우리는 DRS에게 감사하고 있습니다. SP Dinesh - 쿠마과 율 리우 TRV - VIGS 벡터, 그리고 DRS. 척 Kenerley, 테리 휠러, 짐 스타와 무명의 씨앗을 제공 바이엘 CropScience. 이 작품은 PH에 LS와 NIH에 NSF에 의해 투자되었다

1. 목화 모종의 성장 <ol><li> 업랜드 커튼 (고십피움 히르수툼) 다양한 Fibermax 832, Phytogen 425RF, Phytogen 480WR 및 지하철 믹스 700 (SunGR, Beavile, WA)이있는 냄비 (지름 7cm)에 Deltapine 90 씨앗을 뿌리다. </li><li> 23 플라스틱 돔 덮여 트레이에서 냄비를 유지 성장 방에 12시간 light/12 시간 어두운 photoperiod 함께 ° C, 120 μE m -2 S -1 빛. </li><li> 이 cotyledons가 등장 돔을 제거합니다. </li><li> 약 2 주후에 VIGS 분석의 두 가지 완전히 확장된 cotyledons과 식물을 사용합니다. 이 단계에서, 진정한 단풍은 아직 (그림 1) 등장하지 않았습니다. </li></ol> 2. GrCLA1​​ 유전자를 운반 건설 VIGS 벡터 <ol><li> G.의 cDNA 라이브러리에서 primers GrCLA1 ​​- F와 PCR, 5&#39; - GCCCTTTGTGCATCTTC - 3 &#39;와 GrCLA1 ​​- R, 5&#39; - CTCTAGGGGCATTGAAG - 3&#39;로 무명의 Cloroplastos alterados 한 유전자 (GrCLA1)를 증폭 raimondii 잎 조직. </li><li> EcoRI과 KpnI과 GrCLA1의 PCR 제품을 다이제스트와 결합하여 pYL156 (pTRV - RNA2) 벡터에 삽입합니다. </li><li> 50 μg / Kanamycin의 ML이 포함된 LB 한천 플레이트에 클론을 화면. 제한 효소 분해 및 시퀀싱하여 구조를 확인합니다. </li><li> electroporation에 의해 Agrobacterium tumefaciens GV3101으로 플라스미드를 변환 28에서 LB 액체 배지에 복구 ° C. LB와 함께 접시에 transformants을 선택 + Kanamycin (50 μg / ML) + Gentamycin (25 μg / ML). 박테리아는 -80 ° C에서 장기 사용을 위해 25 % 글리세롤에 저장할 수 있습니다. </li></ol> 3. VIGS 접종을 수행 <ol><li> pYL192 (TRV)를 들고 Agrobacterium의 tumefaciens의 접종, 탄력 냉동 글리세롤 주식 사흘 전에 : RNA1, pYL156 (TRV) : RNA2 (벡터 혼자)과 pYL156 (TRV) : 50 μg / Kanamycin의 ML이 포함된 LB 한천 플레이트에 RNA2 - GrCLA1 25 μg / Gentamycin의 ML. 24 시간 동안 28 ° C에서 접시를 품어. </li><li> VIGS 침투하기 전에 이틀, 위의 접시에서 각 구조에 대한 하나의 식민지를 선택 50 μg / Kanamycin의 ML 25 μg / Gentamycin의 ML과 보충 LB 매체의 5 ML에 그것을 예방, 28에서 세균성 문화를 성장 ° 50 RPM의 속도로 롤러 드럼에서 하룻밤을위한 C. </li><li> (- (4 morpholino) 에탄 설폰산 2) 및 20 μm의 제품 acetosyringone 50 μg / Kanamycin의 ML 25 Gentamycin의 μg / ML, 추가로 10 MM MES와 보충 LB 매체 50 ML있는 플라스크에 위의 문화를 전송합니다. ° C 50 RPM의 속도로 통에 하루를위한 28의 문화를 성장. </li><li> 다음날, 5 분 4,000 RPM에서 농업 박테리아 세포를 스핀 다운, 10 MM MgCl 2, 10 MM MES와 200 μm의의 acetosyringone를 포함하는 침투 버퍼의 문화를 다시는 - 일시 중지합니다. 1.5 문화의 OD 600 조정합니다. </li><li> 3 시간 동안 실온에서 벤치에 문화를 둡니다. </li><li> Agrobacterial 침투하기 전에, 펀치 cotyledons 통해 피어싱없이 25 G 바늘로 목화 식물의 cotyledons의 아래쪽. 하나 또는 두 개의 구멍 (조직의 부드러움에 따라 다름) 떡잎의 각 섹션 (그림 2)에 펀치했다. </li><li> 1:1 비율 RNA2 - GrCLA1, 손 1을 사용하여 입힐 수있는 총알 사이트를 통해 cotyledons의 아래쪽에서 혼합물에 침투해 : RNA1 및 pYL156 (TRV) : RNA2 또는 pYL156 (TRV) pYL192의 Agrobacterial 문화 서스펜션 (TRV)를 혼합 ML needleless 주사기 (그림 3). </li><li> 플라스틱 돔과 함께 식물을 커버하고 야간에 대한 희미한 빛 조건하에 상온에서 침입 식물을 두십시오. </li><li> 23 온도와 성장 방에 식물을 전송 ° C, 120 μE m 12 시간 조명 / 12시간 어두운주기 -2 S -1 빛. </li></ol> 참고 : 식물 23 ° C 또는 글래스 하우스 (25-28 ° C) 중 아래의 경우 VIGS가 작동합니다. 온도 (23-25​​ ° C) 상대적으로 낮은 경우, 그것은 접종을 위해 쉽게하고 표현형 높은 (28-30 ° C) 또는 오르 내렸는지를 보여준다 온도에 비해 더 균일합니다. 돔과 식물을 포함하면 VIGS가 더 효율적입니다. <ol start="10"><li> 7 ~ 8 일 게시 침투에 입을 표현형을 검사합니다. pYL156 (TRV)에 의해 입막음을 식물의 진정한 잎 : RNA2 - GrCLA1는 흰둥이 표현형를 (그림 4)을 표시하기 시작했습니다. Agrobacteria 운반 pYL156 (TRV)와 침투 식물 : RNA2는 컨트롤 역할을합니다. </li><li> 제어 및 침묵 면화 공장으로부터 격리 RNA와 리버스 전사 중합 효소의 연쇄 반응 (RT - PCR)을 사용하여 내생 유전자의 표현 수준을 검사하여 유전자 입을 효율성을 확인합니다. </li></ol> 참고 : TRV는 광범위한 호스트 범위를 가지고 있으며 신고 대상 병원체이므로 VIGS - ed를 식물이 포함된 검역 조건과 식물에 따라 보관해야 제대로 autoclaved 및 assays 후 처리하여야한다. 4.대표 결과 : 약 이주 Agrobacterial 혼합 손으로 침투 후 GrCLA1 ​​입을 의한 흰둥이 표현형 분명 진정한 나뭇잎에서 관찰되었다. 입을 효율 50 개 이상의 식물을 여러 실험을 거의 100 %에 도달합니다. 입을 식물 새롭게 엘더 단풍 (그림 4)에 모자이크와 나뭇잎을 개발에 균일하게 배포하고 강한 흰둥이 표현형를 표시합니다. <img src="/files/ftp_upload/2938/2938fig1.jpg" alt="그림 1" /> 그림 1. Agrobacterial 침투에 사용이 완전히 확장 cotyledons 두 주간 올드 스테이지에 대해에서 무명 세들링. <img src="/files/ftp_upload/2938/2938fig2.jpg" alt="그림 2" /> 그림 2. Agrobacterial 침투를 용이하게하기 위해 25 G 바늘을 사용하여 목화 식물의 cotyledons의 아래쪽에 작은 구멍을 펀칭. <img src="/files/ftp_upload/2938/2938fig3.jpg" alt="그림 3" /> 그림 3. 입힐 수있는 총알 사이트를 통해 무명의 cotyledons에 Agrobacterial 혼합의 핸드 침투는 needleless 주사기를 사용하여 <img src="/files/ftp_upload/2938/2938fig4.jpg" alt="그림 4" /> 그림 4. 알비노 표현형은 면화 공장을 침묵 VIGS에 나타났습니다. 네 cultivars가 표시됩니다. A) Fibermax 832, B) Phytogen 480WR, C) Phytogen 425RF, D) Deltapine 90.

<ol>
	<li>Chen, Z. J., Scheffler, B. E., Dennis, E., Triplett, B. A., Zhang, T., Guo, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+sequencing+cotton+(Gossypium)+genomes.">Toward sequencing cotton (Gossypium) genomes.</a> Plant Physiol. 145, 1303-1310 (2007).</li><li>Dinesh-Kumar, S. P., Anandalakshmi, R., Marathe, R., Schiff, M., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-induced+gene+silencing.">Virus-induced gene silencing.</a> Methods Mol Biol. 236, 287-294 (2003).</li><li>Hamilton, A. J., Baulcombe, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+species+of+small+antisense+RNA+in+posttranscriptional+gene+silencing+in+plants.">A species of small antisense RNA in posttranscriptional gene silencing in plants.</a> Science. 286, 950-952 (1999).</li><li>Burch-Smith, T. M., Anderson, J. C., Martin, G. B., Dinesh-Kumar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+and+advantages+of+virus-induced+gene+silencing+for+gene+function+studies+in+plants.">Applications and advantages of virus-induced gene silencing for gene function studies in plants.</a> Plant J. 39, 734-746 (2004).</li><li>Liu, Y., Schiff, M., Dinesh-Kumar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-induced+gene+silencing+in+tomato.">Virus-induced gene silencing in tomato.</a> Plant J. 31, 777-786 (2002).</li><li>Estevez, J. M., Cantero, A., Romero, C., Kawaide, H., Jimenez, L. F., Kuzuyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+expression+of+CLA1,+a+gene+that+encodes+the+1-deoxyxylulose+5-phosphate+synthase+of+the+2-C-methyl-D-erythritol-4-phosphate+pathway+in+Arabidopsis.">Analysis of the expression of CLA1, a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis.</a> Plant Physiol. 124, 95-104 (2000).</li><li>Mandel, M. A., Feldmann, K. A., Herrera-Estrella, L., Rocha-Sosa, M., Leon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLA1,+a+novel+gene+required+for+chloroplast+development,+is+highly+conserved+in+evolution.">CLA1, a novel gene required for chloroplast development, is highly conserved in evolution.</a> Plant J. 9, 649-658 (1996).</li><li>Gao, X., Wheeler, T., Li, Z., Kenerley, C., He, P., Shan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+GhNDR1+and+GhMKK2+compromised+cotton+resistance+to+Verticillium+wilt.">Silencing GhNDR1 and GhMKK2 compromised cotton resistance to Verticillium wilt.</a> Plant J. 66, 293-305 (2011).</li></ol>

관심 없음 충돌 선언하지 않습니다.

VIGS은 transiently 내생 유전자의 표현을 쓰러뜨린하여 기능적 게놈 분석에 강력한 도구로 입증되었습니다. 본 연구에서는, 우리는 TRV 기반 이진 벡터를 이용하여 Agrobacterium - 중재 VIGS을 개발했습니다. 커튼 CLA1 (GrCLA1) 유전자는 입을 효율성을 모니터링하는 시각 마커로 개발되었습니다. 우리는 지속적으로 모든 종류의 진정한 나뭇잎에 나타나는 흰둥이 표현형은 약 2 주 게시물 침투로부터 시...

<table border="1"><tbody><tr><th> 시약 및 장비의 이름 </th><th> 회사 </th><th> 카탈로그 / 일련 번호 </th><th> 댓글 </th></tr><tr><td> 롤러 드럼 </td><td> 글라스 - 콜, LLC. </td><td> 099A TC108 </td><td> 농업 세균 문화 </td></tr><tr><td> 보육기 </td><td> 셸던 제조 주식 회사 </td><td> 01046209 </td><td> 농업 세균 문화 </td></tr><tr><td> UV / 비스 분광 광도계 </td><td> 베크맨 쿨터 </td><td> 모델 : DU530 </td><td> 측정 OD </td></tr><tr><td> 진 Pulser </td><td> BioRad </td><td> 모델 : 1652076; 직렬 : 154BR3880 </td><td> Electroporation </td></tr><tr><td> Pulser 컨트롤러 </td><td> BioRad </td><td> 모델 : 1652098; 직렬 : 232BR4833 </td><td> Electroporation </td></tr><tr><td> Micropulser Electroporation 쿠베​​트 </td><td> BioRad </td><td> 165-2081 </td><td> Electroporation </td></tr><tr><td> 1 ML 주사기 </td><td> BD Biosciences </td><td> 30,962 </td><td> 농업 세균의 접종 </td></tr><tr><td> 메트로 믹스 700 </td><td> SUNGR </td><td> SKU # 553001 </td><td> 성장 세들링 </td></tr><tr><td> 테라 코타 냄비 </td><td> TOPlastics </td><td> GPS 3001B2 </td><td> 성장 세들링 </td></tr><tr><td> MES 일수화물 </td><td> USB </td><td> 18,886 </td><td> 침투 버퍼 </td></tr><tr><td> Acetosyringone </td><td> 시그마 </td><td> D134406 </td><td> 침투 버퍼 </td></tr></tbody></table>

agrobacterium-mediated virus-induced gene silencing assay in cotton

커튼 (고십피움 히르수툼)는 세계적으로 가장 중요한 작물 중 하나입니다. 상당한 노력은 새로운 종류의 분자 육종에되었습니다. 면화의 대규모 유전자 기능 분석은 게놈, 유전자 복제 및 polyploidy, 유전자 변형 1 길이 성장주기와 말을 안들음 자사의 대형에 의한 가능성이 현대 식물 종, 대부분의 뒤에서 느껴지되었습니다. 커튼 높은 처리량 기능 유전자 / 게놈 연구를 촉진하기 위해, 우리는 면화 유전자 기능을 평가하기 위해 신속하고 효율적인 과도 assays을 개발하려고합니다. 바이러스 유도된 유전자 입을 (VIGS)은 바이러스 확산 2,3을 주체할 수있는 호스트 포스트 Transcriptional 진 입을 (PTGS)을 기반으로 개발된 강력한 기술입니다. Agrobacterium - 중재 VIGS가 성공적으로 같은 Solanaceae, Arabidopsis과 콩과의 식물 수종으로 dicots 종의 다양한 적용 및 monocots 수종 다양한 기능적 게놈 연구 3,4을 위해 보리, 밀 및 옥수수를 포함하고 있습니다. 이 신속하고 효율적인 접근법 식물 변환을 피할 수와 기능 중복을 극복로서, 그것은 특히 매력적이고 변화에 대한 의무가없는 목화 같은 작물 종의 기능 게놈 연구에 적합합니다. 본 연구에서는, 우리는 목화의 Agrobacterium - 중재 VIGS 시스템의 상세한 프로토콜을보고합니다. 여러 바이러스 VIGS 벡터 중에서 담배 래틀 바이러스 (TRV)는 호스트의 다양한 침공하고 아직 hosts5에 가벼운 증상을 생산하여 전체 공장을 통해 적극적으로 전파 수 있습니다. 입을 효율, GrCLA1, alterados 1 면화의 유전자 (AtCLA1). CLA1 유전자가 엽록체 개발 6 관여 복제 및 VIGS 이진 벡터 pYL156에 삽입되었으며, 이전의 연구 보여주 Arabidopsis Cloroplastos의 상동체 유전자를 모니터링하는 것을 AtCLA1의 손실 기능은 효율을 입을 수있는 훌륭한 시각적인 마커를 제공하고, 진정한 나뭇잎 7 흰둥이 표현형 결과. 약 이주에 게시 Agrobacterium의 침투, 흰둥이 표현형은 모든 복제 실험에서 100 % 입을 효율, 진정한 나뭇잎에 표시하기 시작했습니다. 내생 유전자 표현의 입을는 RT - PCR 분석에 의해 확인되었다. 크게, 입을는 potently 텍사스 다양한 상업적으로 재배 품종을 포함하여 우리가 테스트한 모든 cultivars에 발생할 수 있습니다. 이것은 신속하고 효율적인 Agrobacterium - 중재 VIGS 분석은 목화의 게놈 - 넓은 수준에서 유전자 기능을 신속하게 대규모 분석을위한 매우 강력한 도구를 제공합니다.

Watch this Scientific Journal Video about Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton at JoVE.com

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

우리는 목화의 Agrobacterium - 중재 바이러스 유발 유전자 입을 (VIGS) 분석에 대한 상세한 프로토콜을 제시한다. 담배 래틀 바이러스 (TRV) - 파생 VIGS 벡터는 면화 GrCLA1​​, Cloroplastos alterados 1 유전자의 입을 RNA를 유도하기 위해 배치되었다. 입을 GrCLA1​​로 인한 흰둥이 표현형은 접종 후 2 주 이내에 세들링 단계에서 관찰되었다.

커튼에서 Agrobacterium - 중재 바이러스 유발 유전자 입을 분석

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

agrobacterium-mediated-virus-induced-gene-silencing-assay-in-cotton

Research

JoVE Journal

Biology

24.2K 조회수.  Texas A&M University. 우리는 목화의 Agrobacterium - 중재 바이러스 유발 유전자 입을 (VIGS) 분석에 대한 상세한 프로토콜을 제시한다. 담배 래틀 바이러스 (TRV) - 파생 VIGS 벡터는 면화 GrCLA1​​, Cloroplastos alterados 1 유전자의 입을 RNA를 유도하기 위해 배치되었다. 입을 GrCLA1​​로 인한 흰둥이 표현형은 접종 후 2 주 이내에 세들링 단계에서 관찰되었다.

비디오: Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

바이러스 유도 진 입을 (VIGS)에 Nicotiana benthamiana 토마토

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

연구

생물학

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

siRNA의 중재 진 입을와 결합 라이브 세포 칼슘 영상은 CA를 나타냅니다 2 + ER 막의 누출 채널과 규제 메커니즘

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

세포 배양의 유도와 Hypoxia의 시험

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Ribosome 바운드 초기 Polypeptides의 분리<em&gt; 체외에서</em&gt; mRNA 함께 Translational 일시 중지 사이트를 식별하려면

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Ribosome 바운드 Polypeptides을 파악하는 도구로 SecM 체포 시퀀스를 사용하여

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

최적화 및 활용<em&gt; 아그로 박테 리움</em에서&gt; 매개 과도 단백질 생산<em&gt;는 담배</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

질병 벡터 모기 유충의 키토산 / 간섭 RNA 나노 입자 중재 유전자 음소거

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

siRNA를 매개 음소거로 마우스 난 모세포의 감수 스핀들 평가

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

불꽃을 침묵 : 약한 전기 물고기에서 CRISPR / Cas9 게놈 편집

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

아그로박테리움-형태유전자를 이용한 리칼시트 옥수수 근친선 미성숙 배아 변형 중재

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) ...