나는 기술 지원을 위해 올가 드 수자 실바을 인정하고 싶습니다. 이 작품은 스위스 국립 과학 재단 (National Science Foundation) (SNSF) 부여 31003A_127029에 의해 지원되었다.

TransFLP 방법 (그림 1)은 세 가지 다른 방법으로 예시되어 있습니다 : I) V.의 독성의 determinants를 인코딩 두 개의 인접한 유전자의 삭제 cholerae (예를 들어, ΔctxAB), II) pathogenicity 섬 (예 : ΔVPI-1)의 제거 및 III) T7 RNA의 효소에 의존 발기인 시퀀스의 통합은 업스트림 유전자 수준의 관심, 이후 수의 각각의 변형을 배경으로 인공 표현 (예를 들어, T7-tfoX) (그림 2). 1. 키틴 플레이크의 작성 <ol><li> 표준 1.5 ML 플라스틱 관 키틴 조각의 무게 50-80 밀리그램. 이것은 대량으로 준비 할 수 있습니다. 키틴 조각은 시그마 (cat. 번호 C9213)에서 상업적으로 이용할 수 있습니다. </li><li> 관의 뚜껑이 열려 유지 그 조각을 압력솥. </li><li> 압력솥이 냉각 된 후 즉시 뚜껑을 닫습니다. </li><li> 스토어 autoclaved 키틴 조각상온에서. </li></ol> 2. 선택 사항 : V.의 인공 변환 Flp Recombinase 인코딩 플라스미드로 cholerae <ol><li> electrocompetent 준비 V. 표준 방법가 11를 사용하여 cholerae 세포. -80에서 관할 세포의 aliquots를 저장 ° C. </li><li> electrocompetent에 플라스미드 PBR-flp 5 일반 미니 준비 1-2 μl를 추가 V. cholerae 세포와는 electroporation 큐벳 (0.2 cm 간격 폭)에 혼합물을 전송합니다. </li><li> 1.6 KV에서 펄스을 적용합니다. </li><li> 0.9 ML SOC 매체를 추가 부드럽게 표준 14 ML 튜브에 세포를 전송할 수 있습니다. 30에서 2.5-3 시간에 대한 비 이동 품다 ° C. </li><li> 판 100 μl와 30 ° C 하룻밤 100 μg / ML 암피실린 및 배양을 포함하는 LB 플레이트에 300 μl. </li><li> 미래 TransFLP 실험을위한 글리세롤 주식 등의 단일 클론과 상점을 정화. </li></ol> 3. Oligonucleotide 설계 및 중합 효소 연쇄 반응 <ol><li> 최소 6 oligonucleotides는 관심의 DNA 영역 (들)뿐만 아니라 PCR에 의한 FRT-둘러싸인 항생제 카세트를 (그림 3) 증폭 할 필요가 있습니다. 또한 삽입 된 PCR 조각 이외의 oligonucleotides의 프라이밍 한 쌍을 포함하는 것이 좋습니다. 이 통합 및 FRT-둘러싸인 항생제 저항 카세트 (예 : 그림 3-5의 &#39;확인해 업&#39;과 &#39;해봐 다운&#39;프리 머으로 묘사)의 정확한 절단을 확인 할 수 있습니다. </li><li> oligonucleotides # 2, # 3, # 4, # 5주의를 디자인합니다. 그들은 충분히 템플릿 DNA에 (예를 들면 ~ 28 BP) 단련 할 수 있어야합니다. 템플릿 DNA는 프리 머에 대한 게놈 DNA (gDNA) 등 pROD17 12 PBR-FRT 관 - FRT (본 연구) 프리 머에 대한 # 3와 # 4 (등 # 2와 # 5 FRT - 칸 FRT - 포함 플라스미드 DNA입니다 그림 3A). </li><li> 수 # 5 프리 머에게 # 2 디자인을 각각 # 2 / # 3와 # 4 / # 5 사이의 5&#39;-끝에서베이스 페어링 다양한 (그림 3A </strong&gt; 삽입). PCR의 두 발은 다음과 같습니다. </li><li> PCR의 1 라운드 3 개 독립 PCR 반응을 수행합니다. 첫번째 oligonucleotides # 1과 # 2의 도움을 유전자 / DNA 지역 수준의 관심 상류 지역을 증폭됩니다. PCR은 세포 내에서 상동 재조합을 허용하는 길이 최소 500 BP의 조각 될 것입니다. 짧은 조각 (~ 250 BP)은 충분한 팔 수 있지만 권장되지 않습니다. </li><li> 병렬로 FRT-둘러싸인 항생제 카세트를 증폭 두 번째 PCR을 수행합니다. 예제는 여기에 제공을 위해 주로 aph (관 R)를 사용했습니다. 사용 oligonucleotides이 반응 (그림 3A)에 # 3와 # 4. </li><li> Concomitantly, PCR (3 샘플)로부터 하류 DNA 지역을 증폭. PCR 조각은 길이가 적어도 500 BP해야합니다. 템플릿 및 oligonucleotides # ​​5와 # 6 (그림 3A)과 같은 gDNA로 PCR을 수행합니다. </li><li> 세 PCR의 정화 후조각은 PCR의 2 차 라운드를 수행합니다. 템플릿으로 첫 라운드에서 얻은 세 조각 같은 혼합을 사​​용합니다. 증폭은 oligonucleotides # 1과 # 6 촉매 있습니다. 그 결과 PCR 조각 (그림 3B)는 자연 변화 실험 (그림 1)에서 DNA를 변형 역할을합니다. </li></ol> 4. 키틴 유발 자연 변화 <ol><li> 성장 V. 30에서 풍부한 미디어에서 aerobically cholerae 세포 ° C는 약 0.5의 600 nm의에서 광학 밀도에 도달 할 때까지. </li><li> 원심 분리하여 박테리아를 수확. DASW 2 권에있는 셀을 resuspending하기 전에 정의 인공 매체 (DASW 6)에 한 번 펠렛을 씻으십시오. 멸균 키틴 조각 (지점 1 아래에 설명)의 50-80 MG에 문화의 1 ML을 추가합니다. 문화 경우 세균 항구 플라스미드 PBR-flp (선택 사항 점 2), 추가 암피실린 (50 μg / ML). 운동없이 16-24 시간에 30 ° C에서 알을 품다. </li><li> g 추가 (&amp; A)전자, 2 차 라운드 PCR-파생 조각 (지점 3에서 설명) 200 것이다. 광범위하게 키틴 표면에서 박테리아를 분리하지 않고 조심스럽게 섞는다. 운동없이 24 시간을위한 30 ° C에서 알을 품다. </li><li> ≥ 30 초에 광범위하게 소용돌이 문화입니다. 선택 LB 매체 플레이트 (여기에 제공된 예제에 대한 예를 kanamycin 또는 gentamicin 함유 LB 플레이트)에 100-300 μl를 벌리고. 박테리아가 다른 항생제 concomitantly 암피실린을 추가하여 PBR-flp 플라스미드을 가지고 있고, 경우에 두 번 선택 접시를 사용합니다. 16-24 시간에 30 ° C에서 번호판을 길러 나 식민지가 표시 될 때까지. </li><li> 선택적 판에서 단일 transformants를 분리합니다. 이러한 transformants는 더블 크로스 오버 이벤트로 인해 PCR 조각하여 원본 염색체 궤적을 대체하고 있습니다. 항생제 카세트의 제거가 필요하지 않습니다 경우에 이러한 변종을 직접 추가 실험을 위해 사용될 수 있습니다. </li></ol> 5. Flp의 선택적 카세트의 제거 (들)- 재조합 <ol><li> 자연 변형 분석 해본하지 않을 경우 지점이 아래에 설명 된대로 인공 PBR-flp 플라스미드를 사용하여 transformants을 변환 할 수 있습니다. </li><li> 16-24 시간에 37 ° C에서 암피실린을 포함하는 LB 한천 플레이트에 박테리아를 성장. 옵션 : 40 2-3 시간에 대한 사이의 온도를 변경 ° C 플라스미드 PBR-flp에서 flp의 표현으로 높은 기온 5,10에 드 억압되어, 약 한 후 새로운 접시로 전송 세균. 부화 8 시간. </li><li> 항생제 함유하고 항생제 무료 한천 플레이트에 병렬로 클론을 restreaking하여, 항생제 - 감도 (그림 1 kanamycin 여기를 시연)을 테스트합니다. 하나의 중요한 식민지를 분리합니다. 당신이 더 다른 삭제 / TransFLP를 사용하여 삽입과 변형을 수정하고자하는 경우 글리세롤 주식 등의 암피실린 방지 복제를 고정. </li></ol> 6. 플라스미드 경화 <ol><li> 에어로빅 조건에서 하루 아침에 문화를 성장하고30의 ° C. 모든 항생제의 추가없이 다양한 매체를 사용하십시오. </li><li> 선택 사항 : 신선한 항생제없는 미디어의 밤 문화 1:100을 diluting하여 3-6 시간에 신선한 문화를 성장. </li><li> 판이나 연속으로 일반 LB 한천 플레이트 (들)에 문화의 희석 (들)과 30에서 배양 ° 8-16 시간에 대한 C (식민지을 볼 때까지). </li><li> 항생제 함유하고 항생제 무료 한천 플레이트 (그림 1)에 병렬로 클론을 restreaking하여 클론 암피실린 감도를 테스트합니다. 글리세롤 주식 등의 암피실린에 민감한 부담을 멈춰 봐. </li></ol>

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관심 없음 충돌이 선언 없습니다.

TransFLP 방법은 5 광범위하게 저희 연구실에서 사용 된 이상, 다른 곳에서 설명했다. 가능한 아르 유전자 조작의 종류는 다른 이들과 같습니다 : 하나의 유전자 및 유전자 클러스터의 삭제, 게놈 섬의 삭제 (예 : VPI-1) 관심 유전자 (예를 들어, 발기인 시퀀스)의 상류 시퀀스의 삽입과 삽입을 특정 유전자 (예를 들어, 인코딩 연관성 태그)의 끝 부분에 시퀀스. <p class="jove_...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> 키틴 조각 </td><td> 시그마 </td><td> C9213 </td><td> 필요 Autoclaving </td></tr><tr><td> 선택 사항 : Micropulser (경우에 항생제 카세트는 PBR-flp에 인코딩 Flp의 recombinase에 의해 excised 수 있습니다) </td><td> Biorad </td><td> 165-2100 </td><td> 또는 비교 electroporators </td></tr></tbody></table>

transflp &#x2014; a method to genetically modify vibrio cholerae based on natural transformation and flp-recombination

몇 가지 방법이 박테리아 염색체 1-3 조작 할 수 있습니다. 이러한 프로토콜의 대부분은 (예 : PIR에 따라 달라 나 온도에 민감한 replicons에게 1,2을 품고) 조건 replicative의 plasmids의 삽입에 의존하고 있습니다. 이러한 plasmids은 호몰 로지로 인한 재조합에 따라 세균​​ 염색체에 통합되어 있습니다. 이러한 insertional 돌연변이는 종종 직접 실험 설정에 사용됩니다. 또는 손실에 이어 플라스미드 절단에 대한 선택은 종종 sacB 유전자 넷으로 인코딩 된 카운터 선택 레반의 sucrase 효소에 의존 그람 음성 박테리아있는 수행 할 수 있습니다. 절단 중 하나 미리 삽입 유전자형 또는 염색체와 수정 된 유전자의 플라스미드 인코딩 복사본 사이의 교류의 결과를 복원 할 수 있습니다. 이 기법의 단점은 시간이 많이 걸리는 점입니다. 플라스미드는 먼저 복제 할 수 있습니다, 그것은 V.으로 수평 이동이 필요합니다 cholerae (대부분 N<html> otably E.와 결합하여 대장균의 기증 부담) 또는 후자의 인공 변환 및 플라스미드의 절단은 임의입니다하거나 초기 유전자형을 복원하거나 더 긍정적 인 선택이 가해되지 않을 경우 원하는 수정을 만들 수 있습니다. 여기, 우리는 V.의 빠른 조작하기위한 방법을 제시 cholerae의 염색체 (들) 5 (그림 1). 이 TransFLP 방법은 같은 V.으로 속 장염의 유기체 6 다른 대표의 자연 능력의 최근에 발견 된 키틴로 인한 유도를 기반으로 fischeri 7. 자연 능력은 PCR 생성 된 DNA 조각을 포함한 무료 DNA의 이해 할 수 있습니다. 일단 차지하고, DNA가 일치하는 지역 8 측면을 노릴의 250-500 BP의 최소의 존재 주어진 염색체와 recombines. 이러한 측면을 노릴 지역에서 중간 선택 마커를 포함하면 자주 발생 transformants을 쉽게 감지 할 수 있습니다. t는 &quot;&gt;이 방법은 V. cholerae의 다른 유전자 조작에 사용되며 잠재적으로 다른 자연적 능력이 박테리아 할 수 있습니다. 우리는 이전에 다음 ID로 출판 단일 유전자 삭제에 대한 연구와에 추가이 방법에 의해 수행 될 수있는 세 개 소설 예제를 제공합니다 연관성 태그 시퀀스 5 추가가. 키틴 유발 자연 변화 6의 초기 프로토콜에 관한 몇 가지 최적화 단계는이 TransFLP 프로토콜에 통합되어 있습니다.이 다른 사람들 사이에 상업적으로 이용 가능한 키틴 조각 8, PCR의 기부에 의한 게 껍질 조각의 교체를 포함 자료 9, FLP - 재결합 대상 사이트 (FRT) 5의 추가를 변형 등의 파생 DNA. FRT 사이트는 Flp recombinase (10)에 의해 중재 선택 마커의 사이트 감독 절단 할 수 있습니다.

세 가지 예 대표 결과는 6도 4에 표시됩니다. 첫 번째 접근 방식은 이웃 한 유전자에게 ctxA과 ctxB을 삭제하기위한. 이들은 V.의 주요 독성 계수를 인코딩 cholerae, 콜레라 독소. 위에서 설명한 3 그림에 따라 우리는 TransFLP 방법에 대한 oligonucleotides를 설계했습니다. 부모의 변형은 ctxAB의 원래 DNA의 궤적과 ctxAB에 대한 삭제 저항 카세트의 해제 ...

Watch this Scientific Journal Video about TransFLP â€” A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination at JoVE.com

TransFLP &amp;#x2014; A Method to Genetically Modify &lt;em&gt;Vibrio cholerae&lt;/em&gt; Based on Natural Transformation and FLP-recombination

의 게놈을 수정하는 빠른 방법<em&gt; V. cholerae</em&gt;이 설명되어 있습니다. 이 수정 한 유전자, 유전자 클러스터와 게놈 섬뿐만 아니라 짧은 시퀀스 (예 : 프로모터 요소 또는 선호도 태그 시퀀스)의 통합의 삭제가 포함되어 있습니다. 방법은 자연의 변화와 FLP-재조합에 기반을두고 있습니다.

TransFLP - 유 전적으로 수정하는 방법<em&gt; 장염 cholerae</em&gt; 자연 변화와 FLP-재조합에 근거

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

transflp-method-to-genetically-modify-vibrio-cholerae-based-on

Research

JoVE Journal

Immunology and Infection

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

14.5K 조회수.  Ecole Polytechnique Fédérale de Lausanne (EPFL). 의 게놈을 수정하는 빠른 방법 V. cholerae이 설명되어 있습니다. 이 수정 한 유전자, 유전자 클러스터와 게놈 섬뿐만 아니라 짧은 시퀀스 (예 : 프로모터 요소 또는 선호도 태그 시퀀스)의 통합의 삭제가 포함되어 있습니다. 방법은 자연의 변화와 FLP-재조합에 기반을두고 있습니다.

비디오: TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

의 식민지 Euprymna scolopes 오징어에 의한 장염 fischeri</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

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면역학 및 감염병학

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

세포에 붙어 Antileishmanial 검사에 대한 기생충 구조 및 변화 분석<em&gt; Leishmania donovani</emTHP1 인간 급성 백혈병 Monocytic 셀 라인에서&gt; Amastigotes

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

임상 신청<em&gt; 잠자는 숲속의 미녀</em유전자 주변과 배꼽 탯줄 혈액에서 T 세포를 수정하는 셀을 제시&gt;과 인공 항원

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

유리 - 지원 평면 지질 이중층에 자연 살해 세포 면역 시냅스의 슈퍼 해상도 이미징

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

투과성 막 삽입 기반 감염 시스템의 구현은 포유 동물 숙주 세포에서 분비 세균 독소의 효과를 연구하기

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

의 검출을위한 더 민감하고 특정 원체 한천 배지의 개발<em&gt; 장염 비브리오 균</em&gt; 및 기타<em&gt; 비브리오</em&gt; 종

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

천연 화합물 라이브러리의 탐험에 적합 안티 바이오 필름 분석 실험의 플랫폼

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

형광 기반의 뉴 라미니다 아제 억제 분석은 항 바이러스제의 뉴 라미니다 아제 억제제 클래스에 인플루엔자 바이러스의 감수성을 평가하기 위해

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

생물학적 유형을 유지하고 차별화하기 위해 사용되는 실험실 기술<em&gt; 콜레라 비브리오</em&gt; 임상 적 및 환경 적 분리 물

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

소설 급지대 무료 시스템 대량 생산을 위한 생체 외에서 Murine 자연 킬러 세포의

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...