이 연구는 NIH R00HL102154 지원 및 P 지행 혈액 학자 수상의 미국 사회되었다.

이 프로토콜에 기술 된 실험은 노스 웨스턴 대학 기관 동물 관리 및 사용위원회에 의해 명시된 지침과 규정에 따라 수행되었다. 문화 매체의 1 준비 <ol><li> 피브로넥틴 솔루션을 준비합니다. 인간 피브로넥틴 (1 mg)을 중 하나를 유리 병에 물 1 ㎖를 추가합니다. 교반없이 30 분 동안 조직 문화 후드에서 솔루션을 둡니다. 20 μg / ML의 최종 농도를 만들어 PBS 50 ㎖로 전체 액체를 전송하고 부드럽게 잘 섞는다. 판 (아래 참조)을 코팅하기 전에 피브로넥틴에 신선한 용액을합니다. </li><li> EPO 배지 (SCF 매체) 50 ㎖를 준비합니다. 신선한했을 때 표 1에 열거 된 성분을 결합합니다. 매체 1 달 동안 4 ° C에 저장할 수 있습니다. </li><li> EPO 함유 배지 50 ㎖를 준비합니다. 신선한했을 때 표 2에 나열된 성분을 결합합니다. 매체 1 달 동안 4 ° C에 저장할 수 있습니다. </li></올&gt; 2 피브로넥틴 코팅 플레이트 <ol><li> 6 - 웰 플레이트 (또는 12 웰 플레이트의 각 웰에 0.5 ml)을 각 웰에 1 ml의 피브로넥틴 용액을 추가한다. 1 시간 동안 조직 문화 후드에서 판을 둡니다. </li><li> 두 번 멸균 물 우물을 씻어 공기 판을 건조. </li><li> 접시를 싸서 4 ° C의 추운 방에 저장합니다. </li></ol> 태아 간 적혈구 모세포의 3 정화 참고 : E15에 E13에서 사용 태아의 간은 임신 한 쥐 (C57BL / 6) 시간이 초과되었습니다. E13.5 간은이 CFU-E 전구 세포의 수율을 최대화하기 위해 바람직하다. 시간이 초과 임신 한 쥐를 얻으려면 6~8주 세 남성과 여성의 마우스는 한 케이지 (하나 또는 두 개의 여성 대개 한 남성)에 배치되었다. 여성 질 플러그 결합이 성공적인지 여부를 판별하기 위해 다음 날 아침을 조사 하였다. 플러그가 발견 된 후 임신의 첫 번째 날 일이었다. <ol><li> 총 태아 간 세포의 준비 <ol><li> 양해 각서 (MOU)를 희생CO 2 흡입 자궁 전위에 의해 그 자체. 소독 용 70 % 에탄올로 복부를 스프레이. 자궁을 제거하기 위해 가위를 해부와 복부를 엽니 다. 1X PBS에서 자궁을 씻으십시오. </li><li> 조직 문화 후드 및 멸균 도구를 사용하여 멸균 조건에서 태아를 해부하고 1X PBS에 세척. </li><li> 태아에서 태아의 간을 해부하다. 조심스럽게 다른 집게와 떨어져 몸에서 태아의 간 (컬러 핑크)를 당겨, 한 집게로 태아의 몸체를 잡고. 관련 섬유 성 조직을 제거하기 위해 집게로 태아의 간을 청소합니다. 10 % 소 태아 혈청을 함유하는 신선한 1X PBS로 모든 태아 간을 놓습니다. </li><li> 기계적 피펫 팅 아래에 의해 태아의 간을 방해. </li><li> 50ml의 원뿔형 튜브에 40 μm의 셀 스트레이너를 통해 세포 현탁액을 필터. 5 분 동안 800 XG에서 세포 펠렛. </li><li> FBS를 뜨는을 기음 10 %로 1 ml의 PBS에 세포를 재현 탁 (약 8 E13.5 태아 간) ML 당. </li></ol></li><li> TER119 네거티브 적혈구 모세포의 정화 <ol><li> 15 분 동안 얼음에 쥐 IgG를 가진 세포 (0.2 ㎎ / ㎖)을 막기. </li><li> 세탁기 또는 원심 분리없이 세포 현탁액 (1 μg / ㎖)에 비 오티 닐화 항-TER119 항체를 추가한다. 얼음에 15 분 동안 품어. </li><li> 5 분 동안 800 XG에 10 % FBS (1시 10분)와 원심 분리기 1X PBS에 세척 할 것. 10 % FBS와 1 ㎖의 PBS에 세포를 재현 탁. </li><li> 추가 75 μL은 자성 입자가 결합과 얼음에 10 분 동안 품어 스트렙 타비 딘. </li><li> PBS는 10 % FBS를 함유하는 2.5 ml의 볼륨을 추가합니다. 바닥 튜브 라운드 5 ㎖의 폴리 프로필렌에 세포 현탁액을 전송합니다. </li><li> 자기 세포 정렬 장치에 튜브를 삽입하고 10 분 동안 배양한다. TER119 양성 세포는 관의 측면에 부착된다. 무소속 TER119 부정적인 적혈구 모세포는 서스펜션에 남아있을 것이다. </li><li> 바닥 튜브 라운드 새로운 5 ㎖의 폴리 프로필렌에 세포 현탁액을 붓고. </li><li><html> 반복 잔류 TER119 양성 세포를 제거하는 3.2.6와 3.2.7 단계를 반복합니다. </li><li> 5 분 동안 800 XG에서 세포 펠렛. 뜨는을 대기음. 10 % FBS를 함유하는 1 ml의 PBS에 세포를 재현 탁하고 트리 판 블루 생균 카운트. </li></ol></li></ol> 바이러스 인코딩 된 cDNA 나 shRNA를 가진 4 형질 도입 <ol><li> 3-5에서 정제 TER119 네거티브 태아 간세포 판형 × 105 / 웰의 12 - 웰 플레이트를 코팅 피브로넥틴 (다른 플레이트를 사용할 경우 셀 수를 조정)한다. </li><li> 바이러스의 형질 도입을 촉진하기 위해 10 μg / ㎖의 최종 농도로 폴리 브렌을 추가한다. 회전은 37 ° C에서 1.5 시간 동안 800 XG에, lentiviruses 또는 레트로 바이러스, 인코딩하는 cDNA 나 shRNA를 가진 세포를 감염. </li></ol> 형질 TER119 네거티브 마우스 태아 간 적혈구 모세포의 5 문화 <ol><li> (그림 1은 점선) 단자 적혈구의 초기 단계에서 유전자의 기능을 테스트합니다.&quot;&gt; </ol.><ol><li><ol. li=""> 농경 12 시간 동안 EPO 배지 (SCF 배지)에 세포를 형질 도입 형질 도입 유전자와 전구의 유지 상태의 발현을 허용한다. </ol.></li><li> 5 분 동안 800 XG에서 세포를 원심 분리기. 뜨는을 대기음. 12 - 웰 플레이트의 각 웰에서 배지를 함유하는 1 ml의 EPO에서 세포를 재현 탁. </li><li> 24 또는 48 시간 동안 배양 한 후, 추가 분석을 위해 세포를 수확. </li></ol></li><li> 터미널 적혈구 (그림 1 실선)의 후반 단계에서 테스트 유전자의 기능을합니다. <ol><li> 문화 EPO 포함하는 배지에서 바로 형질 세포. </li><li> 24 또는 48 시간 동안 배양 한 후, 추가 분석을 위해 세포를 수확. </li></ol></li></ol> 유세포 분석을위한 배양 된 태아 간 세포의 스테 이닝 (6) <ol><li> 세척하고 1X PBS에 세포를 재현 탁. 수확 유세포 분석을 위해 2 × 105 세포를 배양 하였다. </li><li> 품다어둠 속에서 실온에서 20 분 동안 형광 공역 항체와 세포. 차별화를 테스트하려면 APC 복합 항 CD71 및 PE 복합 항 TER119를 사용합니다. 안구 적출술을 테스트하려면 다른 항체에 추가 훽스트 33342 얼룩을 사용합니다. </li><li> 1X PBS로 세포를 씻으십시오. 죽은 세포를 염색 및 분석에서 제외하는 1 % FBS 및 요오드화 프로피 듐 (PI) (1 μg / ㎖)를 함유하는 0.5 ml의 PBS에 세포를 재현 탁. </li><li> 행동 흐름 cytrometric 분석. 흐름 cytometer의 조정 및 보상을위한 최초의 단일 컬러 염색 흠없는 대조군 세포를 분석 할 수 있습니다. </li></ol>

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저자는 그들이 더 경쟁 금전적 이해 관계가 없다고 선언합니다.

여기에서 우리는 시간 순으로 마우스 태아 간 터미널 적혈구를 분석 할 수있는 독특한 시스템을 제시한다. 다른 배양 조건의 적용을 통해, 우리는 성공적으로 초기와 후기 단계에서 터미널 적혈구 해부. 이는 다양한 기능을 가진 유전자의 메카니즘을 결정하는데 중요하다. 예를 들어, 라 세미 GTPases 단자 적혈구 생성의 다른 단계에서 중요한 역할을한다. 터미널 적혈구에 영향 세포 분화와 증식의...

적혈구는 적혈구 성숙 다 능성 조혈 줄기 세포의 분화 과정이다. 이 단계적인 과정은 최선을 다하고 적혈구 버스트 형성 단위 (BFU-ES), 빠른 속도로 나누어 적혈구 콜로니 형성 단위 (CFU-ES) 및 형태 학적 인식 적혈구 모세포 1,2의 형성을 포함한다. CFU-E의 전구 세포에서 터미널 적혈구는 순차적 에리트로 포이 에틴에 의존하고 독립적 인 단계 2,3을 포함한다. 단말기 적혈구 생성의 초기 단계에서, 에리트로 포이 에틴 (EPO)가 세포 표면 수용체에 결합하고 세포 사멸을 방지하고 빠른 세포 분열 및 유전자 발현을 촉진 1,4 하류 신호 전달 경로의 시리즈를 유도한다. 터미널 적혈구의 후반 단계에서 적혈구 모세포는 매우 응축 핵 5 단자 세포주기의 종료, 크로 마틴과 핵 응축, 압출을받을. 터미널 우리의 이해적혈구가 크게 때문에 체외에서 여러 가지의 성공적인 사용에 생체 마우스 모델 6-9에서 크게이다 지난 수십 년에 향상되었습니다. 이러한 모델 중 마우스 태아 간 적혈구 모세포의 체외 배양 세포 정화, 빠른 증식과 분화의 용이성 등 많은 장점을 제공하며, 인간의 적혈구 10, 11에 가까운 모방. 이 시스템에서, 마우스 태아 간에서 적혈구 전구 세포 다수 용이 TER119의 단일 단계 정제 (성숙한 적혈구 세포에 대한 마커) 네거티브 적혈구 모세포에 의해 단리 될 수있다. 적혈구 모세포의 이틀 동안 배양이 세포의 분화가 트랜스페린 수용체 (CD71) 및 TER119 항원 (12)의 표면 발현에 기초하여 유세포 분석으로 모니터 할 수있다. 또한, 말기 분화 적혈구 모세포 적출술 1 DNA 머신 (훽스트 33342)에 의해 검출 될 수있다3. 또한, 정제 된 전구 세포는 유전자의 cDNA 또는 적혈구 11,13,14에 유전자 발현 기능 역학적 연구를 용이 관심 유전자에 대한 작은 헤어핀 RNA들 (shRNA를)의 외인성 발현에 의해 변형 될 수있다. 그것이 단말 적혈구 생성의 다른 단계에서 유전자의 기능을 특성화하는 것이 곤란하기 때문에, 다른 한편으로는, 고속 셀 성장률 양면성 수있다. 대부분의 경우에, 그것의 cDNA 또는 shRNA를 발현 시간까지 보낸 단말 적혈구 생성의 초기 단계에서 특정 유전자의 기능, 세포가 이미 초기 단계를 통과했는지 여부를 결정하는 것은 곤란하다. 이 문제를 해결하기 위해서, 우리는 터미널 적혈구 생성의 초기와 후기 단계를 해부하는 독특한 시스템을 개발 하였다. 단말기 적혈구 생성의 초기 단계를 들면, 유전자 변형 TER119 네거티브 적혈구 모세포는 EPO 배지 함유하지만 줄기 세포 인자 (SCF)에서 IL-6 및 FLT3 배양리간드는 그들의 선조 상태를 유지하고, 식 (13)로 형질 도입 된 cDNA 또는 shRNA를 허용한다. 세포는 세포 증식 및 분화를 유도하기 위하여 12 시간 후에 EPO 함유 배지로 변경 하였다. 세포를 구별하기 시작할 때 이러한 방법으로, 형질 도입 된 cDNA 또는 shRNA를 이미 발현되었다. 터미널 적혈구의 후기를 들어, TER119 부정적인 적혈구 모세포는 EPO가 전달 후 즉시 매체를 포함에서 배양 하였다. 따라서, 하나의 단말은 적혈구 생성의 후반 단계에서의 관심의 유전자의 기능을 분석 할 수있다. 요약하면, 이러한 시스템의 광범위한 애플리케이션 단말 적혈구 생성의 다른 단계에서의 해부 유전자 기능을 도울 것이다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s MediumIMDM (IMDM)</td>
			<td>Gibco</td>
			<td>12440-053</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>GEMINI Bio-product</td>
			<td>700-102P</td>
			<td></td>
		</tr>
		<tr>
			<td>b-mecaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td>0.1 M in IMDM,&#160; 4&#8451;stock</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td>100 X</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Hyclone</td>
			<td>SH30034.01</td>
			<td>200 mM</td>
		</tr>
		<tr>
			<td>Stem cell factor (SCF)</td>
			<td>STEMCELL TECH</td>
			<td>2630</td>
			<td>100 ng/ul, -20&#8451; stock</td>
		</tr>
		<tr>
			<td>Mouse FIT3 Ligand (Flt-3L)</td>
			<td>BD Biosciences&#160;</td>
			<td>14-8001-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Recombinant Interleukin-6 (IL-6)&#160;</td>
			<td>GEMINI Bio-product</td>
			<td>300-327P</td>
			<td></td>
		</tr>
		<tr>
			<td>fibronectin</td>
			<td>BD Biosciences&#160;</td>
			<td>354008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>STEMCELL TECH</td>
			<td>9300</td>
			<td>10% stock in IMDM</td>
		</tr>
		<tr>
			<td>Insulin solution, human</td>
			<td>Sigma</td>
			<td>I9278</td>
			<td>4&#8451; stock</td>
		</tr>
		<tr>
			<td>holo-transferrin human</td>
			<td>Sigma</td>
			<td>T0665</td>
			<td>50 mg/ml in Water -20&#8451; stock</td>
		</tr>
		<tr>
			<td>Erythropoietin(Epo)</td>
			<td>PROCRIT</td>
			<td>NOC 59676-303-00</td>
			<td>3,000 U/ml stock</td>
		</tr>
		<tr>
			<td>Streptavidin particles Plus</td>
			<td>BD Pharmingen</td>
			<td>557812</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>STEMCELL TECH</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Round-Bottom Tube, 5 ml</td>
			<td>FALCON</td>
			<td>352063</td>
			<td></td>
		</tr>
	</tbody>
</table>

mouse fetal liver culture system to dissect target gene functions at the early and late stages of terminal erythropoiesis

적혈구가 빠르게 형성 단위 적혈구 식민지 (CFU-ES)를 나누어 다음에 최선을 다하고 적혈구 버스트 형성 단위 (BFU-ES)로 시작하는 역동적 인 과정을 포함한다. CFU-ES 후, 세포는 형태 학적으로 인식 할 수 있으며 일반적으로 터미널 적혈구 모세포라고합니다. 단말기 적혈구의 연구 과제 중 하나는 연대순 방식 유전자 기능을 해부 실험 방법의 부족이다. 이 프로토콜에서는 단말 적혈구 생성의 초기와 후기 단계에서 유전자의 기능을 결정하기 위해 고유의 전략을 설명한다. 이 시스템에서 마우스 태아 간 TER119 (성숙한 적혈구 세포 마커) 부정적인 적혈구 모세포는 정제하고 cDNA를하거나 관심있는 유전자의 작은 머리 핀의 자형의 RNA (shRNA를)의 외생 적 표현 형질 도입. 세포를이어서 cDNA를 외인성 또는 shRNA를 허용하면서 12 시간 동안 그들의 전구 스테이지를 유지하는 에리트로 포이 에틴 (EPO) 이외의 다른 매체를 포함하는 성장 인자에서 배양 하였다표현. 세포는 외인성 유전 물질이 이미 발현 동안 세포 분화 및 증식을 유도하기 위하여 12 시간 후에 EPO 배지로 변경 하였다. 이 프로토콜은 단말 적혈구 생성의 초기 단계에서 유전자의 기능 분석을 용이하게한다. 후기 단말 적혈구 생성을 연구하기 위해, 세포를 형질 도입 후 즉시 EPO 배지에서 배양 하였다. 형질의 유전 물질이 발현 될 때 이러한 방법으로, 세포는 이미 단말기 적혈구의 말기로 분화되었다. 우리는 터미널 적혈구의 다른 단계에서 세부적인 유전자 기능을 이해하는 데 도움이 될 것이다이 전략의 일반적인 응용 프로그램을 권장합니다.

그림 1은 실험적인 전략을 설명합니다. 프로토콜은 단말 적혈구 생성의 초기와 후기 단계에서 시그널링 분자의 기능을 대상으로 두 가지 독립적 인 상태로 구성된다. TER119 부정적인 태아 간 적혈구 모세포는 E13.5 마우스의 태아에서 정제 하였다. 태아 간 적혈구 세포 전과 정제 후의 유세포 분석은 정제 (도 2a 및도 2b) 효율적임을 보여 주었다. 적혈구 단말 (도 1의</st...

Watch this Scientific Journal Video about Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis at JoVE.com

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

우리는 터미널 적혈구의 초기와 후기 단계를 해부 체외 마우스 태아 간 erythroblast 문화 시스템을 제시한다. 이 시스템은 서로 다른 개발 단계에서 특정 유전자의 기능 분석을 용이하게한다.

마우스 태아 간 문화 시스템은 터미널 적혈구의 초기와 후기 단계에서 대상 유전자의 기능을 해부하기

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

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A Hierarchical Stem-Cell System: Blood Cell Formation

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14.9K 조회수.  Northwestern University.  우리는 터미널 적혈구의 초기와 후기 단계를 해부 체외 마우스 태아 간 erythroblast 문화 시스템을 제시한다. 이 시스템은 서로 다른 개발 단계에서 특정 유전자의 기능 분석을 용이하게한다. 

비디오: Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

세균성 접착 연구에 전단 응력을 소개합니다

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

줄기 세포 기반 유전자 치료 종양 모델로 BLT 인간화 마우스를 사용하여

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

인플루엔자 중합 효소의 유전자에 대한 구조 결정 PB2 소단위를위한 멀티 타겟 병렬 처리 방식

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

C 형 간염 바이러스의 복제를 분석하기위한 프로토콜

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

유전자 발현의 모든 단계에서 바이러스 성 기능을 정량화 우두 바이러스 기자

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

산모 - 태아 인터페이스에서 쥐의 조직에서 백혈구의 분리

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

인간의 태반과 탈락 오르간 문화는 산모 - 태아 인터페이스에서 감염을 연구하는

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

흐름 NK 세포의 기능을 모니터링 분석을 기반 세포 계측법

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

B 형 간염 바이러스 리포터 시스템 개발은 복제주기의 초기 단계를 모니터링하기

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

인플루엔자 바이러스 단일 클론 항 체를 중화의 탈출 변종의 생성

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...