Name: lentiviral mediated production of transgenic mice: a simple and highly efficient method for direct study of founders
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我们感谢 Magali 和罗兰多梅洛尼对手稿和 iVector 和 Phenoparc ICM 核心的关键阅读分别为慢病毒载体向量生产和动物住房提供技术援助。这项工作得到了 Hospitalo-Universitaire de 神经 Translationnelles 巴黎, IHU-ICM, Investissements d ' 艾文莉 ANR-10-IAIHU-06 的支持。该协会为语言法国练习曲 Diabète 等疾病 Métaboliques (ALFEDIAM) 和联合 JDRF/INSERM 赠款提供资金。

包括动物工作在内的所有程序都获得了道德上的批准, 并得到法国研究和教育部的批准, APAFIS#5094-20 16032916219274 v6 和05311.02 号。ICM 动物设施 PHENOPARC 已被法国农业部认可的认证编号 B75 13 19。总体协议要求在图 1中总结的精确时间范围内执行每个过程。
1. 动物购买和基本化合物的制备
<ol><li>动物购买<ol>
<li>命令25输精管切除术男性 B6CBAF1/JRj 8 周的年龄 (F1 ?...

<ol>
	<li>Gordon, J. W., Scangos, G. A., Plotkin, D. J., Barbosa, J. A., Ruddle, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+mouse+embryos+by+microinjection+of+purified+DNA.">Genetic transformation of mouse embryos by microinjection of purified DNA.</a> Proceedings of the National Academy of Science USA. 77 (12), 7380-7384 (1980).</li><li>Bock, T. A., Orlic, D., Dunbar, C. E., Broxmeyer, H. E., Bodine, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+engraftment+of+human+hematopoietic+cells+in+severe+combined+immunodeficient+(SCID)+mice+carrying+human+cytokine+transgenes.">Improved engraftment of human hematopoietic cells in severe combined immunodeficient (SCID) mice carrying human cytokine transgenes.</a> Journal of Experimental Medicine. 182 (6), 2037-2043 (1995).</li><li>Miyakawa, Y., 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=Establishment+of+human+granulocyte-macrophage+colony+stimulating+factor+producing+transgenic+SCID+mice.">Establishment of human granulocyte-macrophage colony stimulating factor producing transgenic SCID mice.</a> British Journal of Haematology. 95 (3), 437-442 (1996).</li><li>Hirabayashi, 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=A+comparative+study+on+the+integration+of+exogenous+DNA+into+mouse,+rat,+rabbit,+and+pig+genomes.">A comparative study on the integration of exogenous DNA into mouse, rat, rabbit, and pig genomes.</a> Experimental Animals. 50 (2), 125-131 (2001).</li><li>Isola, L. M., Gordon, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+animals:+a+new+era+in+developmental+biology+and+medicine.">Transgenic animals: a new era in developmental biology and medicine.</a> Biotechnology. 16, 3-20 (1991).</li><li>Zufferey, R., Nagy, D., Mandel, R. J., Naldini, L., Trono, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiply+attenuated+lentiviral+vector+achieves+efficient+gene+delivery+in+vivo.">Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo.</a> Nature Biotechnology. 15 (9), 871-875 (1997).</li><li>Lois, C., Hong, E. J., Pease, S., Brown, E. J., 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=Germline+transmission+and+tissue-specific+expression+of+transgenes+delivered+by+lentiviral+vectors.">Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors.</a> Science. 295 (5556), 868-872 (2002).</li><li>Ewerling, S., 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=Evaluation+of+laser-assisted+lentiviral+transgenesis+in+bovine.">Evaluation of laser-assisted lentiviral transgenesis in bovine.</a> Transgenic Research. 15 (4), 447-454 (2006).</li><li>Ritchie, W. A., Neil, C., King, T., Whitelaw, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+embryos+and+mice+produced+from+low+titre+lentiviral+vectors.">Transgenic embryos and mice produced from low titre lentiviral vectors.</a> Transgenic Research. 16 (5), 661-664 (2007).</li><li>Park, F. <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:+are+they+the+future+of+animal+transgenesis.">Lentiviral vectors: are they the future of animal transgenesis.</a> Physiological Genomics. 31 (2), 159-173 (2007).</li><li>van Arensbergen, 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+distal+intergenic+region+controls+pancreatic+endocrine+differentiation+by+acting+as+a+transcriptional+enhancer+and+as+a+polycomb+response+element.">A distal intergenic region controls pancreatic endocrine differentiation by acting as a transcriptional enhancer and as a polycomb response element.</a> PLoS One. 12 (2), e0171508 (2017).</li><li>Friedli, 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=A+systematic+enhancer+screen+using+lentivector+transgenesis+identifies+conserved+and+non-conserved+functional+elements+at+the+Olig1+and+Olig2+locus.">A systematic enhancer screen using lentivector transgenesis identifies conserved and non-conserved functional elements at the Olig1 and Olig2 locus.</a> PLoS One. 5 (12), e15741 (2010).</li><li>Sauvain, M. O., 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=Genotypic+features+of+lentivirus+transgenic+mice.">Genotypic features of lentivirus transgenic mice.</a> Journal of Virology. 82 (14), 7111-7119 (2008).</li><li>Punzon, I., Criado, L. M., Serrano, A., Serrano, F., Bernad, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+lentiviral-mediated+human+cytokine+transgenesis+on+the+NOD/scid+background.">Highly efficient lentiviral-mediated human cytokine transgenesis on the NOD/scid background.</a> Blood. 103 (2), 580-582 (2004).</li><li>Zennou, V., 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=The+HIV-1+DNA+flap+stimulates+HIV+vector-mediated+cell+transduction+in+the+brain.">The HIV-1 DNA flap stimulates HIV vector-mediated cell transduction in the brain.</a> Nature Biotechnology. 19 (5), 446-450 (2001).</li><li>Miyoshi, H., Blomer, U., Takahashi, M., Gage, F. 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=Development+of+a+self-inactivating+lentivirus+vector.">Development of a self-inactivating lentivirus vector.</a> Journal of Virology. 72 (10), 8150-8157 (1998).</li><li>Yee, J. 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=A+general+method+for+the+generation+of+high-titer,+pantropic+retroviral+vectors:+highly+efficient+infection+of+primary+hepatocytes.">A general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes.</a> Proceedings of the National Academy of Science USA. 91 (20), 9564-9568 (1994).</li><li>Castaing, 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=Efficient+restricted+gene+expression+in+beta+cells+by+lentivirus-mediated+gene+transfer+into+pancreatic+stem/progenitor+cells.">Efficient restricted gene expression in beta cells by lentivirus-mediated gene transfer into pancreatic stem/progenitor cells.</a> Diabetologia. 48 (4), 709-719 (2005).</li><li>Gradwohl, G., Dierich, A., LeMeur, M., Guillemot, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=neurogenin3+is+required+for+the+development+of+the+four+endocrine+cell+lineages+of+the+pancreas.">neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas.</a> Proceedings of the National Academy of Science USA. 97 (4), 1607-1611 (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> Proceedings of the National Academy of Science USA. 88 (11), 4626-4630 (1991).</li><li>Norrman, 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=Quantitative+comparison+of+constitutive+promoters+in+human+ES+cells.">Quantitative comparison of constitutive promoters in human ES cells.</a> PLoS One. 5 (8), e12413 (2010).</li><li>Vandegraaff, N., Kumar, R., Burrell, C. J., Li, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+human+immunodeficiency+virus+type+1+(HIV)+DNA+integration+in+acutely+infected+cells+as+determined+using+a+novel+assay+for+detection+of+integrated+HIV+DNA.">Kinetics of human immunodeficiency virus type 1 (HIV) DNA integration in acutely infected cells as determined using a novel assay for detection of integrated HIV DNA.</a> Journal of Virology. 75 (22), 11253-11260 (2001).</li><li>Ohtsuka, 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=One-step+generation+of+multiple+transgenic+mouse+lines+using+an+improved+Pronuclear+Injection-based+Targeted+Transgenesis+(i-PITT).">One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT).</a> BMC Genomics. 16, 274 (2015).</li><li>Tasic, B., 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=Site-specific+integrase-mediated+transgenesis+in+mice+via+pronuclear+injection.">Site-specific integrase-mediated transgenesis in mice via pronuclear injection.</a> Proceedings of the National Academy of Science USA. 108 (19), 7902-7907 (2011).</li><li>Sumiyama, K., Kawakami, K., Yagita, K. <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+highly+efficient+transgenesis+method+in+mice+with+the+Tol2+transposon+system+and+cytoplasmic+microinjection.">A simple and highly efficient transgenesis method in mice with the Tol2 transposon system and cytoplasmic microinjection.</a> Genomics. 95 (5), 306-311 (2010).</li><li>Garrels, 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=Cytoplasmic+injection+of+murine+zygotes+with+Sleeping+Beauty+transposon+plasmids+and+minicircles+results+in+the+efficient+generation+of+germline+transgenic+mice.">Cytoplasmic injection of murine zygotes with Sleeping Beauty transposon plasmids and minicircles results in the efficient generation of germline transgenic mice.</a> Biotechnology Journal. 11 (1), 178-184 (2016).</li><li>Ding, S., 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=Efficient+transposition+of+the+piggyBac+(PB)+transposon+in+mammalian+cells+and+mice.">Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice.</a> Cell. 122 (3), 473-483 (2005).</li><li>Aida, T., 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=Cloning-free+CRISPR/Cas+system+facilitates+functional+cassette+knock-in+in+mice.">Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice.</a> Genome Biology. 16, (2015).</li><li>Philippe, S., 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=Lentiviral+vectors+with+a+defective+integrase+allow+efficient+and+sustained+transgene+expression+in+vitro+and+in+vivo.">Lentiviral vectors with a defective integrase allow efficient and sustained transgene expression in vitro and in vivo.</a> Proceedings of the National Academy of Science USA. 103 (47), 17684-17689 (2006).</li></ol>

在这里描述的受精卵母细胞中, 慢病毒载体载体的 perivitelline 注射导致了转基因胚胎的产生, 而相对于采集到的胚胎总数, 转基因胚胎的产量超过了70%。这一结果与以前的报告一致, 体现了程序2、7、10、11、12的特殊性。在比较表 1中显示的所有数据时, 可以突出显?...

戈登等在1980年的开创性工作表明, 在 pseudopregnant 小鼠植入后, 将质粒 DNA 注入受精卵母细胞的雄性pronuclei , 可以产生转基因动物的生产, 整合了质粒 DNA1。转基因哺乳动物的形成对全球生命科学产生了巨大的影响, 为基础科学和转化生物医学科学开辟了新的研究领域。在过去的四年里, DNA 微注射已经成为一种常规的做法。虽然已经生产了大量的转基因小鼠, 但标准方法对所有的老鼠菌株都没有充分的用处, 需要耗费时间 backcrosses2,3。它对其他物种的应用仍然具有挑战性4 , 转基因综合产量仅限于出生动物的少数比例5。此外, 转基因整合的功效是解释显微 DNA 注射液整体产量差的限制因素。在这方面, 集成的病毒载体是最有效的工具, 以货物和集成转基因, 从而可以提供新的手段, 以显著提高整合产量, 唯一的限制是, 转基因大小不能超过 10 kb6.
慢病毒载体载体伪类型与囊性口炎病毒 (VSV) 包络蛋白是以泛热带和高度整合的基因转移工具, 可用于传感器受精卵母细胞7。卵母细胞周围的透明透明是一种天然的病毒屏障, 需要通过它来允许慢病毒载体载体的转导。转基因动物是由传感受精卵母细胞在微钻孔或清除透明透明8,9产生的。然而, 在 perivitelline 空间的透明透明下注射似乎是最简单的方法, 传感器的受精卵, 最初描述的露易丝和同事7。
perivitelline 注射慢病毒载体载体, 可使转基因动物的产量高出70% 以上的出生动物。这种产量超过10倍以上的最佳产量, 可以达到使用标准pronuclei DNA 注射7,10,11。在这种情况下, 单一的注射疗程将产生至少50个转基因创始人 (F0)。因此, 大量的创始人是兼容分型的转基因效果直接执行对 F0 小鼠不需要产生转基因鼠标线。这种优势允许快速筛选的转基因效果, 并特别适应于执行在体内增益和丧失功能研究在几周之内。此外, 调控 DNA 元素也可以快速筛选, 以绘制促进剂和 DNA 图案绑定的转录因子11,12。随着显微注射, 转基因通常集成为多个副本在一个独特的轨迹。使用慢病毒载体向量, 集成在多个位点中作为每个轨迹10、13的单个拷贝出现。因此, 综合基因座的多样性很可能与转基因创始人所观察到的极高表达显性有关, 这使得新生成的模型更加健壮。
重要的是, 当使用显微注射 DNA 时, pronuclei过程中的可视化是绝对必要的。这一技术限制防止了受精卵母细胞的使用来自于大量的小鼠菌株。因此, 在特定菌株中生产一种pronuclei是不可见的转基因模型, 需要在允许的应变下生产动物, 随后至少有10个连续 backcrosses 在所需的小鼠中转移转基因。应变。通过慢病毒载体矢量注射, perivitelline 空间始终可见, 注射不需要高度特异的技能。以14的病毒载体注射液为例, 对不适合pronuclei注射液的转基因小鼠进行了点头/免疫治疗。
在这里, 提出了一个全面的协议, 以允许简单生产的转基因小鼠使用慢病毒载体载体注射在 perivitelline 空间的一个细胞阶段胚胎。本文详细介绍了用无处不在或细胞特异促进剂控制的转基因表达。
pTrip ΔU3 慢病毒载体骨干在本研究中使用15。这个向量允许产生复制缺陷的慢病毒载体向量, 其中 U3 序列被部分删除以移除 U3 启动子活动并生成自失活向量 (SIN)16。慢病毒载体载体是通过对 HEK-293T 细胞的瞬态转染与 p8.91 封装质粒 (ΔVpr ΔVif ΔVpu ΔNef)6, pHCMV g 编码的水泡性口炎病毒 (VSV) 糖蛋白-g17, pTRIP ΔU3重组载体。详细的生产过程作为补充方法提供。
在生物安全二级条件下 (BSL-2) 进行高效价慢病毒载体载体的生产。对于大多数转基因来说, 这是正确的, 除了必须在 BSL-3 中产生的癌基因。因此, 大多数情况下, 生产 BSL-2 条件是足够的。此外, 大多数国家管理机构处理转基因生物 (转基因) 的使用和生产通常是脱节的。有限数量的复制无能的罪慢病毒载体载体 (低于2µg 的 p24 衣壳蛋白) 可在 BSL-1 条件下使用, 如法国转基因机构与欧洲联盟的建议所述。

<table border="0" cellpadding="0" cellspacing="0" width="642">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">PMSG 50UI</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:107px;">G4527</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">hCG 5000UI</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:107px;">CG5-1VL</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">NaCl</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:107px;">7982</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">100 mm petri dish</td>
			<td style="width:143px;">Dutsher</td>
			<td style="width:107px;">353003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">4 wells Nunc dish</td>
			<td style="width:143px;">Dutsher</td>
			<td style="width:107px;">56469</td>
			<td>IVF dish</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">M2 medium</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:107px;">M7167</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">M16 medium</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:107px;">M7292</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">0,22 &#181;m Syringe filter</td>
			<td style="width:143px;">Dutsher</td>
			<td style="width:107px;">146611</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Hyaluronidase Enzyme 30mg</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:107px;">H4272</td>
			<td>mouse embryo tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin serynge</td>
			<td>VWR</td>
			<td>613-3867</td>
			<td>Terumo Myjector</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Curved forceps</td>
			<td style="width:143px;">Moria</td>
			<td style="width:107px;">2183</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Curved scissors</td>
			<td style="width:143px;">Moria</td>
			<td style="width:107px;">MC26</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:229px;">Aspirator tube assemblies for calibrated microcapillary pipettes</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:107px;">A5177-5EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Borosilicate glass capillaries</td>
			<td style="width:143px;">Harvard apparatus</td>
			<td style="width:107px;">GC 100-10</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Horizontal micropipette puller</td>
			<td style="width:143px;">Narishige</td>
			<td style="width:107px;">PN-30</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Microforge</td>
			<td style="width:143px;">Narishige</td>
			<td style="width:107px;">MF-900</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Inverted microscope</td>
			<td style="width:143px;">Nikon</td>
			<td style="width:107px;">Transferman NK2 5188</td>
			<td style="width:164px;">Hoffman modulation contrast illumination is required</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Micromanipulator</td>
			<td style="width:143px;">Eppendorf</td>
			<td style="width:107px;">Celltram air</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Controler of holding pipet</td>
			<td style="width:143px;">Eppendorf</td>
			<td style="width:107px;">Femtojet</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Mineral oil</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:107px;">M8410</td>
			<td>mouse embryo tested</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:229px;">Microinjector</td>
			<td style="width:143px;">Eppendorf</td>
			<td style="width:107px;">Femtojet</td>
			<td style="width:164px;">Can be used to inject DNA or viral vectors</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Dumont # 5 forceps</td>
			<td style="width:143px;">Moria</td>
			<td style="width:107px;">MC 40</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">vannas micro scissors</td>
			<td style="width:143px;">Moria</td>
			<td style="width:107px;">9600</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isoflurane</td>
			<td>centravet</td>
			<td>ISO005</td>
			<td>ISO-VET 100% 250ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">ocrygel</td>
			<td style="width:143px;">centravet</td>
			<td style="width:107px;">OCR002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Povidone iodure</td>
			<td style="width:143px;">centravet</td>
			<td style="width:107px;">VET001</td>
			<td>vetedine 120ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buprenorphine</td>
			<td>centravet</td>
			<td>BUP002</td>
			<td>Buprecare 0,3Mg/ml 10ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris-HCl</td>
			<td>Sigma</td>
			<td>T5941</td>
			<td>Trizma hydrochloride</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SDS</td>
			<td>Sigma</td>
			<td>436143</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>powder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">proteinase K</td>
			<td>Sigma</td>
			<td>P2308&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">oneTaq kit</td>
			<td>NEB</td>
			<td>M0480L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Primers</td>
			<td>Eurogentec</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Strip of 8 PCR tube</td>
			<td>4titude</td>
			<td>4ti-0781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well thermal cycler</td>
			<td>Applied Biosystems</td>
			<td>4375786</td>
			<td>Veriti</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Genomic DNA mini kit</td>
			<td>invitrogen</td>
			<td>K1820-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nanodrop 2000</td>
			<td>Thermo Scientific</td>
			<td>ND-2000C&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">qPCR Master mix</td>
			<td>Roche</td>
			<td>4887352001</td>
			<td>SYBR Green&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Multiwell plate 384</td>
			<td>Roche</td>
			<td>5217555001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">qPCR instrument 384 well</td>
			<td>Roche</td>
			<td>5015243001</td>
			<td>LightCycler 480</td>
		</tr>
	</tbody>
</table>

lentiviral mediated production of transgenic mice: a simple and highly efficient method for direct study of founders

近40年来, 显微 DNA 注射液代表了转基因随机积分法生成转基因小鼠的标准方法。这种常规程序在世界范围内得到了广泛的应用, 其主要局限性在于转基因整合效果较差, 导致了创始人动物的低产量。在注射受精卵母细胞植入后出生的动物中, 只有很少一部分能整合转基因。相比之下, 慢病毒载体载体是整合基因转移的有力工具, 它们用于传感器受精的卵母细胞能够高效地生产出具有70% 以上平均产量的创始人转基因小鼠。此外, 任何小鼠菌株都可用于生产转基因动物, 显性的转基因表达极高, 高于 80%, 慢病毒载体介导转基因与 DNA 显微注射相比。慢病毒载体向量可以是货物的 DNA 片段的大小限制在 10 kb, 代表了该方法的主要局限性。在受精卵母细胞的透明透明下, 使用简单而容易执行的注射程序, 可以在单次显微注射中生产超过50种创建者动物。这种方法是高度适应的, 直接在创始人动物, 快速增益和丧失功能研究或筛选基因组 DNA 区域的能力, 以控制和调节基因表达在体内。

转基因动物是使用这里提出的协议生成的。具有代表性的结果表明, 无处不在和细胞类型的特定转基因表达。
转基因的本构表达
无处不在的发起人是基本的研究工具, 以持续和有效的方式表达转基因。这种促进剂被用于从体外细胞转染到小动物体内转基因...

Watch this Scientific Journal Video about Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders at JoVE.com

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

在这里, 我们提出了一个协议, 以促进转基因基因的整合和生产的创始人转基因小鼠的高效能, 通过简单的注射慢病毒载体载体在 perivitelline 空间的受精卵母细胞。

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

这种方法允许在小鼠体内快速筛选，对转基因的影响，并特别适应进行功能增益或损失的研究，以及答案和DNA动机图谱。该技术的主要优点是转基因动物的预测使用超过70%的新生儿，可以应用于任何小鼠菌株，包括转基因动物。对老鼠实施安乐死后，收集卵巢。要开始手术，使用剪刀将一个大的水平切口放入腹腔。然后，找到子宫和卵巢之间的卵巢。接下来，使用弯曲的钳子去除带有突出血管的肌膜和膜。然后，将卵巢与卵巢分离，从卵巢中解剖卵巢。然后，拖着卵巢，切离子宫。在室温下将所有收集的卵巢转移到M2介质。处理卵巢时，注意避免接触装满受精卵的肿胀的安布拉。接下来，在10厘米的培养皿中加入100微升的海龙酶工作溶液。然后将两个卵巢转移到其中，从卵子中去除积细胞。在立体显微镜下，使用一对胰岛素注射器固定卵巢，同时撕裂安培和分散受精卵。然后，使用准备好的玻璃移液器与管道和喉舌吸气所有的鸡蛋。使用此工具，将它们转移到六滴M2介质之间，以洗掉周围的积液细胞。然后，将洗净的鸡蛋转移到一个10毫米的井中，放在一个四井板中，里面装满了M16的预热。然后，将菜转移到加湿的37摄氏度的孵化器。在制备过程中，将制备的扁病毒载体悬浮液在160克下离心两分钟，以对通常存在于扁病毒库存中的碎片进行颗粒化。在第二类安全柜中，将超级钠转移到新的0.5毫升管中。接下来，将一微升的上流液装入喷射移液器中。然后，将喷射移液器连接到右侧微调节器。并将保持移液器连接到左侧微操纵器。现在，在凹陷滑梯的中心分配八个微升的M2介质。并覆盖轻副鳍油，以避免蒸发。然后，将20个鸡蛋放入下降的浅处，而不产生气泡。接下来，确保注射移液器的尖端是打开的。如果没有，用保持移液器轻点喷射移液器以打开它。然后，将微注射器设置为 20 秒的注射。现在，使用保持移液器和手动压力吸气的受精卵，其中包含两个前核和两个极性体。然后，使用注射移液器注射卵子的环线空间。不要触摸等离子膜。调整压力，使边缘空间充满溶液。压力不应超过600个 hecto pascals。注射20个卵子后，立即将批次转移到预热M16介质的浴缸中。在植入它们之前，先孵育至少30分钟。将正常雌性与男性进行输精管切除术后 16 小时，检查有没有交配塞。使用这些女性进行植入。接下来，制作内径约150微米的植入移液器。和火焰抛光尖端，约四到五毫米长。将带胚胎测试的移液器加载到肩点上方。然后，吸出一个小气泡，然后用 M2 介质进一步加载移液器。接下来，吸进第二个气泡。现在，将胚胎放入另一个后面的移液器中，最大限度地减少胚胎之间的介质体积。完成，通过吸一小滴副鱼油只有一个胚胎宽。重新植入移液器的制备是关键，需要大量的实践。移液器中的鸡蛋必须与它们之间几乎没有介质对齐。接下来，麻醉后，鼠标和确认麻醉与一个头趾捏剃掉两个，两厘米的头发条沿脊髓的最后一根肋骨的水平。现在，将鼠标放在无菌场地的加热垫上，然后披上它，为裸露的皮肤留下一扇窗户。接下来，用交替的碘酮和70%乙醇的交替磨砂来消毒暴露的皮肤。对于植入，首先做一个一厘米的横向切口，然后横向滑动皮肤，直到橙色的卵巢通过身体壁可见。接下来，用细剪刀做一个五毫米的切口，用创伤斗牛犬夹拿起脂肪垫。一旦垫被抓住，拉出卵巢，卵巢和子宫的顶部。接下来，找到安图拉，并在将卵巢与安培的卵巢连接的卵巢段上与凡纳斯剪刀进行下切。然后，引入移植胚胎移液器并弹出卵子。鸡蛋释放后，在第一个气泡停止。准备好所需材料，在第二次风管上重复该过程。完成后，用伤口夹合上皮肤。关闭皮肤后，立即注射镇痛剂，并放置动物在恢复室与39摄氏度的加热元素。监控，直到它完全清醒。转基因动物是用介绍的方法产生的。使用2.3公斤基增强剂为神经根-3驱动eGFP，生产了高滴度扁病毒载体。或者作为一个控制，染色体六的片段来驱动eGFP。进入胚胎是相当成功的。从胚胎，胰腺芽被分段和免疫污，以寻找eGFP的局部表达。超过90%的载体，用神经原宁-3增强剂，在神经g3表达细胞中表达eGFP。然而，在胰腺芽中，没有一个综合控制载体表示eGFP。利用CDX2基因作为对照，通过定量PCR对转基因整合位点进行评估。使用 Neurogenin-3 构造的集成站点的平均数量约为 20 个，使用控制构造的集成站点数约为 10 个。奇怪的是，用于神经基因-3构造的病毒转导滴度，是控制载体的两倍。建议转导滴度与集成数之间的关系。看完这段视频后，你应该对如何利用高疗效的扁病毒载体的近维泰林注射产生大量创始人转基因胚胎有了很好的了解。一旦掌握，这项技术可以产生多达50个创始人动物在一天内的微注射，如果它执行得当。按照这个程序，其他方法，如滴度亲核DNA注射可以执行，以建立转基因小鼠线。不要忘记，使用扁病毒病媒需要获得您当地的转基因委员会的授权。这些病媒可能非常危险，执行此过程时应始终采取预防措施。

Research

JoVE Journal

Genetics

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

野生类型阻止PCR结合直接测序作为低频体细胞突变的检测的高灵敏度的方法

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging ...

科研

遗传学

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

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

真菌快速缺失生产<em&gt;农杆菌</em&gt;介入OSCAR删除构造的转换

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

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

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

整合酶缺陷慢病毒载体载体的制备及其在细胞 CRISPR/Cas9-mediated 基因敲除中的表达

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 ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

斑马斑马模型系统中 CRISPR/Cas9-generated 基因挖空的高效生产与鉴定

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Production and Purification of Baculovirus for Gene Therapy Application

杆状病毒基因治疗应用的生产与纯化

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel

一种快速的银染色协议, 能够使用非变性聚丙烯酰胺凝胶对 SSR 标记进行简单而有效的检测

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

crispr 介导的人类真菌病原体白色念珠菌基因组编辑

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...