Name: preparation of raav9 to overexpress or knockdown genes in mouse hearts
Uploaded: 2016-12-17T13:00:00
Description: Watch this Scientific Journal Video about Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts at JoVE.com

We thank Dr. Zaffar Haque for careful reading of the manuscript. We thank Drs. Masaharu Kataoka and Gengze Wu for discussions and help. Work in the Wang lab is supported by the American Heart Association, Muscular Dystrophy Association, and NIH (HL085635, HL116919, HL125925).

모든 기술 단계는 바이오 안전성위원회와 보스턴 어린이 병원의 기관 동물 관리 및 사용위원회에 의해 승인 된 프로토콜에 따라 수행 하였다. 보스턴 어린이 병원은 규제 빛 / 어둠주기와 온도 조절과 무균 마우스 시설을 갖추고 있습니다. 수의학 및 동물 관리 직원의 변화 케이지는 마우스의 상태를 확인합니다. 시설은 AAALAC 인증을하고 활동적인 동물 복지 보증 인증 (AAALAC의 인증이 1992년 2월 24?...

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	<li>Primrose, S. B., Twyman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of gene manipulation and genomics. , (2013).</li><li>Doudna, J. A., Charpentier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+frontier+of+genome+engineering+with+CRISPR-Cas9.">The new frontier of genome engineering with CRISPR-Cas9.</a> Science. 346, 1258096 (2014).</li><li>Gaj, T., Gersbach, C. A., Barbas, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZFN,+TALEN,+and+CRISPR/Cas-based+methods+for+genome+engineering.">ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering.</a> Trends Biotechnol. 31, 397-405 (2013).</li><li>Hsu, P. D., Lander, E. S., Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+applications+of+CRISPR-Cas9+for+genome+engineering.">Development and applications of CRISPR-Cas9 for genome engineering.</a> Cell. 157, 1262-1278 (2014).</li><li>Sander, J. D., Joung, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas+systems+for+editing,+regulating+and+targeting+genomes.">CRISPR-Cas systems for editing, regulating and targeting genomes.</a> Nat. Biotechnol. 32, 347-355 (2014).</li><li>Szulc, J., Wiznerowicz, M., Sauvain, M. -. O., Trono, D., Aebischer, 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+versatile+tool+for+conditional+gene+expression+and+knockdown.">A versatile tool for conditional gene expression and knockdown.</a> Nat. Methods. 3, 109-116 (2006).</li><li>Nimesh, S., Halappanavar, S., Kaushik, N. K., Kumar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+Gene+Delivery+Systems.">Advances in Gene Delivery Systems.</a> BioMed Res. Int. 2015, 610342 (2015).</li><li>Kamimura, K., Suda, T., Zhang, G., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+gene+delivery+systems.">Advances in gene delivery systems.</a> Pharm. Med. 25, 293-306 (2011).</li><li>Thomas, C. E., Ehrhardt, A., Kay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+problems+with+the+use+of+viral+vectors+for+gene+therapy.">Progress and problems with the use of viral vectors for gene therapy.</a> Nat. Rev. Genet. 4, 346-358 (2003).</li><li>Giacca, M., Zacchigna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-mediated+gene+delivery+for+human+gene+therapy.">Virus-mediated gene delivery for human gene therapy.</a> J. Control Release. 161, 377-388 (2012).</li><li>Witlox, M., Lamfers, M., Wuisman, P., Curiel, D., Siegal, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolving+gene+therapy+approaches+for+osteosarcoma+using+viral+vectors:+review.">Evolving gene therapy approaches for osteosarcoma using viral vectors: review.</a> Bone. 40, 797-812 (2007).</li><li>De Miguel, M. P., Cheng, L., Holland, E. C., Federspiel, M. J., Donovan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+the+c-Kit+signaling+pathway+in+mouse+primordial+germ+cells+by+retroviral-mediated+gene+transfer.">Dissection of the c-Kit signaling pathway in mouse primordial germ cells by retroviral-mediated gene transfer.</a> Proc. Natl. Acad. Sci. USA. 99, 10458-10463 (2002).</li><li>Nagano, M., Shinohara, T., Avarbock, M. R., Brinster, 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=Retrovirus-mediated+gene+delivery+into+male+germ+line+stem+cells.">Retrovirus-mediated gene delivery into male germ line stem cells.</a> FEBS Lett. 475, 7-10 (2000).</li><li>Scharfmann, R., Axelrod, J. H., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+expression+of+retrovirus-mediated+gene+transfer+in+mouse+fibroblast+implants.">Long-term in vivo expression of retrovirus-mediated gene transfer in mouse fibroblast implants.</a> Proc. Natl. Acad. Sci. USA. 88, 4626-4630 (1991).</li><li>Katz, R. A., Greger, J. G., Skalka, 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=Effects+of+cell+cycle+status+on+early+events+in+retroviral+replication.">Effects of cell cycle status on early events in retroviral replication.</a> J. Cell. Biochem. 94, 880-889 (2005).</li><li>Escors, D., Breckpot, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vectors+in+gene+therapy:+their+current+status+and+future+potential.">Lentiviral vectors in gene therapy: their current status and future potential.</a> Arch. Immunol. Ther. Exp. 58, 107-119 (2010).</li><li>M&#225;trai, J., Chuah, M. K., VandenDriessche, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+lentiviral+vector+development+and+applications.">Recent advances in lentiviral vector development and applications.</a> Mol. Ther. 18, 477-490 (2010).</li><li>Miyazaki, Y., Miyake, A., Nomaguchi, M., Adachi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+dynamics+of+retroviral+genome+and+the+packaging.">Structural dynamics of retroviral genome and the packaging.</a> Front. Microbiol. 2, 1-9 (2011).</li><li>Douglas, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-Mediated+Gene+Delivery.">Adenovirus-Mediated Gene Delivery.</a> Gene Delivery to Mammalian Cells: Volume 2: Viral Gene Transfer Techniques. , 3-14 (2004).</li><li>Armend&#225;riz-Borunda, 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=Production+of+first+generation+adenoviral+vectors+for+preclinical+protocols:+amplification,+purification+and+functional+titration.">Production of first generation adenoviral vectors for preclinical protocols: amplification, purification and functional titration.</a> J. Biosci. Bioeng. 112, 415-421 (2011).</li><li>Snyder, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus-mediated+gene+delivery.">Adeno-associated virus-mediated gene delivery.</a> J Gene Med. 1, 166-175 (1999).</li><li>Samulski, R. J., Muzyczka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV-mediated+gene+therapy+for+research+and+therapeutic+purposes.">AAV-mediated gene therapy for research and therapeutic purposes.</a> Annu. Rev. Virol. 1, 427-451 (2014).</li><li>Kaplitt, M. G., 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=Long-term+gene+transfer+in+porcine+myocardium+after+coronary+infusion+of+an+adeno-associated+virus+vector.">Long-term gene transfer in porcine myocardium after coronary infusion of an adeno-associated virus vector.</a> Ann. Thorac. Surg. 62, 1669-1676 (1996).</li><li>Kaspar, B. 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=Myocardial+gene+transfer+and+long-term+expression+following+intracoronary+delivery+of+adeno-associated+virus.">Myocardial gene transfer and long-term expression following intracoronary delivery of adeno-associated virus.</a> J. Gene. Med. 7, 316-324 (2005).</li><li>Ding, 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=Trbp+regulates+heart+function+through+microRNA-mediated+Sox6+repression.">Trbp regulates heart function through microRNA-mediated Sox6 repression.</a> Nat. Genet. 47, 776-783 (2015).</li><li>Lin, 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=Cardiac-specific+YAP+activation+improves+cardiac+function+and+survival+in+an+experimental+murine+MI+model.">Cardiac-specific YAP activation improves cardiac function and survival in an experimental murine MI model.</a> Circ. Res. 115, 354-363 (2014).</li><li>Wahlquist, 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=Inhibition+of+miR-25+improves+cardiac+contractility+in+the+failing+heart.">Inhibition of miR-25 improves cardiac contractility in the failing heart.</a> Nature. 508, 531-535 (2014).</li><li>Carroll, K. 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=A+mouse+model+for+adult+cardiac-specific+gene+deletion+with+CRISPR/Cas9.">A mouse model for adult cardiac-specific gene deletion with CRISPR/Cas9.</a> Proc. Natl. Acad. Sci. USA. 113, 338-343 (2016).</li><li>Jiang, J., Wakimoto, H., Seidman, J., Seidman, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allele-specific+silencing+of+mutant+Myh6+transcripts+in+mice+suppresses+hypertrophic+cardiomyopathy.">Allele-specific silencing of mutant Myh6 transcripts in mice suppresses hypertrophic cardiomyopathy.</a> Science. 342, 111-114 (2013).</li><li>Gibson, D. G., 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=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nat. Methods. 6, 343-345 (2009).</li><li>Rychlik, W., Spencer, W., Rhoads, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+annealing+temperature+for+DNA+amplification+in+vitro.">Optimization of the annealing temperature for DNA amplification in vitro.</a> Nucleic Acids Res. 18, 6409-6412 (1990).</li><li>Allocca, 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=Serotype-dependent+packaging+of+large+genes+in+adeno-associated+viral+vectors+results+in+effective+gene+delivery+in+mice.">Serotype-dependent packaging of large genes in adeno-associated viral vectors results in effective gene delivery in mice.</a> J. Clin. Invest. 118, 1955-1964 (2008).</li><li>Wu, Z., Yang, H., Colosi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+genome+size+on+AAV+vector+packaging.">Effect of genome size on AAV vector packaging.</a> Mol. Ther. 18, 80-86 (2010).</li><li>Li, J., Sun, W., Wang, B., Xiao, X., Liu, X. -. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+trans-splicing+as+a+means+for+viral+vector-mediated+in+vivo+gene+therapy.">Protein trans-splicing as a means for viral vector-mediated in vivo gene therapy.</a> Hum. Gene Ther. 19, 958-964 (2008).</li><li>Piras, B. A., O'Connor, D. M., French, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+delivery+of+shRNA+by+AAV9+provides+highly+efficient+knockdown+of+ubiquitously+expressed+GFP+in+mouse+heart,+but+not+liver.">Systemic delivery of shRNA by AAV9 provides highly efficient knockdown of ubiquitously expressed GFP in mouse heart, but not liver.</a> PLoS One. 8, e75894 (2013).</li><li>Lovric, 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=Terminal+differentiation+of+cardiac+and+skeletal+myocytes+induces+permissivity+to+AAV+transduction+by+relieving+inhibition+imposed+by+DNA+damage+response+proteins.">Terminal differentiation of cardiac and skeletal myocytes induces permissivity to AAV transduction by relieving inhibition imposed by DNA damage response proteins.</a> Mol. Ther. 20, 2087-2097 (2012).</li></ol>

이 플라스미드를 구조 중에 원하지 ITR 재결합을 최소화하는 것이 중요하다. 바이러스를 생성하기 전에, 하나는 항상 제한 분해 및 아가 로스 겔 전기 영동을 사용하여 플라스미드 AAV ITR의 무결성을 모니터링한다. 이는 100 % 그대로 플라스미드를 획득하는 것은 불가능하지만, 재결합 율은 가급적 최소화되어야한다. 20 % 미만에 성공 rAAV9 포장 허용됩니다. 참고로, 저급 진탕 속도 (RPM 180-200)와 하부 ?...

A correction was made to the Authors section in: <a href="http://www.jove.com/video/54787">Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts</a>.
One of the authors names and affiliation was corrected from:
Jian-Ming Jiang3,4 
3 Department of Genetics, Harvard Medical School 
4 Howard Hughes Medical Institute
to:
Jianming Jiang5 
5 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore

A correction was made to the Authors section in: <a href="http://www.jove.com/video/54787">Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts</a>.

Erratum: Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

다양한 생물학적 시스템에서 특정 유전자의 발현 또는 활성을 제어하는 유전자 기능 (1)의 연구에 귀중한 전략이되고있다. 이러한 목적을 달성하는 직접적인 방법은 뉴클레오티드 서열을 조작하여 돌연변이 대립 유전자를 생성한다. 살아있는 세포의 게놈에 정확한 타겟 변경하는 것은있을지라도 a는 시간과 노동 집약적 인 연습, 강력한 TALEN 및 Crispr / Cas9 도구의 개발은 게놈 편집 2-5의 새로운 시대를 열었습니다. 유전자 조작보다 일상적인 실험 방법은 유전 물질 (DNA를 코딩 서열 또는 siRNA를 / shRNA를 포함하는 RNA를) 명시 적 또는 관심 1,6의 유전자 (들)를 넉다운 할 수있는 세포로을의 도입에 초점을 맞추고있다. 많은 경우에, 유전자 조작을위한 주요 병목 세포 내로 DNA, RNA 또는 단백질의 전달이다. 시험관 연구와 관련, 효율적인 transfecti로시스템에 많은 배양 세포 라인에 설립되었습니다. 그러나, 특히 마우스 모델에서 생체 내 유전자 전달은 더 도전이다. 외인성 시약의 효율적인 세포 흡수를 달성하기 위하여 바이 패스 될 필요 역외 세포 내 장벽의 시리즈가있다. 추가 장애물은 신속한 통관 및 전달 물질의 7,8의 짧은 기간을 포함한다. 이러한 문제를 회피하기위한 한 가지 전략은 생체 내 유전자 전달에 대한 &quot;캐리어&quot;또는 &quot;차량&quot;으로 바이러스 벡터를 사용하는 것이다. 바이러스의 자연적 진화 전달 특성은 세포 7,9,10에 관심있는 유전자의 효율적인 전달을 할 수 있습니다. 바이러스 벡터의 많은 종류가 개발 마우스에서 다른 유형의 세포 및 기관의 생체 내 유전자 조작 유연한 사용되었다. 가장 일반적으로 사용되는 바이러스 시스템은 레트로 바이러스, 렌티 바이러스, 아데노 바이러스 및 아데노 - 관련 바이러스 (AAV)를 포함 <s&gt; 11입니다. 레트로 바이러스는 단일 가닥 RNA 바이러스이며 표적 세포 및 기관 12-14에서 형질 도입 유전자의 평생 발현의 가능성을 제공하고, 유사 분열 분할 중에 안정한 방식으로 숙주 세포의 게놈에 유전 물질을 도입 할 수있다. 그러나, 레트로 바이러스의 여러 유형의 세포만을 분할 감염, 비 - 분할 세포의 효능 15 매우 낮다. 이 유전자 전달을 위해 자신의 유틸리티를 제한합니다. 렌티 바이러스는 Retroviridae 가족의 속입니다. 다른 레트로 바이러스와는 달리, 렌티 바이러스 모두 분할 및 비 - 분할 세포를 감염시킬 수있는 널리 포스트 유사 분열 및 고도로 분화 된 세포 (16)에 유전자 전달을 위해 사용되었다. 렌티 바이러스의 생활주기는 숙주 게놈 내로 벡터 DNA의 통합을 포함한다. 따라서,의 Lentivirus이 매개 유전자 전달은 형질 유전 적 요소 16-18의 안정적이고 오래 지속 표현을 할 수 있습니다. 그러나이 기능은 이중 전자를 나타낼 수있다벡터 DNA의 통합 삽입 성 돌연변이를 초래할 수있다 이러한 바이러스의 사용 dged 검, 유전자 발현을 조작 할 및 숙주 세포에 artefactual 효과를 일으킬 수 있습니다. 아데노 다른 널리 사용되는 유전자 전달 시스템이다. 레트로 바이러스 및 렌티 바이러스와는 달리, 아데노 바이러스는 비 통합하고 숙주 세포의 게놈 8,10,11,19 무결성을 방해하지 않는다. 또한, 아데노 바이러스는 많은 종류의 세포로 DNA를 형질 감염 할 수 있으며, 감염 활성 세포 분열 (19)에 의존하지 않다. 바이러스 벡터의 능력 (19, 20)를 복제 할 필요 같이 아데노 바이러스의 또 다른 중요한 특징은, 벡터 정제의 용이성이다. 그러나, 이러한 시스템의 주요주의 할 점은 아데노 바이러스 감염, 특히 유전자 치료 연구에서, 여러 연구에서의 사용을 제한하는 대상 세포 및 기관 (19)의 강한 면역 반응을 유발할 수 있다는 것이다. 서로 다른 유형에 비해바이러스 벡터들, 재조합 아데노 - 관련 바이러스 (rAAV)는 이상적인 유전자 전달 시스템 (21, 22)로 나타난다. 그것은 최소한의 면역 원성 및 병원성 (23, 24)을 나타낸다. 또한 rAAV는 핵공 포함한 세포 유형의 넓은 범위를 감염시킨다. 대부분의 경우, rAAV가 숙주 게놈에 통합되지 않는다; 따라서, 표적 세포의 바람직하지 않은 유전자 또는 게놈 변화의 위험이 낮은 (22)이다. 최근 rAAV 시스템 성공적 쥐 심근 23,25-29 DNA로 인코딩 단백질의 miRNAs, shRNA를하고 Crispr-gRNAs의 생체 내 전달을 위해 사용되었다. 이 방법론은 심혈관 연구 분야의 기초 연구 및 유전자 치료 연구를 촉진했다. 여기서, 자세한 절차는 효율적으로 기술되었다 과발현 또는 마우스 마음에 대한 관심의 유전자를 녹다운 rAAV9 벡터를 생성한다. 프로토콜은 간단하고 효과적인 방법을 제공한다실험 쥐 모델에서 심장 유전자 발현을 조작.

<table border="0" cellpadding="0" cellspacing="0" width="1175">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Polyethylenimine, Linear (MW 25,000)</td>
			<td style="width:241px;">Polysciences, Inc.&#160;</td>
			<td style="width:192px;">#23966-2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Tube, Polypropylene, 36.2 mL, 25 x 87 mm, (qty. 56)</td>
			<td style="width:241px;">Beckman Coulter, Inc</td>
			<td style="width:192px;"># 362183</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Nuclease, ultrapure</td>
			<td style="width:241px;">SIGMA</td>
			<td style="width:192px;">#E8263-25KU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Density Gradient Medium(Iodixanol)</td>
			<td style="width:241px;">SIGMA</td>
			<td style="width:192px;">#D1556-250ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Centrifugal Filter Unit with Ultracel-100 membrane</td>
			<td style="width:241px;">EMD Millipore Corporation</td>
			<td style="width:192px;">#UFC910008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Laboratory pipetting needle with 90&#176; blunt ends,gauge 14, L 6 in., nickel plated hub</td>
			<td style="width:241px;">SIGMA</td>
			<td style="width:192px;">#CAD7942-12EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Poloxamer 188 solution (Pluronic&#174; F-68 solution)</td>
			<td style="width:241px;">SIGMA</td>
			<td style="width:192px;">P5556-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Proteinase K</td>
			<td style="width:241px;">SIGMA</td>
			<td style="width:192px;">3115828001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">DNase I</td>
			<td style="width:241px;">Roche</td>
			<td style="width:192px;">10104159001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Centrifuge machine</td>
			<td style="width:241px;">Thermo Scientific</td>
			<td style="width:192px;">75004260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Centrifuge System</td>
			<td style="width:241px;">Beckman Coulter</td>
			<td style="width:192px;">363118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Ultracentrifuge</td>
			<td style="width:241px;">Beckman Coulter</td>
			<td style="width:192px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">DMEM medium</td>
			<td style="width:241px;">Fisher Scientific</td>
			<td style="width:192px;">SH30243FS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Fetal Bovine Serum&#160;</td>
			<td style="width:241px;">Atlanta Biologicals&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:192px;">S11150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">rAAV9 vector</td>
			<td style="width:241px;">Penn Vector Core</td>
			<td style="width:192px;">P1967</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of raav9 to overexpress or knockdown genes in mouse hearts

뮤린 모델에서 유전 물질의 전달을 통해 심근 발현 또는 특정 유전자의 활성을 조절하는 유전자 기능의 조사를 허용한다. 마음에 그들의 치료 가능성도 결정될 수있다. 마우스 마음에 생체 분자의 개입에 대한 제한된 접근 방법이있다. 재조합 아데노 관련 바이러스 (rAAV) 기반 게놈 엔지니어링은 생체 내 심장 유전자 조작을위한 필수적인 도구로 활용되고있다. 이 기술의 구체적인 장점은 최소한의 높은 효율, 특이성, 낮은 게놈 통합 레이트를 포함 면역 원성, 최소한의 병원성. 여기서, rAAV9 벡터를 구성하는 패키지와 정화 상세한 절차를 설명한다. 신생아 새끼로 rAAV9의 피하 주사는 간 및 기타 조직에서 강력한 발현 또는 마우스 심장에 대한 관심의 유전자 (들)의 효율적인 최저의 결과가 아니라. 심장-보고 특정 사용C TnnT2 프로모터, 마음에 GFP 유전자의 고 발현을 얻었다. 또한, rAAV9-U6-shRNA를가 이용 될 때의 mRNA가 마음에 억제 대상. rAAV9 기술의 지식을 작업 심장 혈관 연구에 유용 할 수있다.

rAAV9.cTNT : GFP 또는 rAAV9.U6 :: shRNA를 플라스미드 rAAV9 구조에 대한 전략은 각각도 1 및도 2에 나타내었다. 예로서, rAAV9 벡터는 마우스의 마음에 GFP 유전자를 과발현하기 위해 생성 하였다. 그 결과 플라스미드이 ITR 사이트 (그림 1) 측면에 나란히 서고 cTnT의 :: GFP 카세트가 포함되어 있습니다. rAAV9.U6 :: shRNA를 벡터가 Trbp의 mRNA를 넉다운하?...

Watch this Scientific Journal Video about Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts at JoVE.com

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

마우스 마음에서 과발현하는 rAAV9의 제조 또는 최저 유전자

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

The overall goal of this procedure is to prepare recombinant adeno-associated virus nine, or rAAV9, to manipulate gene expression in mouse hearts. The method can answer key question in the field of cardiology and molecular genetics, such as how to study gene function in mouse heart in vivo. The main advantage of this technique is that it will allow us to easily manipulate the gene expression and function in vivo in mouse heart, allowing the functional access of gene function without having to rely on the genetically modified animal models.After generating rAAV9 constructs and culturing HEK293 cells according to the text protocol, to transfect the cells on day one, combine 70 micrograms of AAV Rep-Cap plasmid, 70 micrograms of rAAV9 plasmid, and 200 micrograms of Ad helper plasmid in a 50-milliliter centrifuge tube. Add 49 milliliters of room temperature DMEM without FBS to the tube and mix well. Then, to make a four-to-one PEI to DNA solution, add 1, 360 microliters of PEI to the tube and mix well.Incubate the solution at room temperature for 15 to 30 minutes. After the incubation, add five milliliters of the solution to each 150-milliliter dish of cells. Culture the cells at 37 degrees Celsius and 5%CO2 for 50 to 72 hours.To harvest the transfected cells, dislodge and suspend the cells by using the culture medium to pipette up and down. Then transfer the cell suspensions to sterile 50-milliliter tubes. Centrifuge the cells at 500 times G for five minutes.Then add five milliliters of PBS to each tube and re-suspend the cell pellets before combining all the suspensions in one 50-milliliter tube. After spinning the cells at 500 times G for five minutes, discard the supernatant. To purify the AAV, re-suspend the pellet in 10 milliliter of lysis buffer.Freeze the lysate at minus 80 degrees Celsius or in a dry ice ethanol bath. Then thaw it at 37 degrees Celsius. Vortex the lysate for one minute before repeating the freeze and thaw two more times.Next, add one millimolar magnesium chloride to the thawed lysate. Then add nuclease to a final concentration of 250 units per milliliter. Incubate the solution at 37 degrees Celsius for 15 minutes to dissolve the DNA protein aggregation.Centrifuge the sample at 4, 800 times G and four degrees Celsius for 20 minutes. Then collect the supernatant. Meanwhile, to prepare a 17%iodixanol gradient solution, mix five milliliters of 10X PBS with 0.05 milliliters of one molar magnesium chloride, 0.125 milliliters of one molar potassium chloride, 10 milliliters of five molar sodium chloride, and 12.5 milliliters of density gradient medium.Use water to adjust the total volume to 50 milliliters. After preparing 25, 40, and 60%gradient solutions according to the text protocol, use a needle and syringe to load five milliliters of 17%gradient solution into a polypropylene tube. Follow this with five milliliters each of 25, 40, and 60%gradient solutions.Layer the lysate on top of the gradient. Then fill the tube with lysis buffer and cover it with a cork. Centrifuge the gradient at 185, 000 times G and 16%Celsius for 90 minutes.Following the spin, use a syringe to harvest the 40%viral fraction by inserting a 21 gauge needle into the intersection between the 40 and 60%fractions, completely avoiding the 25%layer. Mix the viral fraction with sterilized polyoxyethylene polyoxypropylene, or PEG-PPG block copolymer PBS solution, up to a total volume of 15 milliliters. Load the mixture into a filter tube with a molecular weight cutoff of 100 kilodaltons.Then centrifuge the sample at 2, 000 times G and four degrees Celsius for 30 minutes. Decant the solution from the bottom of the tube. Then use PEG-PPG to refill the filter tube to 15 milliliters.Centrifuge the tube at 2, 000 times G and four degrees Celsius for 20 minutes. After repeating the refilling and spin two more times, collect the purified rAAV9 virus fraction still above the filter. Aliquot 100 to 400 microliters of the purified rAAV9 into 1.7-milliliter tubes, depending on the viral titer, and store the virus at minus 80 degrees Celsius.After determining the viral titer according to the text protocol, prepare rAAV9 working solutions with virus titers of one to seven times 10 to the 12th particles per milliliter. To treat neonatal mice, use the rAAV9 solution to pre-fill a 0.33 by 12.7-milliliter insulin syringe with a 29 gauge one-half inch needle, avoiding air bubbles. After cryo-anesthetizing a pup, hold the pup in one hand with thumb and forefinger, then swipe the back skin of the pup with a swab stick saturated with 70%isopropyl alcohol.Insert the syringe into the anterior dorsal subcutis of the animal at an angle of five to 10 degrees before injecting 50 to 70 microliters of the rAAV9 solution. The standard curve for rAAV9 titration was generated with the qPCR data by linear regression. The manipulated variable y represents the log10 value of the DNA molecular concentration of each standard sample, and the corresponding variable x represents the Ct value.The log10 concentration values and Ct numbers exhibit a nice linear correlation and fit with the equation shown here. Titers of rAAV9 samples were calculated based on the linear equation. Using the method demonstrated in this video, a high titer of rAAV9 vectors was obtained in this representative study.To monitor the efficiency and tissue specificity of rAAV9 cTNT vectors, P0.5 pups were treated with the same amount of rAAV9 cTNT luciferase or rAAV9 cTNT GFP by subcutaneous injection. Two weeks after injection, robust expression of GFP was detected in the heart but not in other organs. To monitor the knockdown efficiency and tissue specificity of rAAV9 U6 vectors, P0.5 mice were treated with the same amount of rAAV9 U6 scramble or rAAV9 U6 Trbp shRNA by subcutaneous injection.Two weeks after injection, the mRNA level of Trbp in the heart was substantially reduced by rAAV9 U6 shTrbp. Down-regulation of Trbp was also detected to a lesser extent in the liver. Once mastered, rAAV preparation can be done in five days, if it is formulated properly.While attempting this procedure, it is important to remember to use healthy 293 cells for rAAV packaging. After its development, this technique paved the way for researchers in the field of cardiology and genetics to explore gene functions and gene therapeutic potentials in mouse hearts. After watching this video, you should have a good understanding of how to prepare recombinant AAV9 to manipulate the gene expressing any function in mouse hearts.

preparation-of-raav9-to-overexpress-or-knockdown-genes-in-mouse-hearts

Research

JoVE Journal

Genetics

The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little is known about the molecular control that governs their development. This is in part due to physical, resource and time limitations often associated with the study of secondary growth processes. A number of in vitro techniques have been used involving either plant part or whole plant system in both woody and non-woody plant species. However, questions about their applicability for the study of secondary stem growth processes, the recalcitrance of certain species and labor intensity are often prohibitive for medium to high throughput applications. Also, when looking at secondary stem development and wood formation the specific traits under investigation might only become measurable late in a tree&#39;s lifecycle after several years of growth. In addressing these challenges alternative in vivo protocols have been developed, named Induced Somatic Sector Analysis, which involve the creation of transgenic somatic tissue sectors directly in the plant&#39;s secondary stem. The aim of this protocol is to provide an efficient, easy and relatively fast means to create transgenic secondary plant tissue for gene and promoter functional characterization that can be utilized in a range of tree species. Results presented here show that transgenic secondary stem sectors can be created in all live tissues and cell types in secondary stems of a variety of tree species and that wood morphological traits as well as promoter expression patterns in secondary stems can be readily assessed facilitating medium to high throughput functional characterization.

The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

나무 형성 및 보조 스템 개발에 관여하는 유전자 및 발기인 유학을위한 유도 체세포 업종 분석 (ISSA)의 사용

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little ...

연구

유전학

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 ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

양육 및 숨기기 비틀에 RNA 매개 유전자 최저를 두 번 좌초,<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

대상<em&gt; 현장에서</em신진 효모에서&gt; 돌연변이 유발 히스톤의 유전자

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

과도 식의 외국 유전자 단백질 기능 분석 결과 대 한 곤충 세포 (sf9)

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

동시 비디오-뇌 파-ECG 모니터링 Neurocardiac 부전 간 질 마우스 모델에서을 식별 하

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a polymorphic cluster of genes on chromosome 19. Their best-characterized ligands are the human leukocyte antigen (HLA) molecules that are encoded within the major histocompatibility complex (MHC) locus on chromosome 6. There is substantial evidence that they play a significant role in immunity, reproduction, and transplantation, making it crucial to have techniques that can accurately genotype them. However, high-sequence homology, as well as allelic and copy number variation, make it difficult to design methods that can accurately and efficiently genotype all KIR genes. Traditional methods are usually limited in the resolution of data obtained, throughput, cost-effectiveness, and the time taken for setting up and running the experiments. We describe a method called quantitative KIR semi-automated typing (qKAT), which is a high-throughput multiplex real-time polymerase chain reaction method that can determine the gene copy numbers for all genes in the KIR locus. qKAT is a simple high-throughput method that can provide high-resolution KIR copy number data, which can be further used to infer the variations in the structurally polymorphic haplotypes that encompass them. This copy number and haplotype data can be beneficial for studies on large-scale disease associations, population genetics, as well as investigations on expression and functional interactions between KIR and HLA.

Quantitative killer cell immunoglobulin-like receptor (KIR) semi-automated typing (qKAT) is a simple, high-throughput, and cost-effective method to copy number type KIR genes for their application in population and disease association studies.

qKAT: 킬러 세포 같은 면역 글로불린 수용 체 유전자의 양적 반자동 입력

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a ...

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

종양 세포에서 MicroRNA 수준, 기능 및 관련 표적 유전자를 평가하기 위한 시험관 내 프로토콜

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

소독멜라노가스터에서 새로운 신경보호 유전자를 식별하기 위한 생체 전방 유전자 스크린

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, recent evidence indicated that Tau is present also in other subcellular compartments including the nucleus where it is implicated in DNA protection, in rRNA transcription, in the mobility of retrotransposons and in the structural organization of the nucleolus. We have recently demonstrated that nuclear Tau is involved in the expression of the VGluT1 gene, suggesting a molecular mechanism that could explain the pathological increase of glutamate release in the early stages of Alzheimer&#39;s disease. Until recently, the involvement of nuclear Tau in modulating the expression of target genes has been relatively uncertain and ambiguous due to technical limitations that prevented the exclusion of the contribution of cytoplasmic Tau or the effect of other downstream factors not related to nuclear Tau. To overcome this uncertainty, we developed a method to study the expression of target genes specifically modulated by the nuclear Tau protein. We employed a protocol that couples the use of localization signals and the subcellular fractionation, allowing the exclusion of the interference from the cytoplasmic Tau molecules. Most notably, the protocol is easy and is composed of classic and reliable methods that are broadly applicable to study the nuclear function of Tau in other cell types and cellular conditions.

Tau is a neuronal protein present both in the cytoplasm, where it binds microtubules, and in the nucleus, where it exerts unconventional functions including the modulation of Alzheimer&#39;s disease-related genes. Here, we describe a method to investigate the function of nuclear Tau while excluding any interferences coming from cytoplasmic Tau.

질병 관련 유전자의 발현을 조사하는 도구로 타우 세포 국소화의 변조

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, ...