Name: removal of an internal translational start site from mrna while retaining expression of the full-length protein
Uploaded: 2022-03-16T13:30:00
Description: Watch this Scientific Journal Video about Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein at JoVE.com

该项目得到了美国国立卫生研究院（R01HL152691，R01HL138577和R01HL159983）对RMS的支持。

“所有动物护理和研究方案都得到了Cedars-Sinai医疗中心和犹他大学的机构动物护理和使用委员会的批准。在8-9周龄时从商业来源获得的C57BL / 6J雌性小鼠（参见 材料表）用于实验。
1. 基因靶向的准备
<ol><li>使用CRISPR网络算法4，9，10 选择编码M213的靶向突变位点周围的引导靶标序?...

<ol>
	<li>Smyth, J. W., Shaw, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoregulation+of+connexin43+gap+junction+formation+by+internally+translated+isoforms.">Autoregulation of connexin43 gap junction formation by internally translated isoforms.</a> Cell Reports. 5 (3), 611-618 (2013).</li><li>Basheer, W. A., 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=GJA1-20k+arranges+actin+to+guide+Cx43+delivery+to+cardiac+intercalated+discs.">GJA1-20k arranges actin to guide Cx43 delivery to cardiac intercalated discs.</a> Circulation Research. 121 (9), 1069-1080 (2017).</li><li>Fu, 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=Cx43+isoform+GJA1-20k+promotes+microtubule+dependent+mitochondrial+transport.">Cx43 isoform GJA1-20k promotes microtubule dependent mitochondrial transport.</a> Frontiers in Physiology. 8, 905 (2017).</li><li>Xiao, 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=Auxiliary+trafficking+subunit+GJA1-20k+protects+connexin-43+from+degradation+and+limits+ventricular+arrhythmias.">Auxiliary trafficking subunit GJA1-20k protects connexin-43 from degradation and limits ventricular arrhythmias.</a> Journal of Clinical Investigation. 130 (9), 4858-4870 (2020).</li><li>Syvanen, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+gels+to+chips:+&amp;#34;minisequencing&amp;#34;+primer+extension+for+analysis+of+point+mutations+and+single+nucleotide+polymorphisms.">From gels to chips: &amp;#34;minisequencing&amp;#34; primer extension for analysis of point mutations and single nucleotide polymorphisms.</a> Human Mutation. 13 (1), 1-10 (1999).</li><li>Han, 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=Genome-wide+SNP+discovery+in+tetraploid+alfalfa+using+454+sequencing+and+high+resolution+melting+analysis.">Genome-wide SNP discovery in tetraploid alfalfa using 454 sequencing and high resolution melting analysis.</a> BMC Genomics. 12, 1-11 (2011).</li><li>Han, Y., Khu, D. M., Monteros, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+melting+analysis+for+SNP+genotyping+and+mapping+in+tetraploid+alfalfa+(Medicago+sativa+L.).">High-resolution melting analysis for SNP genotyping and mapping in tetraploid alfalfa (Medicago sativa L.).</a> Molecular Breeding. 29 (2), 489-501 (2012).</li><li>Peng, B. 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=A+novel+and+quick+PCR-based+method+to+genotype+mice+with+a+leptin+receptor+mutation+(db/db+mice).">A novel and quick PCR-based method to genotype mice with a leptin receptor mutation (db/db mice).</a> Acta Pharmacologica Sinica. 39 (1), 117-123 (2018).</li><li>Haeussler, 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=Evaluation+of+off-target+and+on-target+scoring+algorithms+and+integration+into+the+guide+RNA+selection+tool+CRISPOR.">Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR.</a> Genome Biology. 17 (1), 148 (2016).</li><li>Concordet, J. P., Haeussler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPOR:+Intuitive+guide+selection+for+CRISPR/Cas9+genome+editing+experiments+and+screens.">CRISPOR: Intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens.</a> Nucleic Acids Research. 46 (1), 242-245 (2018).</li><li>Hsu, P. D., 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=DNA+targeting+specificity+of+RNA-guided+Cas9+nucleases.">DNA targeting specificity of RNA-guided Cas9 nucleases.</a> Nature Biotechnology. 31 (9), 827-832 (2013).</li><li>Jinek, 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+programmable+dual-RNA-guided+DNA+endonuclease+in+adaptive+bacterial+immunity.">A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.</a> Science. 337 (6096), 816-821 (2012).</li><li>Paquet, D., 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+introduction+of+specific+homozygous+and+heterozygous+mutations+using+CRISPR/Cas9.">Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9.</a> Nature. 533 (7601), 125-129 (2016).</li><li>Behringer, R., Gertsenstein, M., Nagy, K., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: A Laboratory Manual. , (2014).</li><li>Pomp, D., Critser, E. S., Rutledge, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+sodium+lactate+in+Whitten's+medium+improves+in+vitro+developmental+capacity+of+one-cell+mouse+embryos.">Lower sodium lactate in Whitten's medium improves in vitro developmental capacity of one-cell mouse embryos.</a> Theriogenology. 29 (5), 1019-1025 (1988).</li><li>Summers, M. C., McGinnis, L. K., Lawitts, J. A., Raffin, M., Biggers, 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=IVF+of+mouse+ova+in+a+simplex+optimized+medium+supplemented+with+amino+acids.">IVF of mouse ova in a simplex optimized medium supplemented with amino acids.</a> Human Reproduction. 15 (8), 1791-1801 (2000).</li><li>Green, M. R. S. <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: A Laboratory Manual (Fourth Edition). , (2012).</li><li>Koh, J. Y., Iwabuchi, S., Huang, Z., Harata, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+genotyping+of+animals+followed+by+establishing+primary+cultures+of+brain+neurons.">Rapid genotyping of animals followed by establishing primary cultures of brain neurons.</a> Journal of Visualized Experiments. 95. (95), e51879 (2015).</li><li>Iyer, 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=No+unexpected+CRISPR-Cas9+off-target+activity+revealed+by+trio+sequencing+of+gene-edited+mice.">No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice.</a> PLoS Genetics. 14 (7), 1007503 (2018).</li><li>Nature Medicine. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keep+off-target+effects+in+focus.">Keep off-target effects in focus.</a> Nature Medicine. 24 (8), 1081 (2018).</li><li>Mayer, H. <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+EcoRI-activity+of+EcoRI+endonuclease.">Optimization of the EcoRI-activity of EcoRI endonuclease.</a> FEBS Letters. 90 (2), 341-344 (1978).</li><li>Kozak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point+mutations+close+to+the+AUG+initiator+codon+affect+the+efficiency+of+translation+of+rat+preproinsulin+in+vivo.">Point mutations close to the AUG initiator codon affect the efficiency of translation of rat preproinsulin in vivo.</a> Nature. 308 (5956), 241-246 (1984).</li><li>Kozak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point+mutations+define+a+sequence+flanking+the+AUG+initiator+codon+that+modulates+translation+by+eukaryotic+ribosomes.">Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes.</a> Cell. 44 (2), 283-292 (1986).</li><li>Basheer, W. A., 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=Stress+response+protein+GJA1-20k+promotes+mitochondrial+biogenesis,+metabolic+quiescence,+and+cardioprotection+against+ischemia/reperfusion+injury.">Stress response protein GJA1-20k promotes mitochondrial biogenesis, metabolic quiescence, and cardioprotection against ischemia/reperfusion injury.</a> JCI Insight. 3 (20), 121900 (2018).</li><li>Shimura, D., 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=Protective+mitochondrial+fission+induced+by+stress-responsive+protein+GJA1-20k.">Protective mitochondrial fission induced by stress-responsive protein GJA1-20k.</a> Elife. 10, 69207 (2021).</li></ol>

基因修饰小鼠模型是了解基因功能的常用方法。然而，由于GJA1-20k内部翻译的同种型是从与全长Cx43相同的 Gja1 mRNA翻译而来的，因此设计了一种创造性的策略来保留全长Cx43表达，同时抑制GJA1-20k表达。该方法基于GJA1-20k内部起始密码子的突变。通过单点突变，M213在 Gja1 mRNA上切换为L，成功抑制GJA1-20k表达，但保留了全长Cx43表达4。
然而，单个内部?...

全长连接蛋白43（Cx43）和N-末端截断亚型GJA1-20k，由相同的GJA1 mRNA编码，但利用不同的起始密码子1来启动翻译。Cx43翻译发生在第一个AUG起始密码子，而GJA1-20k翻译发生在残基213的AUG开始。先前发现GJA1-20k对全身Cx43运输，肌动蛋白稳定和体外线粒体形态调节1，2，3具有重要作用。
为了了解GJA1-20k 在体内的作用，生成了GJA1-20k“敲除”小鼠模型，该模型保留了创建全长Cx43的能力。方法是使用CRISPR-Cas9系统将213的单个残基从蛋氨酸（M）替换为亮氨酸（L）（Gja1M213L / M213L）4。内部M至L突变大大降低了发生内部翻译的可能性，但保留了全长蛋白4的翻译和功能。由于野生型（WT）和突变等位基因具有相同的大小和几乎相同的mRNA产物，因此在小鼠中确认基因型存在相当大的困难。DNA测序可以识别突变，但对于常规使用来说太昂贵和耗时。通常，已经建立了几种更快的基因分型方法，例如实时聚合酶链反应（RT-PCR），微调序列连接，高分辨率熔解分析和四引物扩增难治性突变系统PCR（ARMS-PCR）5，6，7，8。然而，这些替代方法需要多个步骤，独特的资源和/或几个特定的引物集，可以诱导非特异性PCR产物。
该协议通过CRISPR-Cas9引入了详细的基因靶向和编辑方法，以创建单个氨基酸突变，并提出快速基因分型以确认突变。基因型鉴定涉及创造性地使用限制性内切酶，仅使用一组引物来鉴定靶基因。读者参考参考文献4 ，以观察由一个残基Gja1M213L / M213L 取代突变引起的心源性猝死的深刻电生理效应，该突变仍然产生全长蛋白质，但未能产生较小的内部翻译截断亚型。该方案将有助于使其他小鼠模型使用点突变来降低目标亚型的内部翻译，同时保留内源性全长蛋白的表达。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 mL 8-Strip PCR tube</td>
			<td>Thomas Scientific</td>
			<td>1148A28</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA pH 8.0</td>
			<td>Invitrogen</td>
			<td>15575-038</td>
			<td>For pronuclear micorinjection buffer</td>
		</tr>
		<tr>
			<td>10x CutSmart buffer</td>
			<td>New England Biolabs</td>
			<td>B7204S</td>
			<td>Digestion buffer for NlaIII</td>
		</tr>
		<tr>
			<td>1 M Tris-HCl pH 7.5</td>
			<td>Invitrogen</td>
			<td>15567-027</td>
			<td>For pronuclear micorinjection buffer</td>
		</tr>
		<tr>
			<td>50x TAE Buffer</td>
			<td>Invitrogen</td>
			<td>24710-030</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Thermal cycler</td>
			<td>Applied Biosystems</td>
			<td>Model # 9902</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J</td>
			<td>The Jackson Laboratory</td>
			<td>664</td>
			<td>mouse strain</td>
		</tr>
		<tr>
			<td>Chemidoc MP imaging system</td>
			<td>Bio-rad</td>
			<td></td>
			<td>To take gel image by 302 nm UV light</td>
		</tr>
		<tr>
			<td>eSpCas9 protein</td>
			<td>MilliporeSigma</td>
			<td>ESPCAS9PRO-50UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Lading Dye Purple (6x)</td>
			<td>New England Biolabs</td>
			<td>B7024S</td>
			<td>Loading buffer for electrophoresis</td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>hCG (human chorionic gonadotropin)</td>
			<td>MilliporeSigma</td>
			<td>23-073-425G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>MilliporeSigma</td>
			<td>H3884-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Lab software</td>
			<td>Bio-rad</td>
			<td></td>
			<td>To analyze gel image</td>
		</tr>
		<tr>
			<td>Inverted stereo microscope whit plasDIC</td>
			<td>Zeiss</td>
			<td>Axiovert A1</td>
			<td></td>
		</tr>
		<tr>
			<td>KAPA Mouse Genotyping Kits</td>
			<td>Roche Diagnostics</td>
			<td>KK7352</td>
			<td>For tissue or cell lysis, DNA extraction, and PCR master mix</td>
		</tr>
		<tr>
			<td>KSOM</td>
			<td>LifeGlobal</td>
			<td>ZEKS-050</td>
			<td>embryo culture medium</td>
		</tr>
		<tr>
			<td>Lab coart</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M2 medium</td>
			<td>MilliporeSigma</td>
			<td>MR015D</td>
			<td></td>
		</tr>
		<tr>
			<td>NlaIII</td>
			<td>New England Biolabs</td>
			<td>R0125S</td>
			<td>To digest PCR product</td>
		</tr>
		<tr>
			<td>Nuclease-Free Water</td>
			<td>Ambion</td>
			<td>AM9937</td>
			<td>To dilute reagents</td>
		</tr>
		<tr>
			<td>Paraffin oil</td>
			<td>Nacalai USA</td>
			<td>2613785</td>
			<td></td>
		</tr>
		<tr>
			<td>Plugged 20 &#956;L fine pipette tip</td>
			<td>FisherScientific</td>
			<td>02-707-171</td>
			<td>To pick up blastocytes</td>
		</tr>
		<tr>
			<td>PMSG (pregnant mare&#8217;s serum gonadotropin)</td>
			<td>ProSpec-Tany</td>
			<td>HOR-272</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Invitrogen</td>
			<td>AM9760G</td>
			<td>For pronuclear micorinjection buffer</td>
		</tr>
		<tr>
			<td>SYBR safe DNA Gel Stain</td>
			<td>Invitrogen</td>
			<td>S33102</td>
			<td>To detect bands in gel</td>
		</tr>
		<tr>
			<td>tracrRNA</td>
			<td>Dharmacon</td>
			<td>U-002000-120</td>
			<td>Guid RNA</td>
		</tr>
	</tbody>
</table>

removal of an internal translational start site from mrna while retaining expression of the full-length protein

CRISPR-Cas9基因编辑系统基于基因组修复机制，与传统的同源重组相比，可以更快，更轻松地生成基因修饰的小鼠模型。CRISPR-Cas9系统在需要单点突变时特别有吸引力。间隙连接蛋白Connexin 43（Cx43）由基因Gja1编码，该基因具有单个编码外显子并且无法剪接。然而，Gja1不仅产生全长Cx43蛋白，而且通过称为内部翻译的过程产生多达六个N-末端截断亚型，这是内部AUG（蛋氨酸）起始位点核糖体翻译启动的结果。GJA1-20k是Cx43最常生成的截断亚型，在Gja1 mRNA的213位置的AUG密码子处引发。由于残基213发生在Cx43的最后一个跨膜结构域的末端，因此GJA1-20k实际上是Cx43的20 kDa C末端尾部，作为独立的蛋白质。先前的研究者在细胞中发现，GJA1-20k的关键作用是促进全长Cx43间隙连接半透明体到质膜的运输。为了 在体内检查这种现象，产生了具有Gja1点突变的突变小鼠，该突变用TTA（亮氨酸，M213L突变）替换残基213处的ATG（蛋氨酸）。M213L的结果是Gja1 mRNA和全长Cx43仍然产生，但Gja1-20k的翻译显着降低。本报告重点介绍如何选择限制性内切酶位点来开发一种氨基酸突变（Gja1M213L/M213L）小鼠模型。该协议描述了通过CRISPR-Cas9系统进行的转基因小鼠，以及通过结合PCR和限制性内切酶治疗进行快速基因分型。

CRISPR/ Cas9基因编辑系统在小鼠10号染色体上分别产生ATG至TTA突变和56，264，279至56，264，281和56，264，291至56，264，293的ATG至TCG突变和TCG的沉默TCC突变，或在Gja1 mRNA上分别产生869至871和881至883的TCC至TCG突变。这些突变导致GJA1蛋白上的蛋氨酸213到亮氨酸（M213L）突变，并且TTC到TTG突变破坏了附近的PAM序列以避免不需要的基因版本（图1）。突变的细节在上一份报告4</su...

Watch this Scientific Journal Video about Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein at JoVE.com

Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein

本方案描述了Gja1中的单个M213L突变，该突变保留了全长Connexin43生成，但阻止了较小的GJA1-20k内部翻译亚型的翻译。

从mRNA中去除内部翻译起始位点，同时保留全长蛋白的表达

The CRISPR-Cas9 gene-editing system, based on genome repair mechanisms, enables the generation of gene-modified mouse models more quickly and easily ...

单个残基取代的困难在于，尽管单个残基存在差异，但要有效地对动物进行基因分型。限制性内切酶的特殊使用仅为典型的基于PCR的基因分型增加了单一、快速和低成本的步骤。如果目标序列被限制性内切酶识别，则该协议可以应用于任何其他具有突变点的小鼠模型，通常情况就是这样。用干净的剪刀切一到三毫米的尾尖，并将其转移到0.2毫升八条PCR管中。按照手稿中的描述包含尾部样品后，将其储存在T-minus 20摄氏度直至提取，最多约一周。每管加入100微升组织裂解液并充分混合，然后使用台式小型离心机在室温下以2， 200倍G旋转10秒。确保尾部样品浸没在溶液中。通过对组织裂解、灭活和保温的参数进行编程，将试管设置到热循环仪上。如果不能立即进行PCR，请将组织裂解物在四摄氏度下储存长达一周。制备PCR溶液，其中含有5微升无核酸酶水，0.75微升10微摩尔正向和反向引物，以及每个样品7.5微升PCR预混液到新的PCR管中。将一微升先前制备的组织裂解物加入PCR溶液中。充分混合PCR溶液，并在室温下用台式微型离心机以2， 200倍G离心10秒。将试管安装到编程的热循环仪上。反应完成后，将PCR产物在4摄氏度下储存一至两个月或在零下20摄氏度下储存约一年。制备含有七微升无核酸酶水，两微升10倍强度的CutSmart缓冲液和一微升NlaIII限制性内切酶的酶溶液。如果有几个样品，将每种含量相乘以制成混合物，每管等分10微升。每管向酶溶液中加入10微升先前获得的PCR产物。充分混合并使用台式小型离心机旋转，如前所述。将管子设置在编程的热循环仪或加热块上。在寒冷条件下孵育后储存产品。在50毫升单强度TAE缓冲液中加入0.75克琼脂糖。在微波炉中混合并加热琼脂糖，直到其完全溶解。冷却后，加入五微升DNA染色剂，轻轻混合。将25毫升琼脂糖凝胶倒入凝胶模具中，使凝胶固化。将10微升消化的PCR产物加载到孔中。用100伏特电泳凝胶35分钟。在紫外光下对琼脂糖凝胶进行成像。使用消化的PCR产物进行的琼脂糖电泳在每个基因型中产生了几个不同大小的条带，其中两条条带分别为野生型，三条为杂合子条带，一条为纯合条带。使用囊胚分析进行测试注射后，每微升供体寡核苷酸注射50纳克的成功率为76.9%，同一供体寡核苷酸每微升注射25纳克的成功率为25%。最后，每微升供体寡核苷酸注入50纳克乙醇沉淀，得到成功率为50%的创始动物。通过乙醇沉淀纯化寡核苷酸供体降低了毒性，同时保持了较高的基因组编辑效率。小心处理和添加少量试剂非常重要。确保正确添加并混合良好。

Research

JoVE Journal

Biology

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

科研

生物学

Homemade Site Directed Mutagenesis of Whole Plasmids

自制的网站定向诱变整个质粒

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

GST-他的净化：一个两步亲和纯化协议屈服全长纯化的蛋白质

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Identification of Post-translational Modifications of Plant Protein Complexes

植物蛋白复合体的翻译后修饰的鉴定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

在体外合成在人体细胞修饰蛋白表达的mRNA的感应

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...