Staffordshire, United Kingdom</td>
			<td>SSM3</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock Microcentrifuge Tubes (1.5 mL)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock Microcentrifuge Tubes (2.0 mL)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel Pipette (0.1-10 &#181;L)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel Pipette (100-1000 &#181;L)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel Pipette (10-200 &#181;L)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo Transfer System</td>
			<td>Bio-Rad Laboratories, Feldkirchen, Germany</td>
			<td>1704150</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0,05&#160;%)</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>25300054</td>
			<td></td>
		</tr>
	</tbody>
</table>

electroporation-based genetic modification of primary human pigment epithelial cells using the sleeping beauty transposon system

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 &#181;L of resuspension buffer and combined with 2 &#181;L of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

Cultivation and electroporation of primary human RPE cells 
We have shown that seeding of a sufficient number of primary RPE cells of animal origin allows for the cultivation and growth to an integrated monolayer of pigmented, hexagonally shaped cells36,37,48. Their capability to form tight junctions, to exhibit phagocytic activity, and to express specific marker genes in vi...

Watch this Scientific Journal Video about Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System at JoVE.com

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately ...

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