Name: retroviral transduction of bone marrow progenitor cells to generate t-cell receptor retrogenic mice
Uploaded: 2016-07-11T15:00:00
Description: Watch this Scientific Journal Video about Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice at JoVE.com

이 작품은 MLB, BCM에서 당뇨병 연구 센터 (P30-DK079638)의 파일럿 / 타당성 프로그램, JDRF 1-FAC-2014-243-APF, ADA에 NIH (5K22A1119151-01 및 1R56DK104903-01)에서 보조금에 의해 지원되었다 1-15-JF-07, 면역학 MB에 친목, 그리고 로버트와 제니스 맥 네어 재단 AAI 채용.

윤리 정책 : 모든 노력이 조사와 꼬리 정맥 주사시 최소한으로 동물의 불편이나 스트레스를 유지한다. 마우스는 실험에서 세포의 공급원으로서 사용된다; 같은 어떠한 절차 나 조작은 안락사 따로 없습니다. 마우스는 죽음을 확인하기 위해 자궁 경부 전위 다음 CO 2 흡입에 의해 안락사됩니다. 이 절차는 미국 수의학 협회의 안락사에 대한 패널의 권고와 일치한다. 1. 레트...

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프로토콜에서, 최적의 골수 건강, 전달 효율 및 재구성을 보장하기 위해 여러 세부 중요한 단계를 우리. 첫 번째 중요한 단계는 생성 및 GP + E86 바이러스 생산자 세포의 적절한 유지된다. 초기 통로 생산 세포주를 사용하여 80 % 컨 플루 이하 사용하기 전에에서 유지한다. 48 시간 - 신선한 GP + E86 바이러스 생산 세포를 만드는 경우, 293T 세포가 초기 통로 (24)에 대한 문화에서 성장되어 있는지 확인합?...

인간 및 마우스의 T 세포 수용체 (TCR) 레파토리는 1 × 108, 2 × 106 고유 TCR도 각각 1.2로 추정되었다. 이 넓은 다양성은 T 세포는자가 펩티드에서뿐만 아니라 항원 제시 세포 (APC를)의 주 조직 적합성 복합체 (MHC)에 의해 제공 병원체 유래의 항원 에피토프의 광대 한 배열을 인식 할 수있다. 독특한 펩타이드 MHC 단지와 TCR도의 상호 작용의 미묘한 차이는 T 세포가 사멸, 아네 르기, 활성화, 분화, 사이토 카인 생산 세포 독성을 받아야할지 여부를 지시. 그러나, 큰 TCR 레퍼토리 특정 TCR 특정 항원에 응답하는 방법의 분석은 하나의 TCR 시스템의 사용을 필요로한다. 다양한 TCR 유전자 변형 마우스를 생체 내 모델 3-9 단일 TCR의 기능을 연구하기 위해 생성되었다. 그러나, 포함하는 형질 전환 마우스를 TCR 할주의 사항이있다선정 된 하나의 트랜스 제닉 마우스 생식선 DNA (10)에 임의의 형질 전환 유전자 삽입 소위 창시자 효과를 생성하는 시간의 길이. 따라서, 비교적 적은 TCR 형질 전환 마우스는 임의의 주어진 항원에 대해 생성 된 동일한 에피토프에 대한 고 및 저 TCR 친화력의 기능적 의미는 거의 언급하지 않는다. 화면 빠른 접근을위한 필요성을 해결 개별적으로 또는 조합하여 복수의 TCR도 공부 (레트로 바이러스 &#39;레트로&#39;형질 전환에서 &#39;제닉&#39;) retrogenic 마우스는 형질 전환 마우스 11-13 TCR의 대안으로 이용되어왔다. 여러 바이러스에서 발견 된 2A 펩타이드 합의 모티브 분열은 2A의 글리신과 2B의 프롤린 사이에 발생하는에서, 2A-ASP를-발 / 일드 - 공급 과잉-X-ASN-프로의 Gly-2B-프로 구성 시스 -acting 가수 분해 효소 활동에서 번역 10,14-16 동안 리보솜 건너 뛰기 결과. 은 C를 나타내는 상세한 다이어그램이러한 방식으로, 하나의 벡터 화학 양론 번역의 결과로 연결될 수 2 시스 트론 (TCR 알파와 TCR 베타) (12) -를 다양한 2A 펩티드 (F2A, E2A, T2A 및 P2A)의 leavage 참고 문헌 10를 참조하십시오. 이 방법을 활용하여, 우리는 표현하고 직접 생체 내에서 여러 항원 특정 TCR도 비교 할 수 있습니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM, high glucose + glutamine</td>
			<td>Corning Cellgro</td>
			<td>10-013-CV</td>
			<td>Dulbecco&#39;s Modification of Eagle&#39;s Medium with 4.5 g/L glucose, L-glutamine &#38; sodium pyruvate</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Atlanta Biological</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-Versene</td>
			<td>Lonza</td>
			<td>17-161F</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 um syringe filter</td>
			<td>Thermo Scientific</td>
			<td>194-2545</td>
			<td></td>
		</tr>
		<tr>
			<td>polybrene</td>
			<td>Sigma</td>
			<td>H9268-10G</td>
			<td>Sterile Filtered in dH2O</td>
		</tr>
		<tr>
			<td>Ciprofloxacin&#160;</td>
			<td>VWR</td>
			<td>AAJ61970-06</td>
			<td></td>
		</tr>
		<tr>
			<td>5-fluorouracil (5-FU)</td>
			<td>VWR</td>
			<td>AAA13456-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Corning Cellgro</td>
			<td>25-000-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM nonessential Amino Acids</td>
			<td>Corning Cellgro</td>
			<td>25-025-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1M solution</td>
			<td>Corning Cellgro</td>
			<td>25-060-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco by Life Technologies</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strept</td>
			<td>Corning Cellgro</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Corning Cellgro</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mm tissue culture dishes</td>
			<td>Greiner Bio-one</td>
			<td>639160</td>
			<td></td>
		</tr>
		<tr>
			<td>Tisue culture-treated 6-well flat plate</td>
			<td>Greiner Bio-one</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>70 um nylon cell strainers</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-3</td>
			<td>Invitrogen</td>
			<td>PMC0033</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-6</td>
			<td>Invitrogen</td>
			<td>&#160;PHC0063</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Stem Cell Factor</td>
			<td>Invitrogen</td>
			<td>PMC2113L</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Corning Cellgro</td>
			<td>46-D13-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>HANKS Buffer</td>
			<td>Corning Cellgro</td>
			<td>21020147</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 10 mL Syringe</td>
			<td>BD</td>
			<td>300912</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 1 mL Syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>27G x 1/2 BD Precision Glide Needle</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>30G x 1/2 BD Precision Glide Needle</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
	</tbody>
</table>

retroviral transduction of bone marrow progenitor cells to generate t-cell receptor retrogenic mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

골수 대표 골수 전달도 (도 1a)에있어서 꼬리 정맥에서 정맥 내로 주입되기 전에 골수 전달 효율은 수확 된 골수의 약 10 μL 중 100 ㎕를 첨가하고, 프로토콜의 단계 133에서 판정한다 PBS 및 ametrine 발현을 분석 하였다. 일반적으로 형광 양성 세포의 비율은 구조 및 레트로 바이러스 역가에 따라 25 %에서 70 % 사이이다. 6주 골수 주사 후, 마우스 골수 재구성 (?...

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Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

골수 전구 세포의 레트로 바이러스 형질 도입은 T 세포 수용체 Retrogenic 마우스를 생성하는

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Research

JoVE Journal

Immunology and Infection

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

반대 미리 정의된 T 세포 수용체 Specificities와 Transnuclear 쥐 Toxoplasma gondii SCNT 비아 획득

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

연구

면역학 및 감염병학

Retroviral Transduction of T-cell Receptors in Mouse T-cells

마우스 T - 세포에서 T - 세포 수용체의 Retroviral 전달

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Bone Marrow-derived Macrophage Production

뼈 대식 세포 생산에게 골수 유래

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

만성 EAE와 마우스의 신경 줄기 / 전구 세포의 전신 주입

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

쥐 뒷다리 긴 뼈 해부 및 골수 격리

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

생성<em&gt; 드 노보</em&gt; 중심 Hemichain의 레트로 바이러스 형질 도입에 의해 항원 특이 적 인간 T 세포 수용체

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

공부하는 유전 적 접근으로 헬퍼 T 세포의 레트로 바이러스 형질 도입은 메커니즘 자신의 차별화 및 기능 제어

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

An In Vivo Mouse Model to Measure NaÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¯ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Vivo에서 마우스 모델을 측정 순진한 CD4 T 세포 활성화, 확산 및 골 수 유래 수지상 세포에 의해 유도 된 Th1 차별화

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

호중구 계보 세포의 질량 세포 분석 분석을위한 전체 골수의 준비

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

콜라게나아제 E를 사용하여 뮤린 결장에서 라미나 프로프리아 단핵 세포의 분리

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...