1) Hücre hazırlık <ol><li> Yapışık hücrelere kullanırken, trypsinize ve elektroporasyon önce hücreleri toplamak için gereklidir. </li><li> Üç farklı geçit numaraları temsil eden dört fare embriyonik fibroblast (MEF) kültürleri, arasında transfeksiyon karşılaştırmak için, her bir şişesi aşağıdaki gerçekleştirin. </li><li> Hücre kültür ortamı aspire. </li><li> Hücreleri yıkamak için PBS ekleyin. </li><li> PBS çıkarın; hücreleri kapsayacak şekilde yeterli tripsin ve tripsin hücreleri ayırmak için izin vermek için birkaç dakika bekleyin. </li><li> Hücrelerin durumunu doğrulamak için bir mikroskop ile şişeler kontrol hücreleri ayırmak için balon şaplak ve sonra, emin hücreleri tüm müstakil olmak için tekrar şişeyi kontrol edin. </li><li> Ek süre ve gerekirse tekrar bekleyin. </li><li> Tüm hücreleri müstakil sonra, tripsin nötralize etmek için serum içeren medya ekleyin. </li><li> Hücrelerin bir santrifüj tüpüne aktarın ve santrifüj yoluyla pelet hücreleri (RCF = 300 xg). </li><li> PBS bilinen bir ses supernatant ve tekrar süspansiyon hücreleri çıkarın. </li><li> Hücreleri saymak. </li><li> Uygun hacimli hücre süspansiyonu, deneyler için hücreler (1 x 10 6 hücre / mL yoğunluğu hücreleri de ortalama 150 mcL gerekecektir) gerekli sayıda sağlamak için yeni bir tüpe aktarın . </li><li> Santrifüj hücreleri. </li><li> Gene Verici elektroporasyon 1 x 10 6 hücre / mL bir hücre yoğunluğu elde etmek için tampon uygun hacmi supernatant ve tekrar süspansiyon hücreleri çıkarın. </li><li> Hücre süspansiyonu plazmid başına mL 20 mg ekleyin ve hafifçe karıştırın. </li></ol> 2) Elektroporasyon damar kurulum ve elektroporasyon Plaka kurulum ve elektroporasyon <ol><li> Gene Verici MXcell elektroporasyon sisteminin güç modülü takın plaka odası. </li><li> Pipet 150 mcL hücre karışımı ya da 96-kuyu elektroporasyon plaka kuyu içine tampon. </li><li> Plaka odası ve nabız plaka koyun. </li><li> Odasından plaka çıkarın. </li><li> Her bir kuyunun içinde yukarı ve aşağı pipetleme iyi içeriği karıştırın. </li><li> Hücrelerin her kuyudan 12 kuyucuğu önceden ısıtılmış tampon aktarın. </li><li> Plaka dokunun hücreleri dağıtmak ve inkübatörde koymak. </li><li> Hücreler 24 saat kurtarmanızı sağlar. </li></ol> Küvet kurulum ve elektroporasyon <ol start="9"><li> ShockPod Gene Verici MXcell elektroporasyon sistemi ve fiş ™ küvet kamara güç modülü plaka odasına çıkarın. </li><li> Pipet 600 mcL 0.4 cm boşluk elektroporasyon küvet içine hücre süspansiyonu. </li><li> ShockPod odasına küvet koyun ve elektrik darbe sağlamak. </li><li> Küvet odasından çıkarın. </li><li> Küvet içinde yukarı ve aşağı pipetleme küvet içeriğini karıştırın. </li><li> Hücrelerin her kuyudan 12 kuyucuğu önceden ısıtılmış tampon aktarın. </li><li> Plaka dokunun hücreleri dağıtmak ve inkübatörde koymak. </li><li> Hücreler 24 saat geri alalım. </li></ol> 3) Temsilci Sonuçlar Hücreleri transfecting ve onları kurtarmak için izin sonra, flow sitometri kullanarak epifluorescent mikroskopi kullanılarak kantitatif, kalitatif transfeksiyon verimlilik, analiz ve. <img src="/files/ftp_upload/1662/1662fig1.jpg" alt="Şekil 1" /> Şekil 1. başarıyla electroporated ve şimdi, GFP geninin ifade Hücreler epifluorescent mikroskobu altında görünür. <img src="/files/ftp_upload/1662/1662fig2.jpg" alt="Şekil 2" /> Şekil 2 faz kontrast altında hücreleri görüntüleme hem transfekte ve untransfected hücrelerinin görselleştirme sağlar. Bunlar 200V düşük voltaj elektroporasyon darbe maruz kaldılar hücrelerdir. Hücreler yüksek hücre yoğunluğu nedeniyle büyük ölçüde konfluent. <img src="/files/ftp_upload/1662/1662fig3.jpg" alt="Şekil 3" /> Şekil 3, Epifloresans altında bakış aynı alanda bir dizi hücreleri GFP işaretleyici ifade gösterir, ama bu sadece küçük bir yüzdesi önceki resim görünür hücreleri . <img src="/files/ftp_upload/1662/1662fig4.jpg" alt="Şekil 4" /> Şekil 4 250V, faz kontrast altında görülen canlı hücrelerinin toplam sayısı biraz azalır. <img src="/files/ftp_upload/1662/1662fig5.jpg" alt="Şekil 5" /> Epifloresans altında bir Şekil 5 GFP ifade hücrelerinin sayısının arttığını görebilirsiniz. <img src="/files/ftp_upload/1662/1662fig6.jpg" alt="Şekil 6" /> Şekil 6, en yüksek voltaj 375V uygulanan, daha az görünür canlı hücreler vardır. <img src="/files/ftp_upload/1662/1662fig7.jpg" alt="Şekil 7" /> Şekil 7 Ancak, geri kalan hücreler büyük bir yüzdesi, GFP ifade edilmektedir. Hangi durum optimal deney tasarımı bağlıdır. Bazı deneylerde transfekte hücrelerinin en büyük sayı diğer deneylerde en yüksek yüzde transfeksiyon belki de en iyisi, en uygun olabilir. Biz her koşul altında olumlu GFP ve yüzdeleri hücre yaşla birlikte değişir hücrelerin yüzdesi olarak ilgilenen. Flow sitometri, her farklı elektroporasyon koşullar altında transfeksiyon sonuçları hakkında kantitatif bilgi sağlayabilir. <img src="/files/ftp_upload/1662/1662fig8.jpg" alt="Şekil 8" /> Şekil 8 12 elektroporasyon koşulların her birinin altında 5 hücre gösterilen pasajda olumlu GFP hücre yüzdesi. Durumu 12, maksimum transfeksiyon yüzdesi güçlü kare dalga darbe altında yüksek voltaj üstel çürüme darbe, durumu 6 ve% 70 altında yaklaşık% 80, test. <img src="/files/ftp_upload/1662/1662fig9.jpg" alt="Şekil 9" /> Şekil 9 hücrelerin elektroporasyon önce 9 kez geçti, transfeksiyon yüzde genel desen neredeyse aynıdır, ancak çok hafif bir azalma ile transfeksiyon yüzdeleri. <img src="/files/ftp_upload/1662/1662fig10.jpg" alt="Şekil 10" /> Şekil 10 Burada gösterilen geçit genç hücrelere göre transfeksiyon yüzdesinde belirgin bir azalma göstermektedir 13 hücreler, GFP hücrelerinin yüzdeleri. En yüksek transfeksiyon yüzdeleri yaklaşık yarısı, mümkün olduğu kadar kısa sürede izolasyon sonra sağlıklı hücreleri kullanarak önemini gösteren genç hücreleri ne ile elde edildi. <table border="1"><tbody><tr><td colspan="3"> Gene Verici MXcell kullanarak transfecting MEF hücreleri için kullanılan Elektroporasyon Koşullar </td></tr><tr><td> Durumu (1-6) Üstel Bozunma bakliyat, 350 uF, 1000ohm </td><td> Voltaj (V) </td><td></td></tr><tr><td> 1 </td><td> 200 </td><td></td></tr><tr><td> 2 </td><td> 250 </td><td></td></tr><tr><td> 3 </td><td> 300 </td><td></td></tr><tr><td> 4 </td><td> 326 </td><td></td></tr><tr><td> 5 </td><td> 350 </td><td></td></tr><tr><td> 6 </td><td> 376 </td><td></td></tr><tr><td> Durumu (7-12) Kare Dalga bakliyat, 2000 uF, 1000 ohm ve 1 darbe </td><td> Voltaj (V) </td><td> Darbe Süresi (ms) </td></tr><tr><td> 7 </td><td> 200 </td><td> 10 </td></tr><tr><td> 8 </td><td> 250 </td><td> 10 </td></tr><tr><td> 9 </td><td> 300 </td><td> 10 </td></tr><tr><td> 10 </td><td> 200 </td><td> 20 </td></tr><tr><td> 11 </td><td> 250 </td><td> 20 </td></tr><tr><td> 12 </td><td> 300 </td><td> 20 </td></tr></tbody></table>

Yazarlar, bu yazıda kullanılan reaktifler ve alet üreten Bio-Rad Laboratories tarafından istihdam edilmektedirler

Bu video makale MXcell elektroporasyon sistemi kolayca MEFS veya diğer birincil hücre hatları için optimal elektroporasyon koşulları belirlemek için nasıl kullanılacağını gösterir. 96-plaka biçimi, pek çok, pek çok ayrı deneyler için ihtiyacını ortadan kaldırabilir aynı anda yapılacak deneysel ya da en iyi duruma getirme koşullarını, çoğaltır için izin verir. Bu yordamı yürütürken, bir, mümkün olduğu kadar kısa sürede izolasyon sonra sağlıklı hücreleri kullanmak ve elektroporasyon tampon eşleştirilir elektroporasyon koşulları kullanmak için hatırlamak gerekir.

<table><tbody><tr><td>Gene Pulser&amp;#174; Electroporation Buffer</td><td>Bio-Rad</td><td>165-2676</td><td></td></tr><tr><td>Gene Pulser MXcell&amp;#8482; Electroporation System</td><td>Bio-Rad</td><td>165-2670</td><td></td></tr><tr><td>Gene Pulser MXcell&amp;#8482; ShockPod&amp;#8482; Cuvette Chamber</td><td>Bio-Rad</td><td>165-2673</td><td></td></tr><tr><td>Gene Pulser MXcell&amp;#8482; Electroporation System With ShockPod&amp;#8482; Cuvette Chamber</td><td>Bio-Rad</td><td>165-2674</td><td></td></tr></tbody></table>

using the gene pulser mxcell electroporation system to transfect primary cells with high efficiency

Elektroporasyon birincil hücre içine plazmid DNA veya siRNA tanıtmak için en etkili yolu olduğu giderek daha belirgin hale gelmektedir. Gene Verici MXcell elektroporasyon sistemi ve Gen Verici elektroporasyon tampon (Bio-Rad), özellikle memeli hücrelerinde ve birincil ve kök hücreleri gibi zor transfect hücreleri, nükleik asitler kolayca transfect için geliştirilmiştir. Biz iyi elektroporasyon koşulları hızla tanımlamak için basit bir deney gerçekleştirmek için nasıl göstereceğiz. Biz bir deney optimizasyonu yapılır aynı zamanda yapılabilir, böylece elektroporasyon koşulları bir dizi yoluyla çeşitli örnekleri çalıştırmak için nasıl göstereceğiz. Biz de en uygun koşulları, aynı elektroporasyon verimliliği korurken, standart elektroporasyon küvetler kullanılabilir 96-iyi elektroporasyon plakaları kullanarak elektroporasyon küvetler elektroporasyon plakalardan geçiş kolaylaştırmak tespit nasıl gösterecektir. Video, elektroporasyon deneyler başarı ya da başarısızlığa yol açabilecek bazı önemli faktörler de ele alınacaktır.

Watch this Scientific Journal Video about Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency at JoVE.com

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

Bu prosedür, Gen Verici MXcell elektroporasyon sistemi hızlı ve kolay bir fare embriyonik fibroblastlar (MEFS) veya diğer birincil hücreler için en iyi elektroporasyon koşulları belirlemek için nasıl kullanılacağını gösterir. Sorun giderme için dikkat edilmesi gereken noktalar da ilgili video tartışılmıştır.

Yüksek Verimli Primer Hücre Transfect Gen Verici MXcell Elektroporasyon Sistemi

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

using-gene-pulser-mxcell-electroporation-system-to-transfect-primary

Research

JoVE Journal

Biology

24.1K Görüntüleme.  Bio-Rad Laboratories, Inc.. Bu prosedür, Gen Verici MXcell elektroporasyon sistemi hızlı ve kolay bir fare embriyonik fibroblastlar (MEFS) veya diğer birincil hücreler için en iyi elektroporasyon koşulları belirlemek için nasıl kullanılacağını gösterir. Sorun giderme için dikkat edilmesi gereken noktalar da ilgili video tartışılmıştır.

Video: Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

Gene electrotransfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses, which cause destabilization of cell membrane, making it permeable to small molecules and allows transfer of large molecules such as DNA. It represents an alternative to viral vectors, due to its safety, efficacy and ease of application. For gene electrotransfer different electric pulse protocols are used in order to achieve maximum gene transfection, one of them is changing the electric field direction and orientation during the pulse delivery. Changing electric field direction and orientation increase the membrane area competent for DNA entry into the cell. In this video, we demonstrate the difference in gene electrotransfer efficacy when all pulses are delivered in the same direction and when pulses are delivered by changing alternatively the electric field direction and orientation. For this purpose tip with integrated electrodes and high-voltage prototype generator, which allows changing of electric field in different directions during electric pulse application, were used. Gene electrotransfer efficacy is determined 24h after pulse application as the number of cells expressing green fluorescent protein divided with the number of all cells. The results show that gene transfection is increased when the electric field orientation during electric pulse delivery is changed.

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

Gene transfection by electroporation is improved approximately two times when orientation of electric field is changed during pulse application, while cell viability is not affected. The increase in gene transfection is caused by the increase of the membrane area which is made competent for DNA entry into the cell.

Elektrik Bakliyat Uygulama sırasında Yön ve Elektrik Alan yönelimi değiştirme Plazmid Gen Transferi artırır In vitro</em

Gene electrotransfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses, which cause ...

Tıp

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

Akustik Nanodispensing teknolojisini kullanarak yüksek verim DNA Plasmid multiplexing ve transfeksiyon

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

Genetik

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Hazırlık Elektroporasyon murin Kemik İliği itibaren İlköğretim Hematopoetik Hücre Kültürleri

Biyoloji

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Microscale Vortex destekli Sıralı Moleküler Teslimat için Electroporator

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Biyomühendislik

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

Yenidoğan Pial Yüzey Elektroporasyon

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

Nörobilim

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

Sürekli akışlı, permeabilizasyon algılamalı mikro-elektroporasyon sisteminin imalatı ve işletilmesi

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In the granule cell precursor (GCP), the primary cilium acts as a pivotal signaling center that orchestrates precursor cell proliferation by modulating the Hh signaling pathway. The investigation of primary cilium-dependent Hh signaling machinery is facilitated by in vitro genetic manipulation of the pathway components to visualize their dynamic localization to the primary cilium. However, transfection of transgenes in the primary cultures of GCPs using the currently known electroporation methods is generally costly and often results in low cell viability and undesirable transfection efficiency. This paper introduces an efficient, cost-effective, and simple electroporation protocol that demonstrates a high transfection efficiency of ~80-90% and optimal cell viability. This is a simple, reproducible, and efficient genetic modification method that is applicable to the study of the primary cilium-dependent Hedgehog signaling pathway in primary GCP cultures.

Here, we present a reproducible in vitro electroporation protocol for genetic manipulation of primary cerebellar granule cell precursors (GCPs) that is cost-effective, efficient, and viable. Moreover, this protocol also demonstrates a straightforward method for the molecular study of primary cilium-dependent Hedgehog signaling pathways in primary GCP cells.

Granül Hücre Öncüsünde Birincil Seyuma Bağımlı Sinyal Yollarını İncelemek için Verimli ve Uygun Maliyetli Elektroporasyon Yöntemi

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In ...

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.

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).

Uyuyan Güzel Transpozon Sistemi Kullanılarak Primer İnsan Pigment Epitel Hücrelerinin Elektroporasyon Tabanlı Genetik Modifikasyonu

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

Zinc Finger Nuclease-Based Genome Editing: A Technique for Modifying Genome in Human Pluripotent Stem Cells by Double-Stranded Homology Dependant Repair Mechanism

Source: Blair, J. D., et al. <a href="https://www.jove.com/v/53583">Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation.</a> J. Vis. Exp. (2016).
This video demonstrates a technique of zinc-finger nuclease-based genome editing in human pluripotent stem cells (hPSCs). As hPSCs can potentially be differentiated into various cell types, this technology is helpful for in vitro pathological and pharmacological studies in a patient-specific manner.

Çinko Parmak Nükleaz Tabanlı Genom Düzenleme: İnsan Pluripotent Kök Hücrelerinde Çift Sarmallı Homoloji Bağımlı Onarım Mekanizması ile Genomu Değiştirme Tekniği

Source: Blair, J. D., et al. Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation. J. Vis. Exp. (2016).
This ...

JoVE Encyclopedia of Experiments

Biyolojik Teknikler

Genom Düzenleme Teknikleri

Genin devreye girmesi

Electroporation-Mediated In Vitro Gene Delivery: An Electric Pulse Technique to Introduce Fluorescent Reporter Protein-Encoding Plasmids Into Cultured Cells

Source: Lo, J. C. W. et al., <a href="https://www.jove.com/v/63283">Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor.</a>&#160;J. Vis. Exp.&#160; (2021)
This video demonstrates the electroporation technique that introduces fluorescent protein-encoding plasmid DNA into mouse primary cerebellar granule cell precursors.

Elektroporasyon Aracılı İn Vitro Gen İletimi: Floresan Raportör Protein Kodlayan Plazmitleri Kültürlenmiş Hücrelere Sokmak İçin Bir Elektrik Darbe Tekniği

Source: Lo, J. C. W. et al., Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell ...