1 단계 : lentivirus 생산을위한 293 세포와 플레이트를 녹여. <ol><li> 15 - ML 튜브 DMEM + 중간 9 ML을 준비합니다. </li><li> 액체 질소 탱크에서 냉동 293 주식의 병을 제거하고 (전부는 아니지만) 대부분의 세포가 해동 때까지 37 ° C의 물을 욕조에 병을 넣어. </li><li> 1 단계에서 15ml 튜브에 동결 유리병과 장소에서 세포를 제거합니다. </li><li> 5 분 180g에 원심 분리기 다음 뜨는 폐기하십시오. </li><li> DMEM + 중간의 10 ML있는 세포를 Resuspend 및 젤라틴 코팅 100 - mm 요리로 전송할 수 있습니다. 그들이 80~90%의 합류하기 전까지 37 ° C 5 % CO 2 배양기에서 세포를 품어. </li><li> 293 세포의 통과 : PBS로 세척 세포는 PBS를 대기음, 그리고 0.05 % trypsin/0.53 MM EDTA (에틸렌 다이아 민 테트라 초산) 10 cm 요리마다 4 ML을 추가, 37 1 분 품어 ° C.를 </li><li> 10 ML DMEM + 매체 resuspend 감청하여 요리의 세포, 그리고 15 - ML 튜브로 전송을 분리합니다. 5 분 180g에 원심 분리기 및 뜨는을 대기음. </li><li> DMEM + 매체의 적절한 볼륨을 추가하고 몇 시간을 아래와 pipetting하여 단일 세포 현탁액으로 세포를 헤어지게. </li><li> 세포의 개수를 카운트 및 FP 매체와 ML 당 8x10 5 세포에 농도를 조정합니다. </li><li> 종자 100 - mm 배양 접시 당 8x10 6 셀 (10 ML)에서 세포를, 그리고 37 밤새 품어 ° C, 5 % CO 2. </li></ol> 2 단계 : lentivirus의 plasmids과 수확 바이러스 Transfect 293 세포 <ol><li> 각 10 cm 접시, FUW - 테토 - cDNA 10 μg (Oct4, Sox2, Klf4 또는 C - Myc) pMD - G의 2.5ug와 lentivirus 백본 및 pPax2의 7.5 μg을 사용합니다. </li><li> 튜브로 세 plasmids를 aliquoting 동안 혈청없이 DMEM의 500μl를 포함하는 두 번째 튜브로 Fugene 6 transfection 시약의 30μl를 제공하고 실온에서 5 분 도청 손가락과 부화에 의해 부드럽게 섞는다. </li><li> DNA 믹스 Fugene 6/DMEM-containing 튜브에 드롭하여 놓기를 추가, 20 분 손가락 도청 및 부화하여 부드럽게 섞는다. </li><li> 293의 그릇에 DNA / Fugene 6 복잡한 dropwise을 추가하고 37 밤새 품어 ° C 5 % CO 2. </li><li> 다음날 아침 : transfection을 대기음 시약 함유 매체를 신선한 ES 세포 매체의 5 ML을 추가하고, 배양기에 세포를 반환합니다. </li><li> 48 시간 transfection 후 72시간에서 0.45 - μm의 기공 크기 셀룰로스 아세테이트 필터를 통해 필터링, 15 ML 튜브로 전송할 수, 10 ML 멸균 일회용 주사기를 사용하여 293의에서 매체를 수집합니다. </li><li> ultracentrifugation하여 바이러스 뜨는을 집중. 원하는 볼륨의 바이러스 펠렛을 Resuspend하고 Oct-3/4-, Sox2 -, Klf4 및 C - Myc - lentiviruses을 포함하고있는 매체의 동등한 부분 amixture합니다. </li><li> 바이러스를 만드는 동안, (접시 당 2x10 6 셀) 10 - cm 요리 confluency 90 % Oct4-neo/rtTA 또는 Oct4-GFP/rtTA MEFs을 (통과 &lt;3) 준비 </li><li> PBS 10 ML과 문화 미디어와 세척을 대기음. </li><li> 0.05 % trypsin/0.53 MM EDTA (에틸렌 다이아 민 테트라 초산) 접시 당 1 ML을 추가하고, 37 품어 ° C, 10 분, PBS 폐기하십시오. </li><li> 문화 매체 9 ML 추가, 단일 세포로 세포를 중단하고, 15 ML 튜브로 전송할 수 있습니다. </li><li> 세포의 수를 계산하고, ML 당 8x10 4 세포에 농도를 조정합니다. 젤라틴 코팅 10 cm 요리에 세포 현탁액 10 ML (8x10 5 셀)을 전송합니다. 37 밤새 접시를 품어 ° C, 5 % CO 2. </li><li> fibroblast 요리에서 매체를 대기음, 그리고 바이러스가 포함된 매체 10 ML를 추가합니다. 37 4 H에서 하룻밤에 세포 ° C 5 % CO 2 알을 품다. </li><li> 24 후 fibroblast 요리에서 매체를 대기음, 신선한 ES 세포 매체 10 ML를 추가합니다. </li><li> 네 가지 유전자의 표현을 시작하는 매체를 포함하는 Doxycycline와 함께 정기적으로 ES 세포 매체를 교체, 즉, 프로그래밍을 시작합니다. </li><li> Oct4 - 네오 기자를 사용한다면, 6 구일 사이에 언제든지 약물 섹션을 시작합니다. </li><li> 식민지가 뽑힐 정도로 큰이 될 때까지 매일 매체를 변경합니다. 식민지 먼저 프로그래밍의 개시 후 약 일주일 표시가 될 것입니다. 그들은 일 약 20 뽑힐 정도로 커질 것입니다. </li></ol> 3 단계 : IPS 식민지를 따기 따기 전에 일 : <ol><li> γ - DR4 조사 MEFs과 젤라틴 - 코팅 24 - 잘 접시의 필요한 번호 씨앗 </li></ol> 1 일에 클론을 따기 : <ol><li> 1-2시간 따기 전에 접시에 식민지를 공급. </li><li> V - 바닥 96 잘 요리의 우물에 PBS 15 μl를 추가합니다. </li><li> 식민지는 PBS로 한번 잡았 수가 들어있는 접시를 씻고 요리에 PBS의 10ml를 추가합니다. </li><li> 작은 5μl pipet 팁 (4 μl에 P20 pipetman 설정)와 식민지를 선택합니다. 전송 공동96 - 잘 접시에 잘하는 로니스. </li><li> 잘 멀티 채널 (12) pipetor를 사용하여 각 트립신 20 μl를 추가합니다. Pipet 위아래로 3 회. 37 품어 ° C를 4 분간. </li><li> trypsinized 식민지로 ES 매체 100 μl를 추가합니다. Pipet 위, 아래로 10 회. 12 장소 pipetor의 모든 다른 위치에 팁을 사용하여 24 잘 요리에 96 잘 요리에서 한 번에 6 클론을 전송합니다. </li><li> 세포가 ES 세포 매체와 함께 매일 먹이 confluency 80-90%에 도달할 때까지 37 ° C 5 % CO 2 배양기에서 24 잘 접시에 세포를 성장. 세포는 아마 3-7일의 확장 준비됩니다. </li></ol> 4 단계 <ol><li> IPS 세포의 확장 <ol><li> 매체 기음, 그리고 PBS 1 ML로 세포를 씻어. </li><li> 완전히 PBS를 제거, 0.25 % 트립신 / 37 1 밀리미터 EDTA (에틸렌 다이아 민 테트라 초산) 및 부화 ° 10 분 C의 0.1 ML를 추가합니다. </li><li> ES 매체의 0.4 ML를 추가하고 단일 세포 현탁액과 위아래로 pipetting하여 세포를 일시 중지합니다. </li><li> 6 - 잘 접시의 잘하는 세포 현탁액을 전송, 1.5 ML의 ES 세포 매체를 추가하고, 37 품어 ° C 5 % CO 2 배양기 세포는 6 - 잘 접시에 confluency 80-90%에 도달할 때까지. 이 시점에서 다음과 같이, 세포의 냉동 재고를 준비합니다. </li></ol></li><li> 프리즈 주식의 준비 <ol><li> 매체 기음, 그리고 PBS 2 ML으로 세포를 씻으십시오. </li><li> 완전히 PBS를 제거, 0.25 % 트립신 / 37 1 밀리미터 EDTA (에틸렌 다이아 민 테트라 초산) 및 부화 ° 10 분 C 0.5 ML를 추가합니다. </li><li> ES 매체 2 ML 추가 및 단일 세포 현탁액과 위아래로 pipetting하여 세포를 일시 중지합니다. </li><li> 15 ML 튜브에 세포 현탁액을 전송, 세포의 수를 계산하고 세포를 스핀 다운. </li><li> , 뜨는을 삭제 ML 당 2x10 6 세포에서 농도 ES 매체와 세포 resuspend. </li><li> 이 ES 세포 냉동 매체 (ES 매체 20 % DMSO)와 유리병 당 0.5 ML로 나누어지는를 준비합니다. </li><li> 튜브를 고정하고 부드럽게 혼합하는 세포 현탁액의 0.5 ML을 전송합니다. </li><li> 세포 동결 용기에 튜브를 넣고 ° C 야간은 -80에서 보관, 장기 보관을 위해 다음날 액체 질소로 전송할 수 있습니다. </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>

이 비디오에서는, 우리는 Oct4-GFP/R26-M2rtTA과 Nanog-GFP/R26-M2rtTA 생쥐에서 추출한 MEFs에서 GFP 양성, pluripotent IPS 세포를 생성하는 inducible, lentiviral 시스템을 사용하는 방법을 보여줍니다. 그것은 프로그래밍 과정 transgene 표현의 제어를위한 수 있기 때문에이 절차는 IPS 세포를 생성하는 유용한 방법입니다. IPS 세포의 생성에 바이러스의 사용은 임상 환경에서 이러한 세포의 사용을 막는 있지만,이 절차는 우리가 아직 정의 프로그래밍 작업을 밑줄 주요 생물 학적 질문 중 일부에 대답 수 없습니다.

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

Pluripotency는 Oct4의 바이러스 도입하여 차별 murine에서 유도된 수 있습니다 Sox2, Klf4, 그리고 C - Myc (타카하시와 야마 나카, 2006; Wernig, 외, 2007;. 오키타, 외, 2007;.. Maherali, 외, 2007). 우리는이 네 전사 요소 doxycycline에서 표현되는 프로그래밍 전략을 고안해야 (독스) lentiviral 벡터 (Brambrink 외., 2008) - inducible. 이러한 inducible 구조를 사용하여, 우리는 마우스 배아 섬유아 세포 (MEFs)에서 유도된 pluripotent 줄기 (IPS) 세포를 유도하실 수 있습니다. 이 비디오에서는, 우리는 네 개의 전사 요소를 표현하고 IPS 세포를 생산하기 위해이 바이러스를 감염 MEFs하는 방법을 보여줍니다 inducible의 lentiviruses의 생성에 대한 절차를 설명합니다. inducible의 lentiviruses를 사용하여, 문화 매체 doxycyline의 추가에 의해 제어의 4 가지 요소의 표현. 전통 retroviral 감염을 통해이 시스템의 장점은 프로그래밍과 유전자 발현 요구 사항의 반응 속도론은 세부 분석 수 있도록 해제에 대한 유전자를 켜고있는 능력입니다.

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

이 동영상은 inducible의 lentivirus를 사용하여 유도된 pluripotent 줄기 세포를 생성하는 절차를 보여줍니다 표현 Oct4, Sox2, C - Myc와 Klf4.

삭스 - 2의 강제 표현을 통해 MEFS에서 IPS 세포를 생성, 10 - 4, C - Myc, 그리고 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., ...

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36.1K 조회수.  Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology. 이 동영상은 inducible의 lentivirus를 사용하여 유도된 pluripotent 줄기 세포를 생성하는 절차를 보여줍니다 표현 Oct4, Sox2, C - Myc와 Klf4.

비디오: 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.

MEFs부터를 Matrigel하기 : MEFs의 면전에서 Passaging hESCs를

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

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생물학

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.

Matrigel에 MEFs에서 분리 hESCs : MEFs에서 Matrigel 2

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.

MEFs에서 Matrigel 3 : Matrigel에 Matrigel에서 Passaging hESCs

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.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

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.

유기 용매 및 초산없이 쿠매시 G - 250로 젤의 단백질 염색법

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.

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

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.

유전자 발현 연구를 단순화하는 자동 셀 카운터를 사용하여 : Namalwa 셀에서 IL - 4 종속 유전자 발현의 siRNA의 최저

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 ...

Feeder-Free Adaptation, Culture and Passaging 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 cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

완료 녹아웃 세럼 교체 피더 - 무료 매체를 사용하여 인간의 IPS 세포 피더 - 자유 적응력, 문화와 Passaging

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 ...

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.

Cryopreserving와 인간 IPS 세포 복구 완료 마네에게 세럼 교체 피더 - 무료 매체를 사용하여

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.

인간의 세포에서 분비 단백질의 생산을위한 편리하고 일반적인 표현 플랫폼

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