저자는 Fusarium verticillioiodes 에서 OSCAR 돌연변이 생성을위한 학부 및 고등학생에게 Anjellica Miller, Athar Naseer, Xiu Lin, Katelyn Woodburry, Chelsea Patterson, Kathleen Robertson, Krystina Bradley, Ashton Rogers, Alexis McKensie, Manny Hernandez , Ashli ​​Crepsac, Jeff Delong, 크리스 킹, Gi Jeong, Maria Belding, Christy Burre, Daniel O&#39;Meara, Lauren (빅토리아) Cook, Jake Goodman, Sampriti De, Oge Okoye, Alyssa Beckstead, Garrett Hibbs, Nick Goldstein, Caroline Twum , 크리스 벤슨, 루니 스토크 스, 한나 이텔, 제인 헐스, 자시 모하메드, 제임스 로긴스, 켈리 러셀, 그레 니 샤 존스, 크리스틴 셰퍼, 마리암 함머디, 에이브 윌슨, 카트리나 바스 모어, 토니 하퍼, 카린 맥기, 모흐메 모민, Thi Ngoc Le와 Angel Pham.

1. 유전자 측면의 PCR 증폭을위한 프라이머 디자인 <ol><li> 오픈 리딩 프레임 (ORF)과 FungiDB 또는 기타 게놈 데이터 자원의 양 측면에있는 유전자 2K 이상을 포함하여 관심 유전자 (GOI)의 게놈 영역을 워드 프로세싱 파일로 다운로드합니다. </li><li> 삭제하고자하는 ORF를 강조 표시하고 코돈을 시작 및 중지합니다. </li><li> 다운로드 된 시퀀스 내에서 인접한 ORF를 식별하고 강조 표시하십시오. </li><li> GOI ORF의 2 kb 5 &#39;말단과 프라이머 디자인 도구 (Materials List 참조)를 사용하여 PCR 프라이머 쌍 O1과 O2를 설계하여 1 kb의 최소 크기의 생성물을 생성하십시오. 인접한 ORF에 영향을 미치지 않도록주의하십시오. <ol><li> 2 kb 5 &#39;측면을 Sequence Entry 창에 붙여 넣습니다. &quot;Show Custom Parameters&quot;상자를 클릭하십시오. 다음 맞춤 설계 매개 변수를 입력하십시오 : primer TM 58 (min), 60 (opt) and 62 (max); 프라이머 GC 40 (min), 50 (opt) 및 60 (max); 프라이머 크기 22 (분), 24 (선택) 및 26(최대); Amplicon 크기 1250 (분), 1500 (선택) 및 2000 (최대). </li><li> ORF에 가장 가까운 역방향 프라이머 (O2)를 배치하는 결과를 선택하십시오. assays의 스크린 샷을 캡처하고 프라이머 시퀀스를 복사하여 워드 프로세싱 문서에 붙여 넣습니다. </li><li> 프라이머 O3와 O4를 생성하기 위해 3 &#39;플랭크에 대해이 과정을 반복한다. 이번에는 타겟 ORF에 가장 가까운 포워드 프라이머 (O3)를 배치 한 결과를 선택한다. </li></ol></li><li> 프라이머 1 ~ 4의 5 &#39;말단에 적절한 부착 사이트를 추가하십시오 ( 표 1 참조). </li><li> 또한 아래 4 절에서 삭제 확인을 위해 ORF 특정 프라이머를 설계하고 주문하십시오. 필요한 경우 Amplicon Size 500 (min), 750 (opt) 및 1000 (max)의 ORF 길이 조정을 제외한 매개 변수는 1.4.1 단계와 동일해야합니다. 마지막으로 상 동성 재조합 체를 확인하기 위해 염색체 내의 5 &#39;및 3&#39;바깥쪽에 프라이머를 생성한다. 이 &quot;out&quot;프라이머는hygR (210) 및 hygF (850)에 의해 억제되었다 ( 도 2 ). </li><li> 프라이머 주문. </li></ol> 2. OSCAR 구축물 제작 <ol><li> Hi-fidelity Taq을 사용하여 한 반응에서 프라이머 (100 μM) O1과 O2를 사용하고 두 번째 반응에서는 프라이머 O3와 O4를 사용하여 5 &#39;와 3&#39;측면 각각에 대해 두 개의 반응을 수행하십시오. 반응 혼합물은 29.5 μL 멸균 증류수 (SDW), 5 μL 10X LA PCR 버퍼, 8 μL 2.5 MM dNTP 믹스, 5 μL 25 MM MgCl 2 , 1 μL 템플릿 (50 NG / μL), 0.5 μL 하이 - 충실도 Taq , 0.5 μL Primer O1 및 5 &#39;측면 (0.5 μL Primer O3 및 0.5 μL 3&#39;측면에 대한 Primer O4)에 대한 0.5 μL Primer O2 사용 조건은 다음과 같습니다 : 94 ° C에서 1 분, 30 초 동안 94 ℃, 30 초 동안 60 ℃, 2 분 동안 72 ℃에서, 마지막 단계는 72 ℃에서 5 분간 그리고 나서 10 ℃에서 무기한 유지. </li><li> 0.8 % 뛰기제품 크기 및 상대 농도를 결정하기 위해 표준 미니 겔 장치에서 약 125Vh 동안 아가로 오스 1x TAE (40mM 트리스 아세테이트 pH 8.3, 1mM EDTA) 겔 전기 영동을 수행 하였다. 게시물은 제조 업체의 지침에 따라 비 ethidium 얼룩으로 젤을 얼룩. UV 조명기로 시각화하십시오. </li><li> 5 &#39;와 3&#39;측면 제품이 대략 균등 농도 인 경우, 이들을 조합하고 친 화성 칼럼 키트 (또는 PEG 침전물 90 μL 결합 PCR 제품, 240 μL TE 완충액 pH 7.5, 160 μL 30 % 폴리에틸렌 글리콜 PEG8000 30 mM MgCl 2 ) PCR 산물을 제조자의 프로토콜에 따라 동시 정제한다. 이들이 동등한 농도가 아닌 경우, 저농도 생성물 모두를 고 수율 반응으로부터 산출 된 동등한 양의 산물과 결합시킨다. </li><li> 분광 광도계를 사용하여 정제 된 DNA 농도를 측정하십시오. </li><li> 다음을 혼합하여 BP 반응을 수행하십시오 : 1 μL의 60 ng / μL pOSCAR, 2 μL의 60 ng /μL pA-Hyg-OSCAR, 1 nL / 60 ng / μL 조합 측면, 1 μL의 clonase. 25 ° C에서 16 시간 동안 품어 낸다. 0.5 μL proteinase K를 첨가하고 37 ℃에서 10 분 동안 항온 처리하여 반응을 종결시킨다. </li><li> 높은 역량을 개편 E. 대장균 . 상업적으로 이용 가능한 적격 세포와 집에서 만든 DH5α CaCl2 적격 세포 모두를 제조자의 지시에 따라 수령자로서 성공적으로 사용 하였다. 100 μg / mL spectinomycin (스펙)을 포함하는 낮은 나트륨 (0.5 g / L NaCl) LB 플레이트. </li><li> 위에 생성 된 식민지의 DNA minipreps 10 을 수행하여 올바른 구조를 식별합니다. 다음과 같이 Hin dIII와 Kpn I로 이중 분해를 수행하십시오. 피펫 5 μL DNA, 2 U의 각 효소, 2 μL 적절한 10x 완충액 (SDW 포함)으로 20 μL 총 부피. 이것은 벡터 ( 약 7kb)로부터 인서트 (1.5kb 유전자 옆구리를 갖는 대략 4.5kb)를 방출하고예측 가능한 밴드 크기를 줄 수있는 측면. </li><li> 서열 11 은 적절한 줄무늬 패턴을 나타내는 클론을 선택한다. </li></ol> 3. 진균의 Agrobacterium tumefaciens 매개 변형 (ATMT) <ol><li> OSCAR 결실 구조로 Agrobacterium tumefaciens AGL1 균주를 형질 전환 12 . </li><li> GOI OSCAR 결실 플라스미드를 함유하는 AGL1로 곰팡이를 형질 전환시킨다. 이렇게하려면 다음 단계를 수행하십시오. <ol><li> 2 x 10 6 포자 / mL의 멸균 수로 곰팡이 포자 현탁액을 준비하십시오. hemocytometer 또는 자동 셀 카운터에서 포자를 정량. 1.5 ML microcentrifuge 튜브 에이 500 μL를 놓으십시오. 이 셋업은 4 개의 변형 플레이트 용입니다. </li><li> 2 일 2-3 일 오래된 LB - 사양 100 매체에서 AGL1을 포함하는 삭제 플라스미드의 쉽게 보이는 gob (루프 직경의 약 20 %를 커버한다)을 추가하려면 푸른 살균 일회용 루프를 사용하십시오 (Materials List for composition) 플레이트를 포함하고 포자 현탁액에 혼합합니다. 박테리아 세포가 잘 분산 될 때까지 와동 ( 약 5 분 중간 속도). </li><li> 분양 배지가 들어있는 4 개의 6cm 페트리 접시에 셀룰로오스 멤브레인 필터의 중심에 분주 - 아크로 서스펜션 100μL를 피펫으로 넣는다. </li><li> 멤브레인 필터의 전체 표면을 덮기 위해 멸균 유리 비드 (약 4)로 현탁액을 퍼지십시오. 또는 기존 스프레더 도구를 사용하여 서스펜션을 펼칩니다. 멤브레인 필터를 약 10 분 동안 두건에서 건조시키고 파라핀 필름으로 감 쌉니다. 3.2.2 및 3.2.3 단계를 반복하여 여러 변형을 설정합니다. </li><li> 접시를 상온에서 2 일 동안 품어 둔다. </li><li> 하이 그로 마이신 B (Hyg) 150 μg / ML, 200 MM cefotaxime 및 100 μg / ML을 포함하는 6 cm Aspergillus 선택 매체 12 접시에 멤브레인 필터를 전송뜸 락탐. Hyg 내성 콜로니를 분리하기 전에 5-7 일 동안 상온에서 인큐베이션하십시오. </li><li> 멸균 이쑤시개로 150 μg / mL Hyg, 100 μg / mL kanamycin이 들어있는 6 cm Potato Dextrose Agar (PDA) 플레이트에 추정 형질 전환 체를 옮깁니다. </li></ol></li></ol> 4. PCR에 의한 결실 돌연변이 확인 <ol><li> 열 분해로 형질 전환 체로부터 PCR 용 DNA를 추출한다. <ol><li> 형질 전환 체가 3.2.7 단계에서 PDA에 콜로니를 형성하면 멸균 된 이쑤시게를 사용하여 소량 (2mm x 2mm)의 균사체 또는 효모 세포를 식민지에서 100μL의 용해 용액 (50mM 인산 나트륨, pH 7.4, 1mM EDTA, 5 % 글리세롤)을 첨가 하였다. </li><li> 85-90 ° C에서 20-30 분 동안 혼합물을 품는다. 단계 4.2에서 사용까지 -20 ° C에서 게놈 DNA를 포함하는 조잡한 추출물을 저장하십시오. </li></ol></li><li> 형질 전환 체 당 4 개의 PCR 반응 수행선택 가능한 마커 (이 경우 하이 그로 마이신 포스 포 트랜스퍼 라제) 및 GOI ORF에 대해 각각 5 &#39;및 3&#39;측면의 상 동성 재조합을 검출한다. 참고 : 우리는 일반적으로 이러한 반응에 대해 저렴한 충실도의 Taq 를 사용합니다. 반응 조건은 상기 2.1 단계에서 설명한 바와 같으며 SDW 20 μL, 10x Taq buffer 2.5 μL, dNTPS (10 mM) 0.5 μL, 수제 Taq 14 , 0.5 μL, Primer A (100 μM) 0.5 μL, Primer B ) 0.5 μL, 템플릿 0.5 μL. 농도가 더 낮은 프라이머 스톡 (10 μM)도 똑같이 사용할 수 있습니다. </li><li> PCR 제품의 아가 로즈 젤 ( 예 : 0.8 %)을 실행하십시오. 삭제 돌연변이 체는 Hyg 밴드를 생성하지만 ORF 생성물은 생성하지 않습니다. 이소 트로픽 적분기는 두 가지 밴드 모두를 보여줄 것입니다. 와일드 타입은 ORF 밴드 만 생성합니다. </li><li> 차후의 사용을 위해 결실 돌연변이 체 (및 대조를위한 이소성 형질 전환 체 또는 2 개)를 영구 보존한다. 참고 : 15 % 글리세롤은-80 ° C에서 장기적으로 우리의 변종을 찢어 냈습니다. </li></ol>

<ol>
	<li>Paz, Z., Garc&#237;a-Pedrajas, M. D., Andrews, D. L., Klosterman, S. J., Baeza-Monta&#241;ez, L., Gold, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+step+construction+of+Agrobacterium-Recombination-ready-plasmids+(OSCAR),+an+efficient+and+robust+tool+for+ATMT+based+gene+deletion+construction+in+fungi.">One step construction of Agrobacterium-Recombination-ready-plasmids (OSCAR), an efficient and robust tool for ATMT based gene deletion construction in fungi.</a> Fungal Genet Biol. 48 (7), 677-684 (2011).</li><li>Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, C. A., Smith, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nat Methods. 6 (5), 343-345 (2009).</li><li>Klosterman, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+genomics+yields+insights+into+niche+adaptation+of+plant+vascular+wilt+pathogens.">Comparative genomics yields insights into niche adaptation of plant vascular wilt pathogens.</a> PLoS Pathog. 7 (7), (2011).</li><li>Xue, C., Wu, D., Condon, B. J., Bi, Q., Wang, W., Turgeon, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+knockout+in+the+maize+pathogen+Setosphaeria+turcica+using+Agrobacterium+tumefaciens-mediated+transformation.">Efficient gene knockout in the maize pathogen Setosphaeria turcica using Agrobacterium tumefaciens-mediated transformation.</a> Phytopathology. 103 (6), 641-647 (2013).</li><li>Xu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+gene+disruption+methodology+for+the+entomopathogenic+fungus+Metarhizium+robertsii.">A high-throughput gene disruption methodology for the entomopathogenic fungus Metarhizium robertsii.</a> PloS One. 9 (9), (2014).</li><li>Crutcher, F. K., Liu, J., Puckhaber, L. S., Stipanovic, R. D., Bell, A. A., Nichols, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FUBT,+a+putative+MFS+transporter,+promotes+secretion+of+fusaric+acid+in+the+cotton+pathogen+Fusarium+oxysporum+f.+sp.+vasinfectum.">FUBT, a putative MFS transporter, promotes secretion of fusaric acid in the cotton pathogen Fusarium oxysporum f. sp. vasinfectum.</a> Microbiology. 161, 875-883 (2015).</li><li>Yu, X., Wang, Y., Pan, J., Wei, D., Zhu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+of+homologous+gene+disruption+by+single-stranded+DNA+in+the+taxol-producing+fungus+Pestalotiopsis+microspora.">High frequency of homologous gene disruption by single-stranded DNA in the taxol-producing fungus Pestalotiopsis microspora.</a> Ann Microbiol. 65 (4), 2151-2160 (2015).</li><li>Korn, M., Schmidpeter, J., Dahl, M., M&#252;ller, S., Voll, L. M., Koch, 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+Genetic+Screen+for+Pathogenicity+Genes+in+the+Hemibiotrophic+Fungus+Colletotrichum+higginsianum+Identifies+the+Plasma+Membrane+Proton+Pump+Pma2+Required+for+Host+Penetration.">A Genetic Screen for Pathogenicity Genes in the Hemibiotrophic Fungus Colletotrichum higginsianum Identifies the Plasma Membrane Proton Pump Pma2 Required for Host Penetration.</a> PloS One. 10 (5), e0125960 (2015).</li><li>Chettri, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Regulation of dothistromin toxin biosynthesis by the pine needle pathogen Dothistroma septosporum: a thesis presented in the partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatu, New Zealand . , (2014).</li><li>Chen Zhou, ., Yujun Yang, ., Jong, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-prep+in+ten+minutes.">Mini-prep in ten minutes.</a> Biotechniques. 8 (2), 172 (1990).</li><li>Sanger, F., Nicklen, S., Coulson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequencing+with+chain-terminating+inhibitors.">DNA sequencing with chain-terminating inhibitors.</a> P Natl Acad SciUSA. 74 (12), 5463-5467 (1977).</li><li>Khang, C. H., Park, S. Y., Rho, H. S., Lee, Y. H., Kang, S., Wang, K. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamentous+fungi+(Magnaporthe+grisea+and+Fusarium+oxysporum).">Filamentous fungi (Magnaporthe grisea and Fusarium oxysporum).</a> Agrobacterium Protocols. 2, 403-420 (2007).</li><li>Zhang, Y. J., Zhang, S., Liu, X. Z., Wang Wen, H. A., M, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+genomic+DNA+extraction+suitable+for+analysis+of+bulk+fungal+strains.">A simple method of genomic DNA extraction suitable for analysis of bulk fungal strains.</a> Lett Appl Microbiol. 51 (1), 114-118 (2010).</li><li>Pluthero, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+purification+of+high-activity+Taq+DNA+polymerase.">Rapid purification of high-activity Taq DNA polymerase.</a> Nucleic Acids Res. 21 (20), 4850-4851 (1993).</li><li>McCluskey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boosting+Research+and+Industry+by+Providing+Extensive+Resources+for+Fungal+Research.">Boosting Research and Industry by Providing Extensive Resources for Fungal Research.</a> Gene Expression Systems in Fungi: Advancements and Applications. , 361-384 (2016).</li></ol>

저자는 공개 할 것이 없습니다.

Agrobacterium의 한 단계 건설 - 재조합 준비 - 플라스미드 (OSCAR)는 지속적으로 증가하는 자낭 균 (Ascomycota) 균류에 성공적으로 사용되었습니다. 이 방법은 Basidiomycota 및 Agrobacterium 매개 형질 전환 및 상동 재조합이 가능하다고 가정하고 다른 진균 성 phyla의 종 (선택 마커 유전자를 유도하는 적절한 프로모터와 함께)에도 쉽게 적용 할 수 있어야한다. 항 곰팡이 화합물의 선택을 다양 화하고 이중...

유전 적 해부는 개인 또는 유전자 조합의 기능적 중요성을 결정하는 강력한 방법입니다. 특정 유전자의 역할을 이해하기위한 표준 접근법은 다른 유전자에서 변경되지 않은 단일 유전자 돌연변이의 생산이다. 가장 강력하고 최소한의 혼란을주는 접근법은 다른 유전자 기능에 손상을주지 않으면 서 관심 유전자의 오픈 리딩 프레임 (GOI ORF) 유전자를 완전하고 정확하게 삭제하는 것입니다. 삭제 플라스미드 생성을위한 표준 라이 게이션 접근법은 여러 단계가 필요하기 때문에 OSCAR 1 에 대한 합리적인 방법은보다 신속한 시험 관내 접근법을 생산하는 것이 었습니다. 그림 1 은 OSCAR 방식의 어셈블리 프로세스를 보여줍니다. 여기에 설명 된 방법은 이후의 Agrobacterium tumefaciens 매개 transfo와 결합하여 단일 multipart 반응에서 개별 유전자 삭제 벡터의 빠른 건설을 결합의 장점이 있습니다(ATMT). OSCAR는 매우 신속하며 효모 2 에서 Gibson assembly 사용과 같은 다른 전략과 잘 비교됩니다. OSCAR 방법은 여러 종류의 진균류와 함께 성공적으로 사용되었습니다. 이러한 종은 다음을 포함한다 : Fusarium verticillioides (미공개), Verticillium dahliae 3 , Setospaeria turcica 4 , Metarhizium robertsii 5 , Fusarium oxysporum f. sp. vasinfectum 6 , Pestalotiopsis microspora 7 , Colletotrichum higginsianum 8 및 Dothistroma septosporum 9 및 Sarocladium zeae (미공개) . 이 프로토콜은 프라이머 디자인, 플랭크 PCR 증폭, OSCAR BP 반응, 결실 구조 구조 확인, 변형을 포함한 방법에 대한 단계별 지침을 제공합니다이온으로 구성 하고, 결실 구조를 곰팡이 세포로 ATMT에 옮기고, 최종적으로 균류 결실 돌연변이를 돌연변이 적으로 결실 된 결실 구조물과 차별화시킨다.

<table><tbody><tr><td>FungiDB</td><td></td><td></td><td>Database/ http://fungidb.org/fungidb/</td></tr><tr><td>IDT PrimerQuest</td><td>IDT</td><td></td><td>Primer design online software/ http://www.idtdna.com/Primerquest/Home/Index</td></tr><tr><td>Microsoft Word</td><td></td><td></td><td>Sequence file manipulation</td></tr><tr><td>Low Na LB Spec 100 medium</td><td></td><td></td><td>&lt;em&gt;E. coli&lt;/em&gt; transformant selection, composition: 1% tryptone, 0.05% NaCl, 0.5% yeast extract, 1.5% agar if for solid medium</td></tr><tr><td>Co-cultivation medium</td><td></td><td></td><td>ATMT transformation induction (Reference 12)</td></tr><tr><td>Aspergillus minimal medium with Hygromycin</td><td></td><td></td><td>Fungal transformant selection</td></tr><tr><td>PDA medium</td><td>Acumedia</td><td>7149A</td><td>Single spore slant tubes</td></tr><tr><td>PDA-Hyg-Kan medium</td><td></td><td></td><td>Fungal ransformant isolation, PDA containing 150 &amp;mu;g/mL hygromycin B and 100 &amp;mu;g/mL Kanamycin</td></tr><tr><td>Glass beads</td><td>Genlantis</td><td>C400100</td><td>Plate spreading</td></tr><tr><td>Nitrocellulose filters (47 mm)</td><td>Fisher</td><td>09-719-555</td><td>Co-culturing for ATMT</td></tr><tr><td>Various centifuge tubes</td><td></td><td></td><td>multiple preps</td></tr><tr><td>Petri plates (various)</td><td></td><td></td><td>Culturing of bacteria and Fungi</td></tr><tr><td>pA-Hyg OSCAR</td><td>Addgene</td><td>29640</td><td>Selectable marker vector</td></tr><tr><td>pOSCAR</td><td>Addgene</td><td>29639</td><td>Assembly vector</td></tr><tr><td>DH5&amp;#945; One Shot Competent &lt;em&gt;E. coli &lt;/em&gt;cells</td><td>Life Technologies&amp;nbsp;</td><td>12297-016</td><td>BP reaction transformation</td></tr><tr><td>ccdB survival E. coli cells</td><td>Life Technologies&amp;nbsp;</td><td>A10460</td><td>Maintenance of pOSCAR</td></tr><tr><td>Wooden transfer sticks</td><td></td><td></td><td>Colony streaking</td></tr><tr><td>Toothpicks</td><td></td><td></td><td>Colony picking</td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td>Pelleting Bacteria, &lt;em&gt;etc.&lt;/em&gt;</td></tr><tr><td>Preparative centrifuge</td><td></td><td></td><td>Fungal spore collection</td></tr><tr><td>Dissecting microscope</td><td></td><td></td><td>Single spore isolation</td></tr><tr><td>Automated Cell Counter</td><td></td><td></td><td>Spore suspension calculation</td></tr><tr><td>Compound microscope</td><td></td><td></td><td>Hemocytometer cell counting</td></tr><tr><td>QIAquick PCR Purification Kit&amp;nbsp;</td><td>Qiagen</td><td>28104</td><td>PCR gene flank produict purification</td></tr><tr><td>TaKaRa LA &lt;em&gt;Taq&lt;/em&gt;&amp;nbsp;</td><td>Takara Bio USA</td><td>RR002A</td><td>Hi Fidelity &lt;em&gt;taq &lt;/em&gt;polymerase for OSCAR flank generation</td></tr><tr><td>Hygromycin B</td><td>InvivoGen</td><td>ant-hg-5</td><td></td></tr><tr><td>Spectinomycin</td><td>Sigma</td><td>22189-32-8</td><td></td></tr><tr><td>Cefotaxime</td><td>TCI America</td><td>C2224</td><td></td></tr><tr><td>Kanamycin</td><td></td><td>11815032</td><td></td></tr><tr><td>Moxalactam</td><td>Sigma-Aldrich</td><td>43963</td><td></td></tr><tr><td>GelRed</td><td>&amp;nbsp;Phenix Research Products</td><td>RGB-4103</td><td>Post staining agarose gels</td></tr><tr><td>Qiagen QIAquick PCR Purification Kit (Cat. No. 28104)&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>(OneShot_ Mach1TM T1R or One Shot_ OmniMAX&amp;trade; 2 T1R from Invitrogen)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>C862003</td><td></td></tr><tr><td>Gateway BP Clonase II Enzyme mix</td><td>Thermo Fisher Scientific</td><td>11789020</td><td>Used to assemble deletion construct in pOSAR</td></tr></tbody></table>

rapid deletion production in fungi via agrobacterium mediated transformation of oscar deletion constructs

게놈의 나머지 부분을 그대로두고 관심있는 유전자를 정확하게 삭제하면 살아있는 유기체에서 특정 유전자의 기능을 결정하는 이상적인 제품을 제공합니다. 이 프로토콜에서 정확하고 신속한 삭제 플라스미드 구성의 OSCAR 방법이 설명됩니다. OSCAR는 관심 유전자의 정제 PCR 증폭 5 &#39;및 3&#39;측면과 두 개의 플라스미드, pA-Hyg OSCAR (마커 벡터) 및 pOSCAR (조립체)를 포함하는 단일 재조합 효소 반응이 수행되는 클로닝 시스템에 의존한다 벡터). 올바르게 조립 된 결실 벡터의 확인은 제한 효소지도 작성과 시퀀싱에 의해 수행됩니다. Agrobacterium tumefaciens 는 곰팡이 포자 (ATMT)에 결실 구조의 도입을 매개하는데 사용된다. 마지막으로, PCR 분석은 상 동성 또는 비 상 동성 재조합에 의해 결실 된 결실 구조가 유전자 결실을 나타내는 지 또는이소성 통합. 이 접근법은 Verticillium dahliae 와 Fusarium verticillioides 에서 수많은 종의 유전자를 삭제하는데 성공적으로 사용되었습니다.

단일 반응에서 OSCAR 방법은 선별 마커 카세트를 둘러싼 삭제 대상 유전자의 플랭크를 포함하는 플라스미드를 생성한다. OSCAR을 사용하여 삭제 구문을 생성하는 것은 매우 효율적입니다. 그러나 시스템은 세 가지 단편 (두 개의 유전자 측면과 선별 마커)이 아닌 일부를 포함하는 부분 구조를 생성 할 수 있습니다. 일반적으로 대부분의 대장균 형질 전환 체는 정확한 O...

Watch this Scientific Journal Video about Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs at JoVE.com

Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

상 동성 재조합을 통해 생성 된 유전자 결실 돌연변이는 유전자 기능 연구의 황금 표준이다. 결실 구조물의 신속한 생성을위한 OSCAR (Agrobacterium-Recombination-ready-plasmids의 One Step Construction) 방법이 기술되어있다. 아그로 박테 리움 매개 곰팡이 형질 전환이 이어진다. 마지막으로, 곰팡이 형질 전환 체에서 PCR을 이용한 유전자 결실 확인 방법을 제시한다.

경유 곰팡이에 의한 신속한 결실 생산<em&gt; 아그로 박테 리움</em&gt; OSCAR 삭제 구성 요소의 중재 변환

Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular ...

rapid-deletion-production-fungi-via-agrobacterium-mediated

Universidad de Cartagena UDEC

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JoVE Journal

Genetics

Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

9.9K 조회수.  NPRC, USDA-ARS.  상 동성 재조합을 통해 생성 된 유전자 결실 돌연변이는 유전자 기능 연구의 황금 표준이다. 결실 구조물의 신속한 생성을위한 OSCAR (Agrobacterium-Recombination-ready-plasmids의 One Step Construction) 방법이 기술되어있다. 아그로 박테 리움 매개 곰팡이 형질 전환이 이어진다. 마지막으로, 곰팡이 형질 전환 체에서 PCR을 이용한 유전자 결실 확인 방법을 제시한다. 

비디오: Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70&#916;. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

신 재생 바이오 연료 및 생물 생산에 대한 자유로운 효모의 유전 공학

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic ...

연구

유전학

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

플랜트 병원체의 유전자 조작<em&gt; Ustilago maydis</em&gt; 곰팡이 생물과 식물 미생물 상호 작용을 연구하는

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

분열 효 모 섬으로 불안정 프로테아좀 돌연변이 선택할 임의의 Mutagenesis 유전자 타겟팅

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol for transferring gene deletion mutations from one Escherichia coli strain to another by using generalized transduction with the bacteriophage P1. This method requires that the mutation be selectable (e.g., based on gene disruptions using antibiotic cassette insertions). Such antibiotic cassettes can be mobilized from a donor strain and introduced into a recipient strain of interest to quickly and easily generate a gene deletion mutant. The antibiotic cassette can be designed to include flippase recognition sites that allow the excision of the cassette by a site-specific recombinase to produce a clean knock-out with only a ~100-base-pair-long scar sequence in the genome. We demonstrate the protocol by knocking out the tamA gene encoding an assembly factor involved in autotransporter biogenesis and test the effect of this knock-out on the biogenesis and function of two trimeric autotransporter adhesins. Though gene deletion by P1 transduction has its limitations, the ease and speed of its implementation make it an attractive alternative to other methods of gene deletion.

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Here we present a protocol for the use of pre-existing antibiotic resistance-cassette deletion constructs as a basis for making deletion mutants in other E. coli strains. Such deletion mutations can be mobilized and inserted into the corresponding locus of a recipient strain using P1 bacteriophage transduction.

Excisable 항 생 저항 카세트와 P1 변환 하 여 대장균 에서 생산 유전자 삭제

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol ...

Direct Liquid-Culture Screening for Evaluating the Production of Heterologous Proteins Using an Auxotrophic Mutant of Aspergillus Oryzae

Aspergillus oryzae, a filamentous fungus, is one of the most widely used hosts for industrial applications including large-scale production of proteins. A polyethylene glycol (PEG)-mediated protoplast transformation method is generally used for the introduction of heterologous genes into A. oryzae. The conventional method typically requires three weeks for the screening of favorable transformants. Here, a new technique, the direct liquid-culture (DLC) screening method, is introduced which reduces the screening time to six days in a 200 mL flask format or to 10 days in a 24 well microplate format. The DLC screening method ensures the acquisition of positive transformants and evaluation of the secretory production of heterologous proteins in a single step, unlike the conventional screening method where two separate steps are required for the same. The protocol for PEG-mediated protoplast transformation of A. oryzae is described, which consists of five steps: preparation of fresh spore suspension, preculture, preparation of protoplasts, introduction of DNA, and DLC screening. For successful results in DLC screening, it is critical to use a nutrient-rich medium with optimized osmotic pressure. The protocol should further popularize the use of A. oryzae as a host of choice in the industrial production of proteins.

Direct Liquid-Culture Screening for Evaluating the Production of Heterologous Proteins Using an Auxotrophic Mutant of Aspergillus Oryzae

A direct liquid-culture (DLC) screening method has been developed which significantly reduces the time required for polyethylene glycol (PEG)-mediated protoplast transformation of the filamentous fungus, Aspergillus oryzae, when employed for evaluation of the secretory production of heterologous proteins. This method dramatically increases the throughput of the evaluation protocol.

Aspergillus oryzae, a filamentous fungus, is one of the most widely used hosts for industrial applications including large-scale production of proteins. A ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

여러 유전자 삭제를 운반 효모 종자의 생성에 대한 초록 괴물 프로세스

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

생물학

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

게놈 전체 유전자 삭제할에<em&gt; 연쇄상 구균 sanguinis</em&gt; 높은 처리량 PCR에 의한

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

CRISPR / Cas9를 통해 포유 동물 세포주의 게놈에 삭제 세대

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

진화 방법에 감독<em&gt; 사카로 마이 세스 세레 비지에</em&gt; : 돌연변이 라이브러리 생성 및 검사

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

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) ...

경유 곰팡이에 의한 신속한 결실 생산

요약

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플랜트 병원체의 유전자 조작

분열 효 모 섬으로 불안정 프로테아좀 돌연변이 선택할 임의의 Mutagenesis 유전자 타겟팅

Excisable 항 생 저항 카세트와 P1 변환 하 여 대장균 에서 생산 유전자 삭제

Direct Liquid-Culture Screening for Evaluating the Production of Heterologous Proteins Using an Auxotrophic Mutant of Aspergillus Oryzae

여러 유전자 삭제를 운반 효모 종자의 생성에 대한 초록 괴물 프로세스

게놈 전체 유전자 삭제할에

CRISPR / Cas9를 통해 포유 동물 세포주의 게놈에 삭제 세대

진화 방법에 감독

아그로박테리움-형태유전자를 이용한 리칼시트 옥수수 근친선 미성숙 배아 변형 중재