Name: production of replication-defective retrovirus by transient transfection of 293t cells
Uploaded: 2007-12-04T04:22:00
Description: Watch this Scientific Journal Video about Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells at JoVE.com

<ol><li>改造，板3 - 5X10 6细胞/ 6厘米的组织cultureplate 293T细胞前的晚上。 注：我们使用易于转染和efficacyas病毒产生细胞的293T细胞 。 这些细胞是缺乏包装病毒的，除非引入一个辅助质粒。 </li><li>第一天上午，轻轻取出介质替换为4毫升293T细胞MED含有氯喹25毫米。培养箱中培养1小时。 注：板应在80％左右融合。 </li><li>同时，病毒混合2板准备的?...

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	<li>DuBridge, R. B., Tang, P., Hsia, H. C., Leong, P. M., Miller, J. H., Calos, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mutation+in+human+cells+by+using+an+Epstein-Barr+virus+shuttle+system.">Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system.</a> Mol Cell Biol. 7, 379-387 (1987).</li><li>Cherry, S. R., Biniszkiewicz, D., van Parijs, L., Baltimore, D., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+expression+in+embryonic+stem+cells+and+hematopoietic+stem+cells.">Retroviral expression in embryonic stem cells and hematopoietic stem cells.</a> Mol Cell Biol. 20, 7419-7426 (2000).</li><li>Finer, M. H., Dull, T. J., Qin, L., Farson, D., Roberts, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=kat:+A+high-efficiency+retroviral+transduction+system+for+primary+human+T+lymphocytes.">kat: A high-efficiency retroviral transduction system for primary human T lymphocytes.</a> Blood. 83, 43-50 (1994).</li><li>Naviaux, R. K., Costanzi, E., Haas, M., 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=The+pCL+vector+system:+rapid+production+of+helper-free,+high-titer,+recombinant+retroviruses.">The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses.</a> J Virol. 70, 5701-5705 (1996).</li><li>Pear, W. S., Miller, J. P., Xu, L., Pui, J. C., Soffer, B., Quackenbush, R. C., Pendergast, A. M., Bronson, R., Aster, J. C., Scott, M. L., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+rapid+induction+of+a+chronic+myelogenous+leukemia-like+myeloproliferative+disease+in+mice+receiving+P210+bcr/abl-transduced+bone+marrow.">Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow.</a> Blood. 92, 3780-3792 (1998).</li><li>Pear, W. S., Nolan, G. P., Scott, M. L., Baltimore, D. <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+high-titer+helper-free+retroviruses+by+transient+transfection.">Production of high-titer helper-free retroviruses by transient transfection.</a> Proc Natl Acad Sci USA. 90, 8392-8396 (1993).</li><li>Stocking, C., Kollek, R., Bergholz, U., Ostertag, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+terminal+repeat+sequences+impart+hematopoietic+transformation+properties+to+the+myeloproliferative+sarcoma+virus.">Long terminal repeat sequences impart hematopoietic transformation properties to the myeloproliferative sarcoma virus.</a> Proc Natl Acad Sci USA. 82, 5746-5750 (1985).</li><li>Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+chronic+myeloid+leukemia.">Models of chronic myeloid leukemia.</a> Curr Oncol Rep. 3, 228-237 (2001).</li><li>Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+the+pathogenesis+of+BCR-ABL++leukemia+in+mice.">Studying the pathogenesis of BCR-ABL+ leukemia in mice.</a> Oncogene. 21, 8643-8651 (2002).</li></ol>

四个关键点，将保证一个良好的病毒股票的成功： <ol><li>生产的293T细胞很健康，这意味着他们已经非常有规律的时间表上的分裂，从来没有杂草丛生，并在组织培养板，每6厘米的3.5-5万的优化密度镀，使他们达到一个密度80％的板转染上午。 </li><li>此外氯喹，提高转染效率和随后的病毒滴度，约3 - 5倍，稳定细胞lysozomes和增加到达细胞核内的DNA的一小部分。丁酸钠（另一种溶酶体稳定）也被用于这一...

在转染所使用的所有试剂必须是无菌的过滤（ 氯化钙 ，2个哈佛商学院，H 2 O miliQ），并保持无菌。

production of replication-defective retrovirus by transient transfection of 293t cells

我们的实验室研究人类Bcr - Abl的或生长因子受体产生的致癌基因（ZNF198 - FGFR1的，BCR -PDGFRα等）等癌基因诱导骨髓增生性疾病。我们能够在我们的小鼠模型，以模型和研究一个与人类类似的的疾病，以前与利益表达的原癌基因的逆转录病毒感染的骨髓细胞移植。复制缺陷的逆转录病毒编码的人类癌基因和标记（GFP，RFP，耐抗生素基因等）是产生一个瞬时转染293T细胞，腺病毒E1A基因产品转化一个人的肾上皮细胞系的协议。 293细胞有高度磷酸三钙（投诉警察课4）转染不同寻常的特性，容易达到50-80％的转染效率。在这里，我们共同利益和质粒表达GAG - POL - ENV包装的功能，如包装构造吉或PCL，在这种情况下的单基因质粒EcoPak，的原癌基因表达逆转录病毒载体转染293细胞。最初的转染，进一步提高使用氯喹。 ecotropic病毒的股票，收集培养上清48小时。转染后，可以储存在-80 ° C和用于感染细胞系，改造和体外研究，或小鼠骨髓细胞如原代细胞，在我们的小鼠模型，然后可以用于移植。

Watch this Scientific Journal Video about Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells at JoVE.com

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

这种技术演示了一个有效的方式来准备复制缺陷型逆转录病毒编码的人类癌基因的股票，随后诱导骨髓增殖性疾病的小鼠模型中使用。

瞬时转染293T细胞复制缺陷型逆转录病毒生产

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

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