이 연구는 네덜란드 기술 재단 STW (12385), 네덜란드 조직에 대 한 과학적 연구 (NWO)의 부분 이다 고는 부분적으로 경제 통상 부 (OTP 부여 12385 ms)에 의해 자금이 지원 됩니다.  우리는 pCH20, BIBAC-GW 벡터의 등뼈를 제공 하기 위한 캐롤 M. 해밀턴 (코넬 대학, 미국) 감사 합니다.

<ol>
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E., 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=Direct+PCR+of+Intact+Bacteria+(Colony+PCR).">Direct PCR of Intact Bacteria (Colony PCR).</a> Curr Protoc Microbiol. , A.3D.1-A.3D.7 (2016).</li><li>Ausubel, F. M., 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=Mol+Biol.">Mol Biol.</a> Current Protocols in Molecular Biology. 1, (2003).</li><li>Edwards, K., Johnstone, C., Thompson, 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+simple+and+rapid+method+for+the+preparation+of+plant+genomic+DNA+for+PCR+analysis.">A simple and rapid method for the preparation of plant genomic DNA for PCR analysis.</a> Nucleic Acids Res. 19 (6), 1349 (1991).</li><li>Clarke, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cetyltrimethyl+ammonium+bromide+(CTAB)+DNA+miniprep+for+plant+DNA+isolation.">Cetyltrimethyl ammonium bromide (CTAB) DNA miniprep for plant DNA isolation.</a> Cold Spring Harb Protoc. (3), (2009).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+Gel+Electrophoresis+for+the+Separation+of+DNA+Fragments.">Agarose Gel Electrophoresis for the Separation of DNA Fragments.</a> J Vis Exp. (62), e3923 (2012).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning. , (2012).</li><li>Sosa, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatography+with+Sephadex+Gels.">Chromatography with Sephadex Gels.</a> Anal Chem. 52 (6), 910-912 (1980).</li><li>Depicker, A., Stachel, S., Dhaese, P., Zambryski, P., Goodman, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nopaline+synthase:+transcript+mapping+and+DNA+sequence.">Nopaline synthase: transcript mapping and DNA sequence.</a> J Mol Appl Genet. 1 (6), 561-573 (1982).</li><li>Feng, J., Vick, B. A., Lee, M. K., Zhang, H. B., Jan, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+BAC+and+BIBAC+libraries+from+sunflower+and+identification+of+linkage+group-specific+clones+by+overgo+hybridization.">Construction of BAC and BIBAC libraries from sunflower and identification of linkage group-specific clones by overgo hybridization.</a> Theor Appl Genet. 113 (1), 23-32 (2006).</li><li>Lee, M. K., 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=Construction+of+a+plant-transformation-competent+BIBAC+library+and+genome+sequence+analysis+of+polyploid+Upland+cotton+(Gossypium+hirsutum+L).">Construction of a plant-transformation-competent BIBAC library and genome sequence analysis of polyploid Upland cotton (Gossypium hirsutum L).</a> BMC Genomics. 14, 208 (2013).</li><li>Wang, W., 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+large+insert+Thellungiella+halophila+BIBAC+library+for+genomics+and+identification+of+stress+tolerance+genes.">A large insert Thellungiella halophila BIBAC library for genomics and identification of stress tolerance genes.</a> Plant Mol Biol. 72 (1-2), 91-99 (2010).</li><li>Wu, 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+BAC-+and+BIBAC-based+physical+map+of+the+soybean+genome.">A BAC- and BIBAC-based physical map of the soybean genome.</a> Genome Res. 14 (2), 319-326 (2004).</li><li>Xu, Z., 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=Genome+physical+mapping+from+large-insert+clones+by+fingerprint+analysis+with+capillary+electrophoresis:+a+robust+physical+map+of+Penicillium+chrysogenum.">Genome physical mapping from large-insert clones by fingerprint analysis with capillary electrophoresis: a robust physical map of Penicillium chrysogenum.</a> Nucleic Acids Res. 33 (5), e50 (2005).</li><li>Zhang, M., 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=Genome+physical+mapping+of+polyploids:+a+BIBAC+physical+map+of+cultivated+tetraploid+cotton,+Gossypium+hirsutum+L.">Genome physical mapping of polyploids: a BIBAC physical map of cultivated tetraploid cotton, Gossypium hirsutum L.</a> PLoS One. 7 (3), e33644 (2012).</li><li>Frary, A., Hamilton, C. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Position-Dependent+Methylation+and+Transcriptional+Silencing+of+Transgenes+in+Inverted+T-DNA+Repeats:+Implications+for+Posttranscriptional+Silencing+of+Homologous+Host+Genes+in+Plants.">Position-Dependent Methylation and Transcriptional Silencing of Transgenes in Inverted T-DNA Repeats: Implications for Posttranscriptional Silencing of Homologous Host Genes in Plants.</a> Cell Biol. 18 (11), 6165-6177 (1998).</li><li>G&#322;owacka, K., Kromdijk, J., Leonelli, L., Niyogi, K. K., Clemente, T. E., Long, S. 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저자 아무 경쟁 금융 관심사 또는 다른 충돌을 선언합니다.

생성에 중요 한 제 닉은 transgene의 단일, 그대로 통합으로 사용 되는 이진 벡터의 선택입니다. BIBAC 가족 벡터 많은 식물 종23,,2425,26,,2728관심사의 순서를 전달 하기 위해 사용 되었습니다. BIBAC 벡터, BIBAC-GW를 포함 하 여 높은 효율으로 단일 복사 통합...

유전자 변형 식물을 생성할 때 일반적으로 목적은 안정적으로 표현 하는 통합된 transgene(s) 것입니다. 이 transgene의 그대로 단일 복사본 통합 하 여 얻을 수 있습니다. 여러 통합은 transgene의 증가 표현 뿐만 아니라 유전자 입을 발생할 수 있습니다. Transgenes의 입을 경우 삽입된 시퀀스 협동 또는 거꾸로 반복1,2,,34에서 배열 된다 확률이 높다. 이진 벡터 Agrobacterium에 셔틀으로 이용 된다-식물 게놈으로 관심의 시퀀스를 제공 하는 변환 실험을 중재. 식물 게놈으로 통합의 수 Agrobacterium tumefaciens5,6. 이진 벡터의 복사본 수에 따라 달라 집니다. 많은 일반적으로 사용 되 이진 벡터는 높은 복사 벡터 및 따라서 높은 평균 transgene 복사본 수를 산출: 애기53.3 4.9 복사본.
BIBAC7또는 T-DNA A. tumefaciens 염색체5에서 시작 하 여 낮은 복사 번호 A. tumefaciens에 있는 이진 벡터를 사용 하 여 T-DNA 통합의 수를 낮출 수 있습니다. 이러한 경우 transgene 통합의 평균 수는 25,8,,910아래입니다. 따른 단일-복사 A. tumefaciens 에 대장균에서 BIBAC 파생 상품 수 유지 및 구문 150 kb11로 제공.
GW 호환 BIBAC 벡터10,12 게이트웨이 복제를 사용 하 여 벡터에 관심사의 유전자의 도입에 쉽게 수 있습니다. 게이트웨이 기술 사용 하 여 복제 프로시저를 단순화 하지만 또한 큰 낮은 복사 번호 벡터13,14, 낮은 DNA 수율 등 독특한 제한의 제한 된 선택과 관련 된 일반적인 문제를 극복 7,11복제에 사용할 수 있는 사이트. PBIBAC-GW 파생 상품 Glufosinate 염화 (pBIBAC-바-GW) 어느 저항 또는 DsRed 형광 씨 외 투 (pBIBAC-RFP-GW) 식물 (그림 1)10,12선택에 사용할 수 있습니다. 두 벡터에 대 한 대 저항 유전자는 박테리아에서 선택 마커로 사용 됩니다.
PBIBAC-GW 벡터 결합: (1) 쉬운 디자인 및 유전자 조작 대장균및 (2) 그대로 단일 복사 통합에서 planta에 높은 효율에. 들고 단일 유전자 변형 식물의 약 절반으로 애기 에 평균 1.7 통합에 pBIBAC GW 벡터 수확량은 통합 T DNA10.
안정적인 표현의 transgenes 대부분 제 닉 생성에 대 한 요구 사항입니다. 안정적인 transgene 식 그대로, 단일 복사 통합 하 여 얻을 수 있습니다. 그러나 유전자 변형 식물 그대로, 단일 복사 통합 운반 작업은,, 훨씬 더 중요 한 경우 예를 들어 목표 mutagenesis, 재결합, 또는 복구, 등 이러한 의존 chromatin 기반 프로세스의 효율성을 연구 하는 것입니다 genomic 위치 및 삽입 사이트에서 chromatin 구조에 처리 합니다. 우리의 관심 지역의 게놈 문맥에 의존 oligonucleotide 감독 mutagenesis (ODM)의 공부 mutagenesis 취재 원 유전자의 손상, 단일 복사 통합 기자 라인의 집합 생성된 (그림 2)10이었다. 이 일련의 줄을 사용 하 여, 보였다 ODM 효율 되 고 오히려 유사한 transgene 식 수준에도 불구 하 고 다른 genomic 위치의 통합 유전자 변형 loci 사이 변화 한다.

<table>
	<tbody>
		<tr>
			<td>Kanamycin sulphate monohydrate</td>
			<td>Duchefa</td>
			<td>K0126</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin sulphate</td>
			<td>Duchefa</td>
			<td>G0124</td>
			<td></td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Duchefa</td>
			<td>R0146</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline hydrochloride</td>
			<td>Sigma</td>
			<td>T-3383</td>
			<td></td>
		</tr>
		<tr>
			<td>DB3.1 competent cells</td>
			<td>Thermo Scientific - Invitrogen</td>
			<td>11782-018</td>
			<td>One Shot ccdB Survival 2 T1R Competent Cells (A10460) by Invitrogen or any other ccdB resistant E. coli strain can be used instead&#160;&#160;</td>
		</tr>
		<tr>
			<td>DH10B competent cells</td>
			<td>Thermo Scientific - Invitrogen</td>
			<td>18290-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Gateway LR clonase enzyme mix&#160;</td>
			<td>Thermo Scientific - Invitrogen</td>
			<td>11791-019</td>
			<td></td>
		</tr>
		<tr>
			<td>tri-Sodium citrate dihydrate</td>
			<td>Merck</td>
			<td>106432</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA disodium dihydrate</td>
			<td>Duchefa</td>
			<td>E0511</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Thermo Scientific&#160;</td>
			<td>EO0491</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto tryptone</td>
			<td>BD</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BD</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Honeywell Fluka</td>
			<td>13423</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Merck</td>
			<td>104936</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Glucose monohydrate</td>
			<td>Merck</td>
			<td>108346</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporation Cuvettes, 0.1 cm gap</td>
			<td>Biorad</td>
			<td>1652089</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporator Gene Pulser</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Calbiochem</td>
			<td>442613</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Maltose monohydrate 90%</td>
			<td>Acros Organics</td>
			<td>32991</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84100</td>
			<td></td>
		</tr>
		<tr>
			<td>Silwet L-77</td>
			<td>Fisher Scientific</td>
			<td>NC0138454</td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige Skoog medium</td>
			<td>Duchefa</td>
			<td>M0221</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Glufosinate-ammonium (Basta)</td>
			<td>Bayer</td>
			<td>79391781</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction enzymes</td>
			<td>NEB</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Bio-Rad</td>
			<td>1610433</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis system</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Merck</td>
			<td>106498</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Merck</td>
			<td>100316</td>
			<td></td>
		</tr>
		<tr>
			<td>Blotting nylon membrane Hybond N+</td>
			<td>Sigma Aldrich</td>
			<td>15358</td>
			<td>or GE Healthcare Life Sciences (RPN203B)</td>
		</tr>
		<tr>
			<td>Whatman 3MM Chr blotting paper</td>
			<td>GE Healthcare Life Sciences</td>
			<td>3030-931</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP</td>
			<td>Thermo Fisher</td>
			<td>R0181</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetylated BSA</td>
			<td>Sigma-Aldrich</td>
			<td>B2518</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Merck</td>
			<td>805740</td>
			<td></td>
		</tr>
		<tr>
			<td>Sephadex G-50 Coarse</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17004401</td>
			<td>or Sephadex G-50 Medium (17004301)</td>
		</tr>
		<tr>
			<td>Dextran sulfate sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>D8906</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate&#160;</td>
			<td>US Biological</td>
			<td>S5010</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon Sperm DNA</td>
			<td>Sigma-Aldrich</td>
			<td>D7656</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate monohydrate</td>
			<td>Merck</td>
			<td>106346</td>
			<td></td>
		</tr>
		<tr>
			<td>Storage Phosphor screen and casette</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28-9564-74</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphor imager</td>
			<td>GE Healthcare Life Sciences</td>
			<td>Typhoon FLA 7000</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Crosslinker</td>
			<td>Stratagene</td>
			<td>Stratalinker 1800</td>
			<td></td>
		</tr>
		<tr>
			<td>cling film (Saran wrap)</td>
			<td>Omnilabo</td>
			<td>1090681</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Thermo Scientific - Invitrogen</td>
			<td>16500</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Merck</td>
			<td>100165</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA marker &#8216;Blauw&#8217;; DNA ladder.</td>
			<td>MRC Holland</td>
			<td>MCT8070</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA marker &#8216;Rood&#8217;; DNA ladder</td>
			<td>MRC Holland</td>
			<td>MCT8080</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexanucleotide Mix</td>
			<td>Roche</td>
			<td>11277081001</td>
			<td></td>
		</tr>
		<tr>
			<td>Large-Construct Kit</td>
			<td>Qiagen</td>
			<td>12462</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat-sealable polyethylene tubing, clear</td>
			<td>various providers</td>
			<td></td>
			<td>the width of the tubing should be wider than that of blotting membrane</td>
		</tr>
		<tr>
			<td>Heat sealer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane filter disk</td>
			<td>Merck</td>
			<td>VSWP02500</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Merck</td>
			<td>105833</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization mesh</td>
			<td>GE Healthcare Life Sciences</td>
			<td>RPN2519</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating transgenic plants with single-copy insertions using bibac-gw binary vector

유전자 변형 식물을 생성할 때 일반적으로 목표는 transgene의 안정적인 식 것입니다. 이 transgene의 단일, 그대로 통합 다중 복사 통합 유전자 입을 대상이 종종 필요 합니다. 다른 pBIBAC 파생 상품 같은 세균성 인공적인 염색체 (pBIBAC-GW)에 따라 게이트웨이 호환 이진 벡터 고효율 단일 카피 transgenes의 삽입을 수 있습니다. 원래 pBIBAC에 개선으로 게이트웨이 카세트는 되었습니다 복제 pBIBAC GW에 관심의 시퀀스 지금 통합 될 수 있다 쉽게 벡터 전송 DNA (T-DNA)에 게이트웨이 복제 하 여 그렇게. 일반적으로, pBIBAC GW와 변환 결과 0.2-0.5 효율 %, 그것에 의하여는 제 닉의 T-DNA의 본래 단일 복사 통합 수행. PBIBAC-GW 벡터 Glufosinate 염화에 저항 또는 선택 식물, 씨 외 투에서 DsRed 형광와 저항 대에 박테리아에서 선택 항목으로 사용할 수 있습니다. 여기, 프로토콜의 시리즈 pBIBAC GW를 사용 하 여 유전자 변형 식물 생성의 과정을 통해 독자를 안내 하는 표시 됩니다: 진상을 공장 로 변환 하는 선택의 pBIBAC GW 벡터에 관심의 시퀀스에서 시작 Agrobacterium, 제 닉, 그리고 테스트 DNA 럽을 사용 하 여 삽입의 intactness 및 복사 번호에 대 한 식물의 선택. 주의는 DNA 더 럽 히 전략을 인식 하는 단일 및 다중 loci에서 단일 및 다중 복사 통합 주어 집니다.

BIBAC-GW 시스템을 사용 하 여, 식물에서 ODM을 공부 기자 구문이 생성된10했다. 구문을은 게이트웨이 항목 벡터 pENTR gm12 설계 되었고에 pBIBAC-바-GW (그림 1) 게이트웨이 LR 재결합 반응을 사용 하 여 삽입.
애기는 pDM19, BIBAC-바-GW 플라스 변환 정지 codon 위치 120 eYFP 독서 프레임...

Watch this Scientific Journal Video about Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector at JoVE.com

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

PBIBAC-GW 이진 벡터를 사용 하 여 생성 유전자 변형 식물을 그대로 단일 복사본 삽입, 쉬운 과정을 만든다. 여기, 프로토콜의 시리즈는 유전자 변형 애기 식물을 생성 하 고 intactness에 대 한 식물을 테스트 과정을 통해 독자를 안내 하 고 삽입의 수를 복사 하 여.

BIBAC-GW 이진 벡터를 사용 하 여 단일 복사본 삽입을 가진 유전자 변형 식물 생성

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

generating-transgenic-plants-with-single-copy-insertions-using-bibac

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

12.4K 조회수.  University of Amsterdam. PBIBAC-GW 이진 벡터를 사용 하 여 생성 유전자 변형 식물을 그대로 단일 복사본 삽입, 쉬운 과정을 만든다. 여기, 프로토콜의 시리즈는 유전자 변형 애기 식물을 생성 하 고 intactness에 대 한 식물을 테스트 과정을 통해 독자를 안내 하 고 삽입의 수를 복사 하 여.

비디오: Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

펩타이드 유래의 방법은 식물의 유전자와 단백질 세포 간 및 Organellar 장벽을 수송하는

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

연구

유전학

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

유전자 교란 mutans 연쇄 상 구 균 긴장 없이 유전자 복제의 생성

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.

We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.

CRISPR/Cas9-중재 유전자 녹아웃 셀 분할에 대 한 Integrase 불충분 한 Lentiviral 벡터의 생산을 위한 프로토콜

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

태아 Microinjection 및 Nasonia vitripennis 게놈 조작을 위한 이식 기술

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

빠른 고 손쉬운 파이프라인 C. 선 충 을 사용 하 여 CRISPR/Cas9 Ribonucleoproteins에서 게놈 포인트 돌연변이 생성에 대 한

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

마우스 배아 줄기 세포 사용 하 여 남부 럽 연쇄 반응에서에서 동종 재결합 이벤트의 식별

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

인간으로 Epigenome 편집 도구의 효율적인 배달 lentiviral 벡터 플랫폼 유도 만능 줄기 세포 유래 질병 모델

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in regulating gene expression at the post-transcriptional level. Sequencing sRNA libraries by Next Generation Sequencing (NGS) methods has been widely employed to identify and analyze miRNA transcriptomes in the last decade, resulting in a rapid increase of miRNA discovery. However, two major challenges arise in plant miRNA annotation due to increasing depth of sequenced sRNA libraries as well as the size and complexity of plant genomes. First, many other types of sRNAs, in particular, short interfering RNAs (siRNAs) from sRNA libraries, are erroneously annotated as miRNAs by many computational tools. Second, it becomes an extremely time-consuming process for analyzing miRNA transcriptomes in plant species with large and complex genomes. To overcome these challenges, we recently upgraded miRDeep-P (a popular tool for miRNA transcriptome analyses) to miRDeep-P2 (miRDP2 for short) by employing a new filtering strategy, overhauling the scoring algorithm and incorporating newly updated plant miRNA annotation criteria. We tested miRDP2 against sequenced sRNA populations in five representative plants with increasing genomic complexity, including Arabidopsis, rice, tomato, maize and wheat. The results indicate that miRDP2 processed these tasks with very high efficiency. In addition, miRDP2 outperformed other prediction tools regarding sensitivity and accuracy. Taken together, our results demonstrate miRDP2 as a fast and accurate tool for analyzing plant miRNA transcriptomes, therefore a useful tool in helping the community better annotate miRNAs in plants.

A bioinformatics pipeline, namely miRDeep-P2 (miRDP2 for short), with updated plant miRNA criteria and an overhauled algorithm, could accurately and efficiently analyze microRNA transcriptomes in plants, especially for species with complex and large genomes.

식물의 MicroRNA 전사체를 정확하고 효율적으로 분석하는 생물정보학 파이프라인

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in ...

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' lines that express the yeast transcription factor GAL4 in a tissue specific manner, with transgenic 'responder' lines carrying a candidate gene/RNA interference construct whose expression is controlled by Upstream Activation Sequences (UAS) that bind GAL4. In the ensuing progeny, the gene or silencing construct is thus expressed in a prescribed spatiotemporal manner, enabling the resultant phenotypes to be assayed and gene function inferred. The binary system enables flexibility in experimental approaches to screen phenotypes generated by transgene expression in multiple tissue-specific patterns, even if severe fitness costs are induced. We have adapted this system for Anopheles gambiae, the principal malaria vector in Africa.
In this article, we provide some of the common procedures used during GAL4-UAS analysis. We describe the An. gambiae GAL4-UAS lines already generated, as well as the cloning of new responder constructs for upregulation and RNAi knockdown. We specify a step by step guide for sexing of mosquito pupae to establish genetic crosses, that also includes screening progeny to follow inheritance of fluorescent gene markers that tag the driver and responder insertions. We also present a protocol for clearing An. gambiae embryos to study embryonic development. Finally, we introduce potential adaptions of the method to generate driver lines through CRISPR/Cas9 insertion of GAL4 downstream of target genes.

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae

The bipartite GAL4-UAS system is a versatile tool for modification of gene expression in a controlled spatiotemporal manner which permits functional genetic analysis in Anopheles gambiae. The procedures described for using this system are a semi-standardized cloning strategy, sexing and screening of pupae for fluorescent protein markers and embryo fixation.

Anopheles 감비아에서 기능 유전학을 위한 GAL4-UAS 시스템을 이용하십시오

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' ...

Site-Directed &#966;C31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the genome of Anopheles mosquitoes. Amongst these methods, the &#966;C31 system allows precise and stable site-directed integration of transgenes, or the substitution of integrated transgenic cassettes via recombinase-mediated cassette exchange (RMCE). This method relies on the action of the Streptomyces &#966;C31 bacteriophage integrase to catalyze recombination between two specific attachment sites designated attP (derived from the phage) and attB (derived from the host bacterium). The system uses one or two attP sites that have been integrated previously into the mosquito genome and attB site(s) in the donor template DNA. Here we illustrate how to stably modify the genome of attP-bearing Anopheles docking lines using two plasmids: an attB-tagged donor carrying the integration or exchange template and a helper plasmid encoding the &#966;C31 integrase. We report two representative results of &#966;C31-mediated site-directed modification: the single integration of a transgenic cassette in An. stephensi and RMCE in An. gambiae mosquitoes. &#966;C31-mediated genome manipulation offers the advantage of reproducible transgene expression from validated, fitness neutral genomic sites, allowing comparative qualitative and quantitative analyses of phenotypes. The site-directed nature of the integration also substantially simplifies the validation of the single insertion site and the mating scheme to obtain a stable transgenic line. These and other characteristics make the &#966;C31 system an essential component of the genetic toolkit for the transgenic manipulation of malaria mosquitoes and other insect vectors.

The protocol describes how to achieve site-directed modifications in the genome of Anopheles malaria mosquitoes using the &#966;C31 system. Modifications described include both the integration and the exchange of transgenic cassettes in the genome of attP-bearing docking lines.

말라리아의 아노필스 벡터에서 사이트 지향 φC31 중재 통합 및 카세트 교환

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the ...