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 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 µL of resuspension buffer and combined with 2 µL 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.
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'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).
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
2. Electroporation of primary human RPE cells
3. Analyses of transfected primary human RPE cells
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 vitro48 reflects substantial tasks of the retinal pigment epithelium in vivo. Cultivated primary RPE cells isolated from human donor eyes also showed the typical cobblestone morphology, regardless of the donor's age (65.3 ± 9.94 a, min: 49 a, max: 83 a, n = 12), the post-mortem time of isolation (37.3 ± 17.0 h, min: 16 h, max: 68 h, n = 12), and the cultivation time (27.6 ± 14.1 d, min: 13 d, max: 61 d, n = 12) (Figure 2, left panel). The application of short-term electrical pulses to primary human RPE cells using the capillary transfection system did not adversely affect the epithelial morphology (Figure 2, right panel). Testing of various electric parameters revealed that two pulses with a pulse voltage of 1,200 V and a pulse width of 20 ms yielded good and stable transfection efficiencies and the highest number of viable RPE cells (unpublished data).
SB100X-mediated insertion of the PEDF gene into cultivated primary human RPE cells
We have tested plasmid DNA ratios of SB100X-to-PEDF transposon ranging from 250 ng (0.08 pmol) SB100X transposase plus 250 ng (0.052 pmol) PEDF transposon to 12.2 ng (0.0039 pmol) SB100X transposase plus 487.8 ng (0.1 pmol) PEDF transposon. This approach identified the two combinations 29.4 ng (0.0094 pmol) SB100X transposase plus 470.6 ng (0.098 pmol) PEDF transposon and 23.8 ng (0.0076 pmol) plus 476.2 ng (0.099 pmol) as the ones that obtained the best transposition efficiencies37. In cultivated primary human RPE cells, delivery of the PEDF transgene using the SB100X transposon system allowed for a continuously increased PEDF gene expression and PEDF protein secretion. For His-tagged recombinant PEDF, western blot analysis of cell culture media from transfected primary human RPE cells demonstrated PEDF secretion at constant levels without transgene silencing for more than 500 days (Figure 3A). For non-tagged PEDF, secretion in transfected primary human RPE cells was also shown to be significantly increased compared to non-transfected cells38. A representative western blot analysis of cell culture media from serially performed transfections clearly indicated the universally higher PEDF secretion rate at 21 days after transfection (Figure 3B), and in a pursued culture, long-term elevated PEDF secretion was proven for at least 165 days (Figure 3C). ELISA-based quantification demonstrated a 20-fold increase of total PEDF secretion in transfected primary human RPE cells compared to respective non-transfected control cells (Figure 3D). This increment was also affirmed on gene expression level, where the total PEDF expression was raised more than 30-fold (Figure 3E).
Figure 1: Workflow for the electroporation-based insertion of the PEDF gene into primary human RPE cells by means of the SB100X transposon system. The scheme describes the chronological course of (A) the isolation of primary human RPE cells and their subsequent cultivation to a confluent monolayer, (B) the single steps of the electroporation process, including the purification of the SB100X transposase and the PEDF transposon plasmid DNA, the preparation of the SB100X transposase/PEDF transposon plasmid mixture, the set-up of the capillary transfection system, the preparation of the cultivated primary human RPE cells, the electroporation, the seeding, and the cultivation of the transfected RPE cells, as well as (C) the analyses of the transfected primary human RPE cells, comprising the preparation of the cell culture supernatants and transfected cell samples, the evaluation and quantification of PEDF secretion, and the analysis of PEDF gene expression. Please click here to view a larger version of this figure.
Figure 2: Phase contrast micrographs of RPE cells isolated from different donor eyes. Cultures of primary human RPE cells, differing in donor age, post-mortem time of isolation, and cultivation time prior to their application for electroporation (left panel), as well as cultures of transfected primary human RPE cells, exposed to the electric parameters 1,100 V (pulse voltage), 20 ms (pulse width), and 2 pulses (number of pulses) using the capillary transfection system (right panel), exhibited a confluent integrated monolayer of pigmented cells in a cobblestone-like pattern (scale bars: 500 µm). Please click here to view a larger version of this figure.
Figure 3: PEDF secretion and PEDF gene expression after SB100X-mediated transfection of primary human RPE cells. (A–C) Immunoblot-based analyses of PEDF secretion stability in cultivated primary human RPE cells transfected with a mixture of 29.4 ng (0.0094 pmol) SB100X transposase plasmid plus 470.6 ng (0.098 pmol) PEDF transposon plasmid using the capillary transfection system. (A) RPE cells from a 26-year-old donor (female, post-mortem time of isolation: 26 h, cultivation time before electroporation: 14 days) were transfected and maintained in culture for more than 500 days. His-tagged recombinant PEDF (~48 kDa) was purified from cell culture media at different time points and evaluated by western blotting using anti-Penta-His antibodies. (B) For non-tagged recombinant PEDF, RPE cells from a 53-year-old donor (female, post-mortem time of isolation: 28 h, cultivation time before transfection: 19 days) were electroporated without plasmid DNA (control cultures #1 and #2) or with the addition of plasmid DNA (PEDF-transfected cell cultures #3 to #7) and maintained in culture for 21 days. Cell culture supernatants were not purified, but directly analyzed by immunoblotting using anti-PEDF antibodies. Western blot analysis of cell lysates showed the level of intracellular PEDF in controls (cultures #1 and #2) and transfected cells (cultures #3 and #4). Loading of similar protein amounts was indicated by the equal density of GAPDH protein bands (~36 kDa). (C) For the analysis of long-term PEDF secretion, RPE cells from a 63-year-old donor (male, post-mortem-time of isolation: 26 h, cultivation time before transfection: 15 days) were electroporated without plasmid DNA (control cultures #1 and #2) or with plasmid DNA (PEDF-transfected cell culture #3) and maintained in culture for at least 165 days. Cell culture media were not purified, but directly analyzed by immunoblotting using anti-PEDF antibodies. (D) ELISA-based quantification of total PEDF secretion in transfected primary human RPE cells (two samples) and non-transfected control cells (four samples). Secretion of the transfected cells was compared to the secretion of the non-transfected cells (*p = 0.0264, unpaired t test with Welch's correction). (E) Endogenous and total (endogenous plus recombinant) PEDF gene expression in transfected primary human RPE cells were related to the expression in non-transfected control cells, whose expression was set to 1 (dashed line). Data is presented as a box-and-whisker plot (whiskers: min to max). Total PEDF gene expression was compared to endogenous PEDF gene expression (not significant, unpaired t test with Welch's correction). The results for the non-tagged recombinant PEDF (B–E) represent two units of the entire data set of primary RPE cells from up to 27 donors transfected with a SB100X-to-PEDF transposon ratio of 29.4 ng (0.0094 pmol) plus 470.6 ng (0.098 pmol), which was described by Thumann et al. 201738. Please click here to view a larger version of this figure.
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 transfect primary human RPE cells using the SB100X transposon system. DNA delivery and introduction into the RPE cells is performed with a pipette-based capillary transfection system, which uses specific tips as electroporation chamber instead of cuvettes.
The enrichment of primary RPE cultures consisting of morphologically well-differentiated cells to be used for subsequent transfections is influenced by several factors related to the human donor (age and general health status) as well as the receipt of the eyes (post-mortem time of cell isolation). Freshly isolated and seeded primary RPE cells need at least 2 weeks to develop a monolayer of hexagonally shaped cells. As a general rule, cell cultures that needed more time to reach confluence prior to transfection also showed a decelerated adherence and spreading after transfection. Cell adherence and spreading is also impeded by free pigment depositing from RPE cells damaged during cell isolation or the transfection process.
The SB transposon system is a widely used genetic tool that enables the efficient and safe delivery of therapeutically relevant nucleotide sequences into various types of cells to be subsequently applied in gene-based cell therapies. Especially, its enhanced variant SB100X combines the advantages of viral vectors and non-viral plasmid DNA. The integrating mode of action ensures a stable genomic integration of the expression cassette into the host cell's genome and enables a sustained expression of the gene or sequence of interest. In contrast to retroviral vectors, which preferentially integrate into active transcription units with a high risk of potential mutagenesis and oncogenesis27,28, the SB-based integration profile is random. This was demonstrated in a large number of studies using various cultured and primary mammalian cells, including human cells49,50,51,52, as well as for primary rat and human RPE cells38,46. Moreover, application of the SB transposon system as plasmid DNA provides reduced immunogenicity and facilitates the manufacturing process.
Delivery of the genetic information of the SB100X transposase and the PEDF transgene on separate plasmids is an important aspect, as it allows for a precise adjustment of the optimal SB100X-to-PEDF transposon ratio. As described earlier, the SB transposon system is sensitive to an excess of transposase expression, which results in inhibition of the transpositional activity53. We also observed this effect for ARPE-19 cells, a spontaneously evolved cell line purified by selective trypsinization of a primary human RPE culture54, and primary RPE cells. Here, transfection with SB100X-to-PEDF transposon ratios up to 55.6 ng (0.018 pmol) SB100X transposase plus 444.4 ng (0.093 pmol) PEDF transposon resulted in clearly diminished PEDF secretion rates37. The ideal transposase-to-transposon ratio has to be determined for every newly constructed transposon plasmid.
Non-viral plasmid-based vectors can be transferred via lipo-/polyplexes, nanoparticles, laser, ultrasound, or electroporation. We have already shown that lipofection-based transfection of primary PE cells was not efficient and thus switched to the electroporation-based gene transfer36. Electroporation is defined as application of a short-duration electric field to cells, which results in an increase of the membrane's permeability and the formation of aqueous pores, through which plasmid DNA can enter the cell. In general, a mixture of cells, plasmid DNA, and conducting buffer is filled into a specific electroporation chamber between two electrodes, which are connected to the electroporation device generating the electrical pulses. In this so-called bulk electroporation setup, a conventional cuvette electroporation chamber is characterized by two parallel plate-type electrodes, whose distance is variable (0.1-0.4 mm), depending on the cell type and the number of cells used per electroporation reaction. Despite good transfection efficiencies, different side effects may negatively affect the survival of the transfected cells, e.g., pH changes, heat development, bubble generation, and turbulent flow. The capillary transfection system at least attenuates the cuvette-based side effects by possessing electrodes with a minimized surface area and a maximized gap size between them42 as well as by using specific resuspension and electrolytic buffers; however, their compositions are not disclosed.
Despite the enhancements within the bulk electroporation set-up, which led to an increased cell survival, irreversible damage to a certain percentage of cells is unavoidable. Moreover, a precise control of the amount of plasmid integrated into the cells is not possible. The more recent development of miniaturized electroporation systems has allowed for additional improvements by further reducing the limitations associated with bulk electroporation. These new devices are based on microelectrodes, microfluidic, or nanostructures. They offer new application perspectives in the fields of gene therapy, regenerative medicine, and in situ intracellular investigation (reviewed by Chang et al. and Shi et al.)55,56.
We have demonstrated that electroporation, which was initially performed using a cuvette-based system and subsequently established using a pipette-based capillary system, is a suitable and efficient method to transfect both the pigment epithelial cell line ARPE-19 as well as primary PE cells36,37,38,57,58. Depending on the cell type used, it is important to define appropriate electroporation protocols applying parameters that allow for both good transfection efficiencies and cell survival. Insufficient electric fields prevent the cells from damage but result in low transfection efficiencies. With the increase in electric field strength, enhanced transfection efficiencies are achieved. However, elevated electric parameters also lead to higher percentages of dead cells because of irreversible cell damage. For the ARPE-19 cell line, using the capillary transfection system, electroporation parameters of 1,350 V, 20 ms, and 2 pulses were shown to result in initial transfection efficiencies of 100%37. The application of these parameters for the transfection of primary RPE cells resulted in good efficiencies, too, but also in a lower number of cultivated cells after transfection when compared to electroporation parameters of 1,100 V, 20 ms, and 2 pulses (data not shown). Therefore, the decision was taken to choose the milder electroporation parameters to prevent cell damage and accept less transfected cells.
The overall aim of this approach lies in the clinical application of PEDF-transfected cells for the treatment of patients suffering from neovascular AMD. The therapy comprises the stable introduction of the PEDF transgene into autologous pigment epithelial cells ex vivo based on the SB100X transposon system, followed by transplantation of the transfected cells to the subretinal space of the patient. Thus, it is important to ensure that the PEDF-transfected cells do not transdifferentiate and do not acquire a fibroblastic shape and growth behavior. It is also important to prove that the transfected cells allow for a sustained and consistent secretion of PEDF. Moreover, it is crucial to know the precise amount of PEDF secreted by a particular number of cells for a specific period of time.
The authors have nothing to disclose.
This work was supported by the European Union's Seventh Framework Programme for research, technological development and demonstration, grant agreement no. 305134. Zsuzsanna Izsvák 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.
Isolation of primary human RPE cells | |||
24-Well Cell Culture Plate | Eppendorf, Hamburg, Germany | 0030722019 | |
Amphotericin B [250 µg/mL] (AmphoB) | Merck, Darmstadt, Germany | A2942 | |
Colibri Forceps | Geuder, Heidelberg, Germany | G-18950 | |
Curved Iris Forceps | Geuder, Heidelberg, Germany | G-18856 | |
Disposable Scalpel (No. 11) | Feather, Osaka, Japan | ||
Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 Nutrient Mixture (DMEM/F12) | PAN-Biotech, Aidenbach, Germany | P04-41150 | |
Extra Fine Pointed Eye Scissor | Geuder, Heidelberg, Germany | G-19405 | |
Fetal Bovine Serum [0.2 µm Sterile Filtered] (FBS) | PAN-Biotech, Aidenbach, Germany | P40-37500 | |
Glass Pasteur Pipettes | Brand, Wertheim, Germany | 747715 | |
Penicillin [10,000 units/mL] and Streptomycin [10 mg/mL] (Pen/Strep) | Merck, Darmstadt, Germany | P0781 | |
Pipette Tips (1000 µL) | Starlab, Hamburg, Germany | ||
Single Channel Pipette (100-1000 µL) | Eppendorf, Hamburg, Germany | ||
Sterile Drape | Lohmann & Rauscher, Rengsdorf, Germany | ||
Sterile Gauze Compress | Fink-Walter, Merchweiler, Germany | 321063 | |
Sterile Gloves | Sempermed, Wien, Austria | ||
Sterile Petri Dish (Falcon 60 mm x 15 mm) | Corning, Corning, NY | 351007 | |
Sterile Surgical Gown | Halyard Health, Alpharetta, GA | ||
Straight Iris Forceps | Geuder, Heidelberg, Germany | G-18855 | |
Electroporation of primary human RPE cells | |||
10 mM Tris-HCl (pH 8.5) | |||
12-Well Cell Culture Plate | Thermo Fisher Scientific, Waltham, MA | 150628 | |
24-Well Cell Culture Plate | Eppendorf, Hamburg, Germany | 0030722019 | |
Amphotericin B [250 µg/mL] (AmphoB) | Merck, Darmstadt, Germany | A2942 | |
Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 Nutrient Mixture (DMEM/F12) | PAN-Biotech, Aidenbach, Germany | P04-41150 | |
Safe-Lock Microcentrifuge Tubes (1.5 mL) | Eppendorf, Hamburg, Germany | ||
Fetal Bovine Serum [0.2 µm Sterile Filtered] (FBS) | PAN-Biotech, Aidenbach, Germany | P40-37500 | |
Inverted Microscope | Leica Mikrosysteme, Wetzlar, Germany | Leica DMi8 | |
Microvolume Spectrophotometer (NanoDrop Spectrophotometer) | Thermo Fisher Scientific, Waltham, MA | ||
Capillary Transfection System (Neon Transfection System) | Thermo Fisher Scientific, Waltham, MA | MPK5000 | |
Neon Transfection System 10 µL Kit | Thermo Fisher Scientific, Waltham, MA | MPK1096 | |
Hemocytometer (Neubauer Chamber) | Paul Marienfeld, Lauda-Königshofen, Germany | 0640110 | |
PBS Dulbecco w/o Ca2+ w/o Mg2+ | Biochrom, Berlin, Germany | L182-50 | |
Penicillin [10,000 units/mL] and Streptomycin [10 mg/mL] (Pen/Strep) | Merck, Darmstadt, Germany | P0781 | |
Pipette Tips (10 µL) | Starlab, Hamburg, Germany | ||
Pipette Tips (1000 µL) | Starlab, Hamburg, Germany | ||
Pipette Tips (200 µL) | Starlab, Hamburg, Germany | ||
Plasmid Maxi Kit | Qiagen, Hilden, Germany | 12163 | |
Single Channel Pipette (0.1-10 µL) | Eppendorf, Hamburg, Germany | ||
Single Channel Pipette (100-1000 µL) | Eppendorf, Hamburg, Germany | ||
Single Channel Pipette (10-200 µL) | Eppendorf, Hamburg, Germany | ||
Trypan Blue Solution | Merck, Darmstadt, Germany | T8154 | |
Trypsin-EDTA (0,05 %) | Thermo Fisher Scientific, Waltham, MA | 25300054 | |
Analyses of transfected primary human RPE cells | |||
10% SDS-Polyacrylamide Gel | |||
1x Incubation Buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) | |||
2x SDS Sample Buffer | |||
4x Incubation Buffer (200 mM NaH2PO4, 1.2 M NaCl, 40 mM imidazole, pH 8.0) | |||
Amersham Protran Supported 0.2 µm Nitrocellulose Blotting Membrane | Cytiva, Marlborough, MA | 10600015 | |
Amphotericin B [250 µg/mL] (AmphoB) | Merck, Darmstadt, Germany | A2942 | |
Anti-PEDF Antibodies (Rabbit Polyclonal) | BioProducts, Middletown, MD | AB-PEDF1 | |
Anti-Penta-His Antibodies (Mouse Monoclonal) | Qiagen, Hilden, Germany | 34660 | |
Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 Nutrient Mixture (DMEM/F12) | PAN-Biotech, Aidenbach, Germany | P04-41150 | |
Elution Buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) | |||
Fetal Bovine Serum [0.2 µm Sterile Filtered] (FBS) | PAN-Biotech, Aidenbach, Germany | P40-37500 | |
Hemocytometer (Neubauer Chamber) | Paul Marienfeld, Lauda-Königshofen, Germany | 0640110 | |
Horseradish Peroxidase-Conjugated Anti-Mouse Antibodies (Rabbit Polyclonal) | Agilent Dako, Santa Clara, CA | P0260 | |
Horseradish Peroxidase-Conjugated Anti-Rabbit Antibodies (Goat Polyclonal) | Abcam, Cambridge, United Kingdom | ab6721 | |
Human PEDF ELISA Kit | BioProducts, Middletown, MD | PED613 | |
LAS-3000 Imaging System | Fujifilm, Minato, Japan | ||
LightCycler 1.2 Instrument | Roche Life Science, Penzberg, Germany | ||
LightCycler FastStart DNA Master SYBR Green I | Roche Life Science, Penzberg, Germany | 12239264001 | |
LightCycler Capillaries (20 μl) | Roche Life Science, Penzberg, Germany | 4929292001 | |
Microvolume Spectrophotometer (NanoDrop Spectrophotometer) | Thermo Fisher Scientific, Waltham, MA | ||
Mini-PROTEAN Tetra Cell Casting Module | Bio-Rad Laboratories, Feldkirchen, Germany | 1658015 | |
Mini-PROTEAN Tetra Vertical Electrophoresis Cell for Mini Precast Gels, 4-gel | Bio-Rad Laboratories, Feldkirchen, Germany | 1658004 | |
Ni-NTA Superflow | Qiagen, Hilden, Germany | 30410 | |
PageRuler Prestained Protein Ladder | Thermo Fisher Scientific, Waltham, MA | 26616 | |
Penicillin [10,000 units/mL] and Streptomycin [10 mg/mL] (Pen/Strep) | Merck, Darmstadt, Germany | P0781 | |
Pipette Tips (10 µL) | Starlab, Hamburg, Germany | ||
Pipette Tips (1000 µL) | Starlab, Hamburg, Germany | ||
Pipette Tips (200 µL) | Starlab, Hamburg, Germany | ||
PowerPac Basic Power Supply | Bio-Rad Laboratories, Feldkirchen, Germany | 1645050 | |
QIAamp DNA Mini Kit | Qiagen, Hilden, Germany | 51304 | |
Reverse Transcription System | Promega, Madison, WI | A3500 | |
RNase-Free DNase Set | Qiagen, Hilden, Germany | 79254 | |
RNeasy Mini Kit | Qiagen, Hilden, Germany | 74104 | |
Rocking Shaker | Cole-Parmer, Staffordshire, United Kingdom | SSM3 | |
Safe-Lock Microcentrifuge Tubes (1.5 mL) | Eppendorf, Hamburg, Germany | ||
Safe-Lock Microcentrifuge Tubes (2.0 mL) | Eppendorf, Hamburg, Germany | ||
Single Channel Pipette (0.1-10 µL) | Eppendorf, Hamburg, Germany | ||
Single Channel Pipette (100-1000 µL) | Eppendorf, Hamburg, Germany | ||
Single Channel Pipette (10-200 µL) | Eppendorf, Hamburg, Germany | ||
Trans-Blot Turbo Transfer System | Bio-Rad Laboratories, Feldkirchen, Germany | 1704150 | |
Trypan Blue Solution | Merck, Darmstadt, Germany | T8154 | |
Trypsin-EDTA (0,05 %) | Thermo Fisher Scientific, Waltham, MA | 25300054 |