우리는 도움이 논의에 대한 우리 연구실의 모든 구성원을 감사드립니다. 우리는 원고의 중요한 읽기 안나 Lisztwan과 크리스티나 Gewinner 감사드립니다. 이 작품은 BBSRC 기금 BB/I021248/1 지원하고 웰컴 트러스트 부여 090090/Z/09/ZAH은 UCL 암 연구소에서 웰컴 트러스트 연구 경력 개발 동료가되었습니다.

1. pcDNA6_TetR_IRES_blast의 복제 <ol><li> 그림 3에 도시 된 바와 같이, 제한 효소 Xba I 및 NCO 나는 TetR 유전자와 blasticidin 저항 (BlastR) 유전자의 프로모터를 제거 할 수있는 플라스미드 pcDNA6/TR (V1025-20, Invitrogen)의 일부 다이제스트를 수행합니다. 4.6 킬로바이트 벡터 조각을 분리합니다. </li><li> TetR 유전자에서 polyadenylation 순서를 제거하려면 Xho I 사이트는 즉시 TetR의 정지 코돈 cDNA의 5&#39;end의 보존 Xba I 사이트 잠시 후 cDNA PCR에서 소개합니다. pMigR1_TetR이 플라스미드 생성하기 위해 제한 효소 Xba I과 Xho I을 사용하여 pMigR1 17으로 인한 조각을 Subclone. </li><li> TetR_IRES 조각을 출시 할 제한 효소 Xba I 및 NCO I로 pMigR1_TetR 벡터를 소화. 1.25 이하 삽입 조각을 분리합니다. </li><li> 4.6 킬로바이트 pcDNA6/TR와 1.25 킬로바이트 TetR_IRES 조각을 Ligate. TR관할 E.coli에 결합 제품을 ansform 및 표준 분자 생물학 기술을 사용하여 원하는 pcDNA6_TetR_IRES_blast의 구조를 확인합니다. </li></ol> 2. 세포 라인의 생성 안정적으로 TetR을 표현 <ol><li> 1 일, 씨 1x10에서 표준 성장 매체를 사용하여 10 cm 조직 배양 접시 당 6 셀 (예 : DMEM 10 % FCS와 보충). 종자 두 개의 플레이트, pcDNA6_TetR_IRES_blast과 transfection 한, 그리고 blasticidin 선택을위한 대조군으로 다른. 가능한 가장 낮은 통로 및 권장 성장 매체를 사용하여 확인하십시오. </li><li> 2 일에서 transfect 제조업체의 지침에 따라 Fugene 6 일 (E2691, Promega)를 사용하여 pcDNA6_TetR_IRES_blast 2 μg과 하나가 판. 두 번째 판을 transfect하지 마십시오. </li><li> 3 일에 두 접시에 5 μg / ML blasticidin를 포함하는 표준 성장 매체를 추가 할 수 있습니다. 최적의 선택 농도가 주어진 셀 라인에 따라 다를 수 있습니다 및 주시기 바랍니다 수도 N사전에 결정해야 eed. </li><li> 일 4 일, 10 cm 접시 5 MG / ML blasticidin를 포함하는 표준 성장 매체를 사용하여 50분의 1과 1 / 100, 25분의 1, 1 / 5 셀을 분할. 제대로 untransfected 또는 transfected 세포를 포함하는 접시에 라벨을해야합니다. 희석 단계마다 두 개의 플레이트를 준비합니다. </li><li> untransfected 접시에있는 모든 세포는 첫 번째 주일 이내에 사망 있도록 매일 셀을 선택합니다. 모든 2-3일 선택 미디어를 변경합니다. </li><li> 1-2주 후, blasticidin 방지 식민지가 표시 될 것입니다. 하나의 식민지가 침투 할 수 있도록 5-50 식민지를 포함하는 접시를 선택합니다. </li><li> 식민지는 약 5mm (또는 더 큰)의 직경에 도달했을 때, 단일 식민지을 선택 복제 실린더를 사용하여 적어도 12 독립적 인 클론을 분리. 24 잘 조직 배양 플레이트 별도의 잘으로 각 복제를 전송합니다. 때 합류, 잘 6 잘 조직 배양 플레이트의 하나에 각도에서 세포를 분리. </li><li> 문화 CL로 이동선택 미디어들. 6 잘 조직 배양 플레이트의 두 단일 우물에 각도에서 때 합류, 분할 세포. </li><li> 각 클론을 테스트하기 위해서는 immunoblotting의 후속 테스트를 위해 다른 잘에 합류 셀 및 수확 세포의 잘 하나를 동결. </li><li> 서양은 마우스 단클론 항 TET02 항체 (MoBiTec GmbH가)를 사용 blotting에 의해 TetR 감지. 대조군으로 부모 세포 라인의 lysate를 포함해야합니다. </li><li> 가장 높은 TetR 식으로 3 클론을 선택하고 각 clonal 문화를 확장합니다. 가능한 한 빨리 각 유망 클론의 주식을 멈춰 봐. 마지막으로, TetR - 표현 클론과 함께 부모 세포 라인을 비교하여 관심의 분자 및 세포 생물학적 매개 변수를 검사합니다. 관심의 분자 및 세포 생물학적 매개 변수에 변경을 표시하지 않습니다 가장 높은 TetR 표현으로 &#39;최고&#39;클론을 선택합니다. </li></ol> 3. pTER와 PT-렉스 DEST30 벡터의 파일럿 테스트 표현각각 ING shRNA 또는 cDNAs, <ol><li> 12에 설명 된대로 shRNA 삽입과 플라스미드 pTER를 생성 할 수 있습니다. PT-렉스 DEST30 벡터 (12301-016, Invitrogen) 게이트웨이 기술 (Invitrogen)를 사용하여 관심있는 cDNA를 포함하는를 구성합니다. cDNA 표현의 경우, cDNA에 태그 추가 나중에 exogenously 표현 단백질의 검출을 용이하게합니다. </li><li> Transiently 선택의 transfection 시약을 사용하여 pTER 또는 PT-렉스 DEST30 plasmids (shRNAs 또는 관심 cDNAs 중 하나를 표현)와 부모 대상 셀 라인을 transfect. shRNA 표현 plasmids의 테스트를 위해 높은 transfection 효율을 달성 할 수 있는지 확인합니다. </li><li> 수확 관심의 유전자의 고갈이나 overexpression을 감지하는 기대, 해당 항체를 사용하여 immunoblotting을위한 세포와​​ 과정을 transfected. </li></ol> 4. 테트라싸이클린-inducible (Tet-에서) pTER 또는 PT-렉스 DEST30 벡터를 사용하여 shRNAs 또는 cDNAs의 표현과 안정적인 세포 라인의 생성 </p&gt; <ol><li> 1 일, 공인 테트라싸이클린 무료 혈청 (; 16000-014 Invitrogen에서 예를 들어 FBS)을 함께 보충 표준 성장 매체를 사용하여 10 cm 판 당 &#39;최고&#39;TetR - 표현 클론의 씨앗 1x10 6 셀 있습니다. pTER (또는 PT-렉스 DEST30) 및 더블 선정 대조군과 같은 하나 판과 transfection을위한 씨앗이 한 판. 가능한 가장 낮은 통로를 이용해야합니다. </li><li> 2 일에서 transfect Fugene 6을 사용 pTER 2 μg (또는 PT-렉스 DEST30)과 하나 판. 두 번째 판을 transfect하지 마십시오. </li><li> 일 3, 5 μg / ML blasticidin 500 μg / ML zeocin (또는 1 MG / ML G418)이 포함 된 표준 성장 매체를 추가 할 수 있습니다. </li><li> 일 4 일, 5 μg / ML blasticidin 500 μg / ML zeocin (또는 1 MG / ML 포함 10 cm 플레이트 및 표준 성장 매체를 사용하여 50분의 1과 1 / 100, 25분의 1, 1 / 5 셀을 분할 G418). 희석 단계마다 두 개의 플레이트를 준비합니다. </li><li> 첫 번째 주일 이내에 untransfected 접시에있는 모든 세포가 죽었을 확인하고, 매일 셀을 선택합니다.blasticidin 및 zeocin (또는 G418) 모든 3~4일를 포함하는 표준 성장 매체를 변경합니다. </li><li> 2-3주 후, blasticidin / zeocin (또는 blasticidin/G418)를 두 번 방지 식민지가 표시 될 것입니다. 하나의 식민지를 선택 5-50 식민지를 포함하는 접시를 선택합니다. </li><li> 식민지는 약 5mm (또는 더 큰)의 직경에 도달했을 때, 단일 식민지을 선택 복제 실린더를 사용합니다. 최소 24 개인 클론을 분리합니다. 24 잘 조직 배양 플레이트 별도의 잘으로 각 복제를 전송합니다. </li><li> blasticidin (5 μg / ML)와 zeocin (250 μg / ML)의 중간 포함 된 유지 보수 농도의 문화 클론 [또는 G418 (0.5 MG / ML)]. 때 합류, 잘 6 잘 조직 배양 플레이트의 하나에 각도에서 세포를 분리. </li><li> 각 클론을 테스트하기 위해서는 잘 6 잘 플레이트의 세 한 우물에 6 잘 판 중 하나가 합류 분할. 때 합류 잘 하나에서 세포를 동결. tetracyclin을 테스트 나머지 2 우물을 사용하여전자 inducible 표현. 두 번째 잘 테트라 사이클린 무료로 떠난 동안 잘 ... 1 μg / ML의 농도에서 테트라 사이클린을 추가합니다. </li><li> 적절한 항체를 사용하여 immunoblotting을위한 테트라 사이클린, 수확 셀 및 절차에 부화의 24-96 시간 후. cDNA 표현의 유도에 대한 심사 할 때 더 이상 테트라 사이클린 치료는 내생 단백질의 shRNA로 인한 고갈을 결정해야 할 것입니다 동안 짧은 테트라 사이클린의 배양 시간을 권장합니다. </li><li> 원하는 고갈 / overexpression과 적어도 2 클론을 선택하고 각각의 문화를 확장합니다. 가능한 한 빨리 각 유망 클론의 주식을 멈춰 봐. 실험 클론 Tet-에 성장하는 경우가 항상 테트라싸이클린 무료 혈청 및 유지 보수 농도에서 각각의 선택 항생제로 보충 미디어를 사용해야합니다. </li></ol>

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관심 없음 충돌이 선언 없습니다.

우리는 Tet-에 시스템이 크게 특히 조작 유전자가 세포 생존에 필수적인 경우 조작 및 / 또는하기가 어렵 전지 시스템에서 유전자 기능의 분석을 용이하게 있다고 생각합니다. 또한, 매우 구체적인 Tet-에 시스템에 의해 유전자 발현을 제어 할 수있는 능력은 다른 단계 (세포주기의 진행이나 ​​잘 정의 된 차별화 프로세스 동안 예를 들어)에 유전자 기능을 연구 할 수있는 기회를 제공합니다. 여기...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> 태아 소 혈청 (FBS) </td><td> Invitrogen </td><td> 16000-044 </td><td> 테스트 Tet 무료 </td></tr><tr><td> Blasticidin </td><td> Invivogen </td><td> 개미-BL-1 </td><td></td></tr><tr><td> Zeocin </td><td> Invivogen </td><td> 개미 - 아연-5 </td><td></td></tr><tr><td> G418 </td><td> PAA 실험실 </td><td> P31-011 </td><td> 미디어 100 MG / ML </td></tr><tr><td> 안티 TET02 </td><td> MoBiTec GmbH의 </td><td> TET02 </td><td> WB에 1,000분의 1 to 2천분의 1에서 사용 </td></tr><tr><td> pcDNA6/TR </td><td> Invitrogen </td><td> V1025-20 </td><td></td></tr><tr><td> PT-렉스 DEST30 </td><td> Invitrogen </td><td> 12301-016 </td><td></td&gt; </tr><tr><td> 테트라싸이클린 </td><td> 시그마 </td><td> 87,128 </td><td> 2 밀리그램 / 에탄올에 ML </td></tr><tr><td> Doxycycline </td><td> 시그마 </td><td> D9891 </td><td> 2 밀리그램 / 물에 ML </td></tr><tr><td> 복제 실린더 </td><td> Bellco 유리 주식회사 </td><td> 2090-00808 </td><td> 다시 사용 가능한 </td></tr></tbody></table>

generation of stable human cell lines with tetracycline-inducible (tet-on) shrna or cdna expression

포유류의 세포 생물학 분야의 주요 접근 방식은 공개 할 목적으로, 선택한 셀 라인에 대한 관심의 유전자의 표현의 조작이 하나 이상의 유전자의 과도 / 안정 overexpression 또는 분해를 사용하여 유전자의 기능 (들)의 여러 관심. 불행하게도, 다양한 세포 생물 조사에 대해이 방법은 적합 할 때 세포의 성장 / 확산 결함이나 원치 않는 세포 분화의 유전자 발현 결과의 조작. 따라서, 연구자들은 E.에서 가져온 테트라싸이클린 억제 단백질을 (TetR), 적응 대장균의 테트라 사이클린 내성 오페론 1,2,3 포유류의 세포에 cDNAs을 표현하는 매우 효율적이고 긴밀한 규제 시스템을 생성합니다. 즉, TetR은의 TO에 테트라 사이클린 (Tet - 오프 시스템이라고한다)의 추가시 발기인 지역의 Tet-연산자 (TO)에 바인딩 또는 (2) 바인드에 의해 전사 (1) 블록 개시 하나에 수정되었습니다 티테트라 사이클린의 그 부재 (시스템 Tet 기능이라고한다) (그림 1). Tet - 오프 시스템은 테트라 사이클린의 지속적인 존재를 (이는 조직 세포 배양 매체에 약 24 시간의 반감기가) 필요하다는 불편을 끼쳐 드려 감안할 때 Tet는 -에서 시스템보다 폭이 매우 촉박 개발의 결과, 최적화 된 및 cDNA 표현 효율적인 벡터 시스템은 여기에 사용 된. 바로 짧은 머리 핀의 자형 RNAs (shRNAs)를 표현하는 4 포유류 세포 벡터에서 유전자 최저에 대한 RNA 간섭 (RNAi)의 설립 이후 siRNAs 5-11와 매우 유사한이 기능은 설명했다. 유전자 타겟 세포 생존을 위해 필수적입니다 때 안정적으로 고갈이 적합하지 않을 때문에 그러나, 이러한 shRNA로 인한 최저의 접근 방법은 기존의 한방 전략과 같은 제한이 있습니다. 이러한 한계를 극복하기 위해, 밴 드 Wetering 외. 12 shRNA 표현 벡터 pSUPER에게 수정 5 </su그들에게 관심의 대상 유전자의 테트라 사이클린-inducible 고갈과 안정적인 셀 행을 생성 할 수있게 발기인 지역에에를 삽입하여 P를&gt;. 여기, 우리는 효율적으로 생성하는 방법을 설명 안정된 인간 Tet-inducible에 안정적으로의 overexpression 또는 관심의 유전자의 고갈 중 하나를 운전 세포 라인. 이 방법 사용, 우리는 성공적으로 생성했습니다 Tet-에 크게 중심체 13,14과 15,16 신호를 apoptosis에 산악 표준시 / hMOB / NDR 폭포의 분석을 촉진 세포 라인. 이 보고서에서 우리는 선택의 우리의 벡터를 설명 효율적으로 생성하는 데 필요한 두 개의 연속 조작 단계를 설명뿐만 아니라 인간의 Tet-에 세포 라인 (그림 2). 또한,의 생성을위한 프로토콜을 요약 외에 인간의 Tet-에 세포 라인, 우리는 중요한 기술적 절차에 관한 부분 및 특성화 세포 Tet 기능을 설명합니다.

안정적 TetR을 표현 RPE-1 세포 라인의 초기 특성에 대한 예는 그림 4에 표시됩니다. 부모 셀 라인 (대조군 역할을 함) 외인성 TetR 단백질을 표현하지 않는 동안 모든 RPE-1 클론은 (그림 4 차선 1), (레인 2, 5 비교) TetR의 수준을 다양한 표현합니다. RPE-1 중 TetR 식의이 유사 Tet이 기능 세포 클론 예상되며, plasmids을 표현 TetR의 표현부터 플라스미드 통합 사이트에 크게 의존합니다. ...

Watch this Scientific Journal Video about Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression at JoVE.com

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

inducible과 관심의 유전자의 치료 cDNA overexpression 또는 shRNA로 인한 똑 다운과 인간의 세포 라인을 생성하기 위해 신속하고 간단한 방법. 이 방법은 일시적 transfection 방법 또는 기존 최저 / 한방 전략에 의해 변경하기가 어렵 안정적으로 높은 reproducibly 조작 세포 라인에 연구자 수 있습니다.

테트라싸이클린-inducible (Tet-에서) shRNA 또는 cDNA 표현과 안정적인 인간 세포 라인의 생성

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

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JoVE Journal

Biology

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

35.0K 조회수.  UCL Cancer Institute. inducible과 관심의 유전자의 치료 cDNA overexpression 또는 shRNA로 인한 똑 다운과 인간의 세포 라인을 생성하기 위해 신속하고 간단한 방법. 이 방법은 일시적 transfection 방법 또는 기존 최저 / 한방 전략에 의해 변경하기가 어렵 안정적으로 높은 reproducibly 조작 세포 라인에 연구자 수 있습니다.

비디오: Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

트립신과 Passaging 색채 인간의 배아 줄기 세포 - 라인

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

연구

생물학

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Microinjection을 사용하여 안정적인 트렌스​​ 제닉 C. 엘레간스의 생성

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

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 ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

인간 CD40 - 활성화된 B 세포의 생성

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

유전자 발현 분석을위한 배아 Zebrafish 및 cDNA 합성에서 RNA 격리

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

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 ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

향상된 Biosafety에 대한 고분자 - complexed Ecotropic Lentivirus과 인간의 세포의 형질 도입

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.