Name: influenza virus propagation in embryonated chicken eggs
Uploaded: 2015-03-19T13:00:00.000+00:00
Duration: 6 min 56 s
Description: Watch this Scientific Journal Video about Influenza Virus Propagation in Embryonated Chicken Eggs at JoVE.com

The present study was supported by the NIH grants HL120947 (P.C.), HL103868 (P.C.), and the American Heart Association Grant-in-Aid (P.C.)

일반 발언 : 주 멸균 조건에서 계란의 조작과 관련된 모든 절차를 수행하고 멸균 기술 규격 제품을 사용한다. 70 % 에탄올로 모든 장비를 사용하기 전에 사전 깨끗한. 일반적으로, 인플루엔자 바이러스 접종 수확 BSL -2- 실험실에서 수행되어야한다. 그러나,이 프로토콜을 전파하는 데 사용되는 경우 훨씬 더 병원성 인플루엔자 바이러스 (예를 들어, 유행성 및 사전 유행성 균주, 가금류 및 가축, 고병원성 조류 인플루엔자 균주, 1918 년 H1N1 균주에 위험을 초래할 균주), 높은 바이오 안전성 수준 어떤 경우에는, 생물 안전주의 사항과 권한이 필요합니다. 지역 기관 바이오 안전성위원회는 인플루엔자 바이러스와 관련된 모든 연구를 승인해야합니다. 예방 접종에 대한 계란 1. 준비 <ol><li> 적합한 공급 업체에서 혈청 무균 (SPF) 갓 수정 된 조류의 알을 얻습니다. 일반적으로 닭고기 달걀 사용된다. 계란은 우리를해야한다바로 도착 에디션. </li><li> 자동 계란 터너와 가습 계란 인큐베이터에 도착 장소 계란은 계란을 정규적으로 회전시. 자동 계란 터너를 사용할 수없는 경우, 수동으로 적어도 2 ~ 3 회 하루 (장갑을 착용하고 가능한 한 무균 모든 항목을 유지하는 확인) 계란을 회전 할 수 있습니다. </li><li> 37 ° C와 10-11일에 대한 55~60% 습도에서 알을 품어. </li></ol> 2. 계란 캔들링 <ol><li> 배양 7, 8 일 후에 캔들링에 의해 불임 달걀을 확인하는 어두운 방에서 밝은 빛에 계란을 검사 할 수 달걀 캔 들러를 사용합니다. <ol><li> 70 % 에탄올로 계란 캔 들러를 닦습니다. 종종 장갑을 변경하고 병원체에 의한 오염의 위험을 최소화하기 위해 70 % 에탄올로 소독. </li><li> 인큐베이터에서 계란을 제거하고 빈 달걀 종이팩에 배치합니다. </li><li> 실내 조명을 끄고. </li><li> 캔 들러에 대해 한 번에 각 계란 하나의 큰 끝을 잡고. </li><li> determin하기 위해 달걀을 준수전자는 비옥 한 또는 불임 인 경우. 참고 : 콩 모양 배에 이르는 얇은 혈관이 명확하게 볼 수 있어야합니다. 미 수정 달걀은 볼 달걀 노른자와 작은 핏자국으로 표시됩니다. </li></ol></li><li> 미 수정있는 계란을 폐기하고, 인큐베이터 가능한 계란을 반환합니다. 30 분 동안 인큐베이터 밖에서 알을 두지 마십시오. 계란의 상태를 확인할 수없는 경우이를 표시하고 나중에 관찰. </li></ol> 바이러스 접종 3. 준비 <ol><li> 10 mM의 HEPES (PH 7.4)를 포함 멸균 PBS에서 104 PFU / ㎖ - 10 3 인플루엔자 바이러스 주식을 희석. 중요한 단계 : 즉시 사용하기 전에 바이러스 주식을 희석하고 얼음에 모든 시간을 희석 바이러스를 유지합니다. </li></ol> 뇨 경로를 통해 4. 인플루엔자 바이러스 예방 접종 <ol><li> 바이러스 접종 (10 일 또는 11)의 날에 다시 계란을 촛불과 죽은 배아를 제거 할 수 있습니다. 살아있는 배아를 구별계란 내부 이동을 찾고. </li><li> 인큐베이터에서 세 알을 제거하고 공기 주머니 위로 별도의 홀더에 계란을 마련. </li><li> 영구 마커와 계란의 공기 공간을 표시합니다. </li><li> 70 % 에탄올로 공기 공간 위에 달걀 껍질을 씻으십시오. </li><li> 계란 판지 및 바이오 안전성 후드 (BSL-2)에 공기 공간을 최대로 계란을 넣습니다. </li><li> 각 계란의 공기 주머니를 통해 껍질에 작은 구멍을 주먹으로 멸균 18G 바늘을 사용합니다. 배아 또는 노른자를 찔러 방지하기 위해 너무 깊이 바늘을 삽입하지 않도록주의하십시오. </li><li> 멸균 1 ML의 주사기에 희석 된 인플루엔자 바이러스를 그려 22G 바늘을 연결합니다. </li><li> 조심스럽게 뇨 공동으로 45 ° 각도로 바늘과 주사기를 진행합니다. </li><li> 희석 인플루엔자 바이러스 0.2ml를 주입한다. </li><li> 다른 두 계란 주사를 반복 한 다음 주사기와 바늘을 폐기​​합니다. </li><li> 글루건 (또는 녹은 파라핀 왁스)에서 접착제의 드롭 구멍을 밀봉합니다. B전자 접착제 계란 내부에 누출되지 않도록주의. </li><li> 공기 공간이 위쪽으로 지적와 계란 인큐베이터에 계란을 다시 배치합니다. </li><li> 반복 4.2 단계 - 4.12 모든 계란 접종 때까지. </li></ol> 5. 보육 시간 <ol><li> 37 ° C와 ~ 60 %의 습도에서 끄지 않고 감염된 계란을 품어. </li><li> 인플루엔자 바이러스 유형에 따라 최적의 배양 시간을 선택한다. 48 시간 동안 인플루엔자를 품어; 72 시간 동안 인플루엔자 B와 C를 품어. </li></ol> 6. 인플루엔자 바이러스 수확 <ol><li> 인큐베이션 기간 후, 배아를 죽이고 피 감염된 막액 오염의 위험을 최소화하기 위해 혈관을 수축 4 시간 (또는 O / N) 이상 동안 39 ° C에서 계란을 냉각. 수율을 감소 바이러스, 흡수로부터 적혈구를 방지하기 위해 이렇게. 또한, 냉각 계란 30 분 -20 ° C에서; 그러나 혈액이 오염의 위험을 증가시킨다. </li><li> 계란이 충분히 냉각 된 후에, 바이오 안전성 후드에 계란을 전송합니다. 공기 주머니가 위로 향하게 회사 홀더에 달걀을 놓습니다. 70 % 에탄올로 달걀 표면을 청소합니다. </li><li> 공기 주머니 위에 달걀 껍질을 열 멸균 가위를 사용합니다. 융모 막을 파괴하지 않도록주의하면서 공기 주머니 주위에 껍질을 제거합니다. </li><li> 멸균 무딘 집게로 융모 막을 엽니 다. 조심스럽게 배아와 작은 주걱이나 숟가락으로 난황을 옆으로 이동합니다. 노른자를 파열되지 않도록주의하십시오. </li><li> 1 에펜 도르프 피펫을 사용하여 조심스럽게 막액를 수집합니다. 필요한 경우, 옆으로 약간 많은 유체 가능한 수집 계란을 기울입니다. </li><li> 50 ㎖ 플라스틱 원뿔 관에 모든 계란에서 막액을 결합합니다. 바이러스 수확하는 동안 항상 얼음에 튜브를 유지합니다. 하나의 계란 생산량 5 - 약간 노란 유체 10 ㎖. </li><li> 4에서 10 분 동안 1,000 XG에서 바이러스 - 함유 막액 스핀6; C 파편 펠렛합니다. 새로운 50 ㎖ 튜브에 맑은 액체를 이동시켜 얼음에 보관. BSL-2 폐기물 용기에 달걀을 폐기하십시오. </li></ol> 7. 저장 <ol><li> 즉시 뇨 명확히 유체 (예를 들어, 50, 100 또는 500 UL 분량 씩) 나누어지는 스냅 동결을 액체 질소에. 냉동 바이러스 주식 장기 저장을 위해 -80 ° C 냉장고에 전송합니다. </li></ol>

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The authors have nothing to disclose.

인플루엔자는 질병의 큰 부담을 글로벌 초래하고 작업 폐 손상 (7)의 병인을 이해로 계속된다. 이 치명적인 질병의 촉진 연구, 다양한 방법 인플루엔자 바이러스 (6)을 전파하기 위해 개발되었습니다. 여기서는 닭고기 계란 인플루엔자 바이러스를 생산하는 방법을 설명한다. 이 방법의 장점은 높은 재현성 있다는 종종 시험 관내 연구에 필요한 높은 역가 인플루엔자 바이러?...

인플루엔자는 인간의 건강에 큰 위협이되고 있습니다. 그것은 큰 글로벌 부담이 매년 전세계에서 1 500000 사망까지 일으키는 잠재적으로 치명적인 호흡기 질환이다. 인플루엔자 바이러스는 가족 오르 쏘 믹소 바이러스과 (orthomixoviridae)에있는 자신의 게놈 1,2 8 음의 감각 단일 가닥 RNA를 수행한다. 바이러스 게놈의 높은 가변성 (즉, 항원 &quot;드리프트&quot;)는 장기 면역을 방지 할 수 있습니다. 또한, 인플루엔자 항 바이러스제 3 더욱 강하다. 2009 H1N1 인플루엔자 대유행 인플루엔자 질병 (유행성 변형, 항 바이러스 성, 지연 백신 생산)과 관련된 모든 어려운 문제를 강조했다. 백신 제제는 대부분 병원성 인플루엔자 균주 4 (H1N1, H3N2 인플루엔자 B 각각 하나씩)에 대한 세계 보건기구 (WHO)에 의해 매년 결정된다. 이 방법은 예측 인플루엔자 균주를 기반으로하기 때문에들, 병원성 균주는 때때로 잘못 파악하고 백신 유효성이 크게 저하된다. 더욱이, 신규 한 인플루엔자 균주의 출현은이 전염병을 일으키는 이러한 예방 프로그램을 회피 할 수있다. 범용 인플루엔자 백신의 생성은 5 애매하고있다. 따라서 연구는 폐 손상의 기전을 이해하기에 계속해야합니다. 시설 실험실 연구에, 여러 가지 방법이 바이러스 분리 및 전파 (6)을 위해 개발되었다. 인간 인플루엔자 바이러스는 포유 동물 세포의 다양한 기판들에서 증폭 될 수있다. 그러나, 대량으로 생성하는 높은 역가의 바이러스는 가장 유정란에 달성된다. 다음 프로토콜은 인플루엔자에 대한 기존의 바이러스 주식에서 바이러스 전파 및 저장 기술에 대해 설명합니다.

<table><tbody><tr><td>Phosphate buffered saline (PBS)</td><td>Cellgro&amp;nbsp;</td><td>21-040-CV</td><td></td></tr><tr><td>serum pathogen-free (SPF) fertilized chicken eggs&amp;nbsp;</td><td>VALO BioMedia</td><td></td><td></td></tr><tr><td>humidified egg incubator&amp;nbsp;</td><td>FARM iNNOVATORS</td><td>Model 2100</td><td></td></tr><tr><td>automatic egg turner</td><td>FARM iNNOVATORS</td><td>Model 3200</td><td></td></tr><tr><td>egg candler</td><td>FARM iNNOVATORS</td><td>Model 3300&amp;nbsp;</td><td></td></tr><tr><td>1 ml syringe</td><td>BD Bioscience</td><td>309659</td><td></td></tr><tr><td>18G needle</td><td>BD Bioscience</td><td>305196</td><td></td></tr><tr><td>20G / 22G needle</td><td>BD Bioscience</td><td>305176 / 305156</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Ethanol&gt;99.5%</td><td>Sigma-Aldrich</td><td>459844</td><td>diluted to 70% using water</td></tr><tr><td>glue gun &quot;Ad tech Hi Temp Project Pro&quot;</td><td>Ad tech, Adhesive Technologies, Inc.</td><td>Model 1105</td><td></td></tr><tr><td>glue &quot;Ad tech MINI SIZE, Multi Temp&quot;</td><td>Ad tech, Adhesive Technologies, Inc.</td><td>220-34ZIP30</td><td></td></tr><tr><td>MDCK cells</td><td>ATCC</td><td>CRL-2935</td><td></td></tr><tr><td>Washed Pooled Turkey Red Blood Cells, 10%</td><td>Lampire Biological Laboratories</td><td>724908</td><td></td></tr></tbody></table>

influenza virus propagation in embryonated chicken eggs

인플루엔자 감염은 약 36,000 명이 사망하고 미국에서 20 만 명 이상의 입원 매년와 연결되어 있습니다. 돌연변이 다시 구색 인한 새로운 인플루엔자 바이러스 균주의 연속 출현 바이러스의 제어를 복잡하게하고 신규 약물과 백신의 개발을 필요로 영구. 인플루엔자의 실험실 기반의 연구는 바이러스의 전파에 대한 안정적이고 비용 효율적인 방법이 필요합니다. 여기에, 포괄적 인 프로토콜은 지속적으로 높은 역가의 바이러스 주식을 산출 비옥 한 닭 계란에 인플루엔자 바이러스 전파, 제공됩니다. 11 일 - 간단히 말해, 혈청 무균 (SPF) 수정 된 닭고기 달걀은 37 ° C와 10 55~60% 습도에서 배양된다. 이 기간 동안, 배아 발달 쉽게 알 캔 들러를 사용하여 모니터 할 수있다. 바이러스 접종은 바늘을 사용하여 공동 내로 뇨 바이러스 스톡의 주사에 의해 수행된다. 37 ° C에서 배양 2 일 후, 계란은4 ° C에서 적어도 4 시간 동안 냉장. 공기 주머니와 상기 멤브레인 융모 껍질을주의 깊게 개방하고, 바이러스를 함유 막액 수확된다. 유체를 원심 분리에 의해 먼지로부터 클리어 분주 장기 저장에 대해 -80 ° C에 전달된다. 다량 보통이 프로토콜에 의해 달성되고 높은 바이러스 역가 (바이러스 - 함유 달걀 당 유체를 5-10 ml)을 대량으로 요구하는 시험 관내 연구를 위해, 특히 바이러스 제제 우리 유리한 방법 계란의 사용량을 만들었다 바이러스의 것과 동일한 바이러스 주식의 높은 복용량이 필요하다.

여러 방법은 인플루엔자 바이러스 역가를 위해 개발되었다. 적절한 방법을 선택하는 하나의 고려 사항은 일부 가능한 비리을 평가하는 관계없이 다른 사람이 감염을 기반으로하는 반면 생존 능력 (예를 들어, 헤 마글 루티 닌 분석)의 총 입자를 결정하는 것입니다 (예를 들면, 상패 분석) 6. 여기서, 마우스 구성된 인플루엔자 바이러스 (A는 / PR / 34분의 8) 접종 닭고기 계란에?...

Watch this Scientific Journal Video about Influenza Virus Propagation in Embryonated Chicken Eggs at JoVE.com

Influenza Virus Propagation in Embryonated Chicken Eggs

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

인간 백신 닭 계란에 인플루엔자 바이러스 전파

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

influenza-virus-propagation-in-embryonated-chicken-eggs

Research

JoVE Journal

Immunology and Infection

43.0K 조회수.  Cedars-Sinai Medical Center. Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

비디오: Influenza Virus Propagation in Embryonated Chicken Eggs

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

플라스미드 DNA의 재조합 독감 바이러스의 생성

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

연구

면역학 및 감염병학

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

사이 Synergism을 맡은 이후로 발광 생물 이미징을 사용하여 연쇄상 구균 pneumoniae</em유아 마우스에서>와 인플루엔자 바이러스

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

의 cDNA에서 재조합 뉴캐슬 질병 바이러스의 구조

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both as an oncolytic virus and a viral-vectored vaccine. NDV is an attractive therapeutic and prophylactic agent due to its well-established reverse genetics system, potent immunostimulatory properties, and excellent safety profile. When administered as an oncolytic virus or a viral-vectored vaccine, NDV elicits a robust antitumor or antigen-specific immune response, activating both the innate and adaptive arms of the immune system. 
Given these desirable characteristics, NDV has been evaluated in numerous clinical trials and is one of the most well-studied oncolytic viruses. Currently, there are two registered clinical trials involving NDV: one evaluating a recombinant NDV-vectored vaccine for SARS-CoV-2 (NCT04871737), and a second evaluating a recombinant NDV encoding Interleukin-12 in combination with Durvalumab, an antiPD-L1 antibody (NCT04613492). To facilitate further advancement of this highly promising viral vector, simplified methods for generating high-titer, in vivo-grade, recombinant NDV (rNDV) are needed. 
This paper describes a detailed procedure for amplifying rNDV in specified pathogen-free (SPF) embryonated chicken eggs and purifying rNDV from allantoic fluid, with improvements to reduce loss during purification. Also included are descriptions of the recommended quality control assays, which should be performed to confirm lack of contaminants and virus integrity. Overall, this detailed procedure enables the synthesis, purification, and storage of high-titer, in vivo-grade, recombinant, lentogenic, and mesogenic NDV for use in preclinical studies.

Here we provide a detailed procedure for production, purification, and quantification of high-titer recombinant Newcastle disease virus. This protocol consistently yields &#62; 6 &#215; 109 plaque-forming units/mL, providing virus quantities appropriate for in vivo animal studies. Additional quality control assays to ensure safety in vivo are described.

Allantoic Fluid로부터의 고역가 재조합 뉴캐슬병 바이러스의 생산

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both ...

암 연구학

Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is well-defined, and it is relatively easy to achieve precise and timely exposure via multiple methods. Moreover, the process of heart development in chicken embryos is similar to mammals, also resulting in a four-chambered heart, making it a valuable alternative model in the assessment of developmental cardiotoxicities. In our lab, the chicken embryo model is routinely used in the assessment of developmental cardiotoxicities following exposure to various environmental pollutants, including per- and polyfluoroalkyl substances (PFAS), particulate matter (PMs), diesel exhaust (DE) and nano materials. The exposure time can be freely selected based on the need, from the beginning of development (embryonic day 0, ED0) all the way to the day prior to hatch. The major exposure methods include air-cell injection, direct microinjection, and air-cell inhalation (originally developed in our lab), and the currently available endpoints include cardiac function (electrocardiography), morphology (histological assessments) and molecular biological assessments (immunohistochemistry, qRT-PCR, western blotting, etc.). Of course, the chicken embryo model has its own limitations, such as limited availability of antibodies. Nevertheless, with more laboratories starting to utilize this model, it can be used to make significant contributions to the study of developmental cardiotoxicities.

Chicken embryos, as a classical developmental model, are used in our lab to assess developmental cardiotoxicities following exposure to various environmental contaminants. Exposure methods and morphological/functional assessment methods established are described in this manuscript.

발달 심장 독성 평가에 강력한 도구로 닭 배아를 사용

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is ...

발생학

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

칙 태아의 단일 세포 Transfection

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

생물학

In Ovo Intravascular Injection in Chicken Embryos

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering exogenous materials into chicken embryos has a great advantage for studying gene function, transgenic breeding, and chimera preparation during embryonic development. Here we show the method of in ovo intravascular injection whereby exogenous materials such as plasmid vectors or modified primordial germ cells (PGCs) can be transferred into donor chicken embryos at early developmental stages. The results show that the intravascular injection through the dorsal aorta and head allows injected materials to diffuse into the whole embryo through the blood circulatory system. In the presented protocol, the efficacy of exogenous plasmid and lentiviral vector introduction, and the colonization of injected exogenous PGCs in the recipient gonad, were determined by observing fluorescence in the embryos. This article describes detailed procedures of this method, thereby providing an excellent approach to studying gene function, embryo and developmental biology, and gonad-chimeric chicken production. In conclusion, this article will allow researchers to perform in ovo intravascular injection of exogenous materials into chicken embryos with great success and reproducibility.

In Ovo Intravascular Injection in Chicken Embryos

The overall goal of this paper is to describe how to perform in ovo intracellular injection of exogenous materials into chicken embryos. This approach is very useful to study the developmental biology of chicken embryos.

오보에서 닭 배아에 혈관 내 주사

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

인간 인플루엔자 바이러스 감염의 Zebrafish의 모델을 사용하여 항 바이러스 약품을 화면 및 호스트 면역 세포 반응의 특성을

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

고병원성 H5N1 및 조류 H7N9 바이러스의 엔벨로프 당단백질을 가진 고적퇴력 감염성 인플루엔자 슈도타입 입자 의 생산

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of interest. Currently, transgenic technology is becoming an indispensable tool for the study of Eimeria, the causative agents of coccidiosis in poultry and livestock. This protocol provides a detailed description of stable transfection in eimerian parasites: purification and nucleofection of sporozoites or second-generation merozoites, and in vivo propagation of transfected parasites. Using this protocol, we achieved transfection in several species of Eimeria. Taken together, nucleofection is a useful tool to facilitate genetic manipulation in eimerian parasites.

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Here, we provided a method to achieve stable transfection of chicken Eimeria parasites by nucleofecting sporozoites or second-generation merozoites. Genetically modified eimerian parasites expressing heterologous antigenic genes could be used as vaccine delivery vehicles.

닭 에이메리아 기생충의 핵 과 생체 내 전파

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of ...