Adım 1: 293 lentivirüs üretim hücreleri ve plaka çözülme. <ol><li> 9 ml, 15 ml tüp DMEM + orta hazırlayın. </li><li> Dondurulmuş 293 hisse senetlerinin bir şişe sıvı nitrojen tankı çıkarın ve (hepsi değil) en hücreleri çözülmüş kadar 37 ° C su banyosunda flakon koymak. </li><li> 1. adımı 15ml tüp içine hücreleri dondurma şişe ve yerden çıkarın. </li><li> 180g az 5 dakika süreyle santrifüj, ve sonra süpernatantı atın. </li><li> DMEM + orta 10 ml hücreleri yeniden süspanse edin ve jelatin kaplı 100 mm çanak transfer. Hücrelerin% 80-90 konfluent kadar 37 ° C,% 5 CO 2 inkübatör inkübe edin. </li><li> Geçiş: 293 hücreler PBS ile yıkama hücreleri, PBS aspirat ve 0.05% trypsin/0.53 mM EDTA 10 cm çanak başına 4 ml ve 37 az 1 dakika boyunca inkübe ° C </li><li> 10 ml DMEM + orta tekrar süspansiyon dokunarak yemeklerinden hücreleri, ve 15 ml tüp transfer ayırın. 180g az 5 dakika süreyle santrifüj, süpernatantı aspirat. </li><li> DMEM + orta uygun hacmi ekleyin ve birkaç kez aşağı yukarı pipetleme hücreleri tek bir hücre süspansiyonu break up. </li><li> Hücrelerin sayısını ve FP orta ml başına 8x10 5 hücre konsantrasyonu ayarlayabilirsiniz. </li><li> Tohum hücreleri 100 mm kültür çanak başına 8x10 6 hücreli (10 ml), 37 o gece inkübe ° C,% 5 CO 2. </li></ol> Adım 2: Lentivirüs plazmid ve hasat virüsü ile Transfect 293 hücreleri <ol><li> Her 10 cm plaka için, FUW-Teto-cDNA 10 mikrogram (Oct4, Sox2, Klf4 veya c-Myc) PMD-G 2.5ug ile lentivirüs omurga ve pPax2 7.5 mikrogram kullanın. </li><li> Bir tüp içine üç plazmid aliquoting ederken, DMEM, serum olmadan 500μl içeren ikinci bir tüp içine Fugene 6 transfeksiyon reaktif 30μl teslim ve oda sıcaklığında 5 dakika boyunca parmak-vurma ve inkübe tarafından hafifçe karıştırın. </li><li> DNA karışımı Fugene 6/DMEM-containing tüpe damla bırak, parmak vurma ve 20 dakika inkübe hafifçe karıştırın. </li><li> 293 Kullanıcı çanak içine DNA / Fugene 6 karmaşık damla damla ekleyin ve 37 ° gece inkübe ° C,% 5 CO 2. </li><li> Ertesi sabah: transfeksiyon aspire reaktif içeren orta, 5 ml taze ES hücre orta ekleyin ve hücrelerin inkübatör dönün. </li><li> 48 saat ve 72 saat sonra transfeksiyon, 0.45 mikron gözenek boyutu selüloz asetat filtre üzerinden filtreleme, ve 15 ml tüp aktarılması, 10 ml steril tek kullanımlık şırınga kullanarak 293 orta toplamak. </li><li> Ultrasantrifügasyon tarafından virüs süpernatant Konsantre. Istenilen hacmi virüs pelletini tekrar Oct-3/4-, Sox2, Klf4-c-Myc-lentiviruses içeren orta eşit parçaya amixture. </li><li> Virüs yaparken, (2x10 6 hücre çanak ortalama) 10 cm yemekleri% 90 confluency Oct4-neo/rtTA veya Oct4-GFP/rtTA MEFS (geçit &lt;3) hazırlamak </li><li> Kültür ortamı ve 10 ml PBS ile yıkama aspire. </li><li> 0.05% trypsin/0.53 mM EDTA çanak başına 1 ml ekleyin ve 37 inkübe ° C 10 dakika, PBS atın. </li><li> 9 ml kültür ortamı, hücreler tek bir hücre için askıya alma, ve 15 ml&#39;lik bir tüp transfer. </li><li> Hücrelerin sayısını ve 8x10 ml başına 4 hücreli konsantrasyonunu ayarlamak. Jelatin ile kaplı 10 cm çanak 10 ml (8x10 5 hücre) hücre süspansiyonu aktarın. 37 geceleme çanak inkübe ° C,% 5 CO 2. </li><li> Fibroblast çanak orta aspire ve virüs içeren orta 10 ml ekleyin. 37 gecede 4 saat hücreleri ° C,% 5 CO 2 inkübe edin. </li><li> 24 sonra bir fibroblast çanak orta aspirat ve 10 ml taze ES hücre orta ekleyin. </li><li> Dört genlerin ifade başlatmak için orta içeren Doksisiklin düzenli ES hücre orta yerine, diğer bir deyişle, yeniden programlama başlatmak. </li><li> Oct4-neo muhabiri kullanıyorsanız, daha sonra 6 ve 9 gün arasında zaman ilaç bölümünü başlatın. </li><li> Koloniler aldı yeterince büyük olana kadar her gün orta değiştirin. Kolonileri ilk reprogramming başladıktan sonra yaklaşık bir hafta görünür hale gelir. Bunlar, yaklaşık 20 gün kadar alınır kadar büyük haline gelmelidir. </li></ol> Adım 3: IPS koloniler toplayıp Toplama önce Gün: <ol><li> Γ-ışınlanmış DR4 MEFS ile gerekli sayıda jelatin kaplı 24 kuyucuğu Tohum </li></ol> Gün 1 Klonlar Toplama: <ol><li> 1-2 saat almadan önce plakalar üzerinde koloniler besleyin. </li><li> Bir V-96-iyi dipli çanak kuyulardan 15 ul PBS ekleyin. </li><li> Kolonileri PBS ile bir kez alınır içeren çanak yıkayın ve çanak PBS 10ml eklemek. </li><li> Koloniler küçük 5μl pipetlemeyin ipuçları (4 ul P20 Pipetman ayarlamak) ile seçin. Transferi co96-iyi yemek, iyi bir lonies. </li><li> Tripsin 20 ul ve multi-kanal (12) pipetor kullanarak her ekleyin. Pipetleyin yukarı ve aşağı 3 kez. 37 ° C&#39;de 4 dakika. </li><li> 100 ES orta ul tripsinize koloniler ekleyin. Pipetleyin yukarı ve aşağı 10 kez. 12 yerde pipetor diğer her konumda bir ipucu kullanarak 24-iyi bir yemek için 96-iyi çanak bir defada 6 klonları aktarın. </li><li> 24 plaka hücreler hücrelerin ES hücre orta ile günlük beslenme,% 80-90 confluency ulaşıncaya kadar 37 ° C,% 5 CO2 inkübatör büyütün. Hücreler muhtemelen 3-7 gün içinde genişletmek için hazır olacak. </li></ol> Adım 4 <ol><li> IPS hücreleri genişletilmesi <ol><li> Orta aspire, hücreler ve 1 ml PBS ile yıkayın. </li><li> Tamamen PBS çıkarın, 0.1 ml% 0.25 tripsin / 37 az 1 mM EDTA ve inkübe ° C 10 dakika ekleyin. </li><li> ES orta 0.4 ml ekleyin ve tek hücre süspansiyonu için yukarı ve aşağı pipetleme hücreleri tarafından askıya. </li><li> 6-plaka iyi bir hücre süspansiyonu Transfer, 1.5 ml ES hücre orta ekleyin ve 37 inkübe ° C,% 5 CO2 inkübatör hücrelerin 6-kuyucuğu% 80-90 confluency ulaşana kadar. Bu noktada, aşağıdaki gibi hücreler dondurulmuş stok hazırlamak. </li></ol></li><li> Dondurma stok hazırlanması <ol><li> Orta aspire hücreleri ve 2 ml PBS ile yıkayın. </li><li> Tamamen PBS çıkarın, 0.5 ml% 0.25 tripsin / 37 az 1 mM EDTA ve inkübe ° C 10 dakika ekleyin. </li><li> ES orta 2 ml ekleyin ve tek hücre süspansiyonu için yukarı ve aşağı pipetleme hücreleri tarafından askıya. </li><li> 15 ml&#39;lik bir tüp hücre süspansiyonu Transfer, hücrelerin sayısını ve hücrelerin aşağı döndürün. </li><li> Süpernatantı atın ml başına 2x10 6 hücre konsantrasyonu ES orta hücreleri tekrar süspansiyon haline getirin. </li><li> 2 ES hücre dondurma orta (ES orta% 20 DMSO) ve flakon başına 0.5 ml kısım hazırlayın. </li><li> Şişeleri dondurma ve hafifçe karıştırın 0.5 ml hücre süspansiyonu aktarın. </li><li> Bir hücre dondurma kabı koyun ve şişeleri ° C gecede -80 tutmak, uzun süreli depolama için ertesi gün sıvı azot transfer. </li></ol></li></ol>

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Wernig, M., 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=et+al+.+In+vitro+reprogramming+of+fibroblasts+into+a+pluripotent+ES-cell-like+state.">et al . In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.</a> Nature. 448, 318-324 (2007).</li><li>Okita, K., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+germline-competent+induced+pluripotent+stem+cells.">Generation of germline-competent induced pluripotent stem cells.</a> Nature. 448, 313-317 (2007).</li><li>Maherali, N., 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=Global+epigenetic+remodeling+in+directly+reprogrammed+fibroblasts.">Global epigenetic remodeling in directly reprogrammed fibroblasts.</a> Cell Stem Cell. 1, 55-70 (2007).</li><li>Brambrink, 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=Sequential+expression+of+pluripotency+markers+during+direct+reprogramming+of+somatic+cells.">Sequential expression of pluripotency markers during direct reprogramming of somatic cells.</a> Cell Stem Cell. 2, 151-159 (2008).</li></ol>

Bu video, Oct4-GFP/R26-M2rtTA ve Nanog-GFP/R26-M2rtTA fareler elde edilen MEFS GFP pozitif, pluripotent iPS hücreleri oluşturmak için bir indüklenebilir lentiviral sisteminin nasıl kullanılacağını göstermektedir. Bu prosedür, yeniden programlama işlemi sırasında transgen ekspresyonu kontrolü sağlar çünkü iPS hücreleri üretmek için yararlı bir yöntemdir. IPS hücreleri nesil virüslerin kullanımı bir klinik ortamda bu hücrelerin kullanımını engellemektedir rağmen, bu prosedür henüz tanımlanan yeniden programlama sürecine altını önemli biyolojik sorulardan bazılarını yanıtlamak için etkinleştirin.

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generating ips cells from mefs through forced expression of sox-2, oct-4, c-myc, and klf4

Pluripotency farklılaştırılmış murin Oct4 viral transdüksiyon tarafından indüklenen olabilir, Sox2, Klf4, ve c-Myc (Takahashi ve Yamanaka, 2006; Wernig ve ark, 2007; Okita, vd, 2007;. Maherali, et al, 2007). Biz bu dört transkripsiyon faktörleri doksisiklin ifade edildiği bir yeniden programlama strateji geliştirdiler (dox) lentiviral vektörler (Brambrink ve ark, 2008) indüklenebilir. Bu indüklenebilir yapıları kullanarak, fare embriyonik fibroblastlar (MEFS) alınan uyarılmış pluripotent kök (iPS) hücreleri elde edebilirsiniz. Bu videoda, biz dört transkripsiyon faktörleri ifade ve iPS hücreleri üretmek için bu virüsleri ile enfekte MEFS nasıl göstereceğim indüklenebilir lentiviruses nesil için prosedürü göstermektedir. Indüklenebilir lentiviruses kullanarak, kültür ortamı Doxycyline ek tarafından kontrol dört faktör ifade. Bu sistemin avantajı, geleneksel retroviral enfeksiyon üzerinde, programlama ve gen ekspresyonu gereksinimleri kinetiği ayrıntılı olarak analiz edilebilir kapalı genler açmak ve kapatmak için yeteneğidir.

Watch this Scientific Journal Video about Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4 at JoVE.com

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Bu video indüklenebilir lentivirüs kullanarak bağlı pluripotent kök hücreleri üretmek için prosedürü ifade Oct4, Sox2, c-Myc ve Klf4 gösterir.

Sox-2 Zorla İfade yoluyla MEFS iPS Hücreler oluşturma, Eki 4, c-Myc ve Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

generating-ips-cells-from-mefs-through-forced-expression-sox-2-oct-4

Nanyang Technological University

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36.2K Görüntüleme.  Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology. Bu video indüklenebilir lentivirüs kullanarak bağlı pluripotent kök hücreleri üretmek için prosedürü ifade Oct4, Sox2, c-Myc ve Klf4 gösterir.

Video: Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.


This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Part 1 of 3.

MEFS I Matrigel: MEFS Durum Pasajlanması hESC

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.
...

Biyoloji

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF plates to feeder cell-free Matrigel plates.


This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

MEFS Matrigel 2 Matrigel üzerine MEFS Yarma hESC:

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF ...

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated.

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 3 of 3.

Matrigel MEFS Matrigel 3: Matrigel üzerine gelen Pasajlanması hESC

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage ...

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

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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Organik Solvent ve Asetik Asit olmadan Jeller Coomassie G-250 ile Proteinlerin Boyama

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

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

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 (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Yüksek Verimli Primer Hücre Transfect Gen Verici MXcell Elektroporasyon Sistemi

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Gen İfadesi Çalışmaları basitleştirin Otomatik Hücre Sayıcı kullanma: Namalwa Hücreler IL-4 Bağımlı Gen İfadesi siRNA demonte

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

İnsan iPS hücreleri dondurularak ve kurtarma Tam nakavt Serum Yedek Besleyici-Alerjik Orta

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

İnsan Hücrelerden salgılanan Proteinlerin Üretimi için Kullanışlı ve Genel Anlatım Platformu

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Kültür Memeli Hücrelerde Biyolojik tanımında ubikitin ve Ubiquitin benzeri Sistemi Regülatörleri tanımak siRNA Eleme

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...