Name: a scalable, cell-based method for the functional assessment of ube3a variants
Uploaded: 2022-10-10T15:00:00
Description: Watch this Scientific Journal Video about A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants at JoVE.com

这项工作得到了西蒙斯基金会独立之桥奖（SFARI奖#387972;J.J.Y.），大脑和行为研究基金会（J.J.Y.）的NARSAD青年研究员奖，阿尔弗雷德·P·斯隆基金会（J.J.Y.）的研究奖学金，以及天使综合症基金会（J.J.Y.），白厅基金会（J.J.Y.）和NIMH（R01MH122786;杰杰）。

1. 诱变克隆生成Ube3a 变体
<ol><li>将所有 Ube3a 变体克隆到pCIG2质粒中（图1A），这是一种双纤载体，其中包含鸡-β-肌动蛋白启动子和用于EGFP表达的内部核糖体进入位点（IRES）19。全长 Ube3a 构建体包含一个N端Myc标签序列，并使用5'SacI位点和3'XmaI位点克隆到pCIG2中。此外， Ube3a 编码序列中天然存在的EcoRI，EcoRV和PstI位点?...

<ol>
	<li>Landrum, M. 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=ClinVar:+Public+archive+of+relationships+among+sequence+variation+and+human+phenotype.">ClinVar: Public archive of relationships among sequence variation and human phenotype.</a> Nucleic Acids Research. 42, 980-985 (2014).</li><li>Lek, 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=Analysis+of+protein-coding+genetic+variation+in+60,706+humans.">Analysis of protein-coding genetic variation in 60,706 humans.</a> Nature. 536 (7616), 285-291 (2016).</li><li>Starita, L. 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=Variant+interpretation:+Functional+assays+to+the+rescue.">Variant interpretation: Functional assays to the rescue.</a> American Journal of Human Genetics. 101 (3), 315-325 (2017).</li><li>Scheffner, M., Huibregtse, J. M., Vierstra, R. D., Howley, P. 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+HPV-16+E6+and+E6-AP+complex+functions+as+a+ubiquitin-protein+ligase+in+the+ubiquitination+of+p53.">The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53.</a> Cell. 75 (3), 495-505 (1993).</li><li>Albrecht, U., 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=Imprinted+expression+of+the+murine+Angelman+syndrome+gene,+Ube3a,+in+hippocampal+and+Purkinje+neurons.">Imprinted expression of the murine Angelman syndrome gene, Ube3a, in hippocampal and Purkinje neurons.</a> Nature Genetics. 17 (1), 75-78 (1997).</li><li>Rougeulle, C., Glatt, H., Lalande, 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+Angelman+syndrome+candidate+gene,+UBE3A/E6-AP,+is+imprinted+in+brain.">The Angelman syndrome candidate gene, UBE3A/E6-AP, is imprinted in brain.</a> Nature Genetics. 17 (1), 14-15 (1997).</li><li>Vu, T. H., Hoffman, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imprinting+of+the+Angelman+syndrome+gene,+UBE3A,+is+restricted+to+brain.">Imprinting of the Angelman syndrome gene, UBE3A, is restricted to brain.</a> Nature Genetics. 17 (1), 12-13 (1997).</li><li>Kishino, T., Lalande, M., Wagstaff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UBE3A/E6-AP+mutations+cause+Angelman+syndrome.">UBE3A/E6-AP mutations cause Angelman syndrome.</a> Nature Genetics. 15 (1), 70-73 (1997).</li><li>Jiang, Y. H., 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=Mutation+of+the+Angelman+ubiquitin+ligase+in+mice+causes+increased+cytoplasmic+p53+and+deficits+of+contextual+learning+and+long-term+potentiation.">Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation.</a> Neuron. 21 (4), 799-811 (1998).</li><li>Mabb, A. M., Judson, M. C., Zylka, M. J., Philpot, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angelman+syndrome:+Insights+into+genomic+imprinting+and+neurodevelopmental+phenotypes.">Angelman syndrome: Insights into genomic imprinting and neurodevelopmental phenotypes.</a> Trends in Neuroscience. 34 (6), 293-303 (2011).</li><li>Hogart, A., Wu, D., LaSalle, J. M., Schanen, 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=The+comorbidity+of+autism+with+the+genomic+disorders+of+chromosome+15q11.2-q13.">The comorbidity of autism with the genomic disorders of chromosome 15q11.2-q13.</a> Neurobiology of Disease. 38 (2), 181-191 (2010).</li><li>Urraca, N., 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+interstitial+duplication+15q11.2-q13+syndrome+includes+autism,+mild+facial+anomalies+and+a+characteristic+EEG+signature.">The interstitial duplication 15q11.2-q13 syndrome includes autism, mild facial anomalies and a characteristic EEG signature.</a> Autism Research. 6 (4), 268-279 (2013).</li><li>de la Torre-Ubieta, L., Won, H., Stein, J. L., Geschwind, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancing+the+understanding+of+autism+disease+mechanisms+through+genetics.">Advancing the understanding of autism disease mechanisms through genetics.</a> Nature Medicine. 22 (4), 345-361 (2016).</li><li>Scheffner, M., Staub, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HECT+E3s+and+human+disease.">HECT E3s and human disease.</a> BMC Biochemistry. 8, (2007).</li><li>Cooper, E. M., Hudson, A. W., Amos, J., Wagstaff, J., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+analysis+of+Angelman+syndrome-associated+mutations+in+the+E3+ubiquitin+ligase+E6-associated+protein.">Biochemical analysis of Angelman syndrome-associated mutations in the E3 ubiquitin ligase E6-associated protein.</a> Journal of Biological Chemistry. 279 (39), 41208-41217 (2004).</li><li>Yi, J. J., Barnes, A. P., Hand, R., Polleux, F., Ehlers, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+signaling+specifies+axons+during+brain+development.">TGF-beta signaling specifies axons during brain development.</a> Cell. 142 (1), 144-157 (2010).</li><li>Yi, J. 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=The+autism-linked+UBE3A+T485A+mutant+E3+ubiquitin+ligase+activates+the+Wnt/beta-catenin+pathway+by+inhibiting+the+proteasome.">The autism-linked UBE3A T485A mutant E3 ubiquitin ligase activates the Wnt/beta-catenin pathway by inhibiting the proteasome.</a> Journal of Biological Chemistry. 292 (30), 12503-12515 (2017).</li><li>Weston, K. P., 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=Identification+of+disease-linked+hyperactivating+mutations+in+UBE3A+through+large-scale+functional+variant+analysis.">Identification of disease-linked hyperactivating mutations in UBE3A through large-scale functional variant analysis.</a> Nature Communications. 12 (1), 6809 (2021).</li><li>Hand, R., Polleux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenin2+regulates+the+initial+axon+guidance+of+cortical+pyramidal+neurons+projecting+medially+to+the+corpus+callosum.">Neurogenin2 regulates the initial axon guidance of cortical pyramidal neurons projecting medially to the corpus callosum.</a> Neural Development. 6, 30 (2011).</li><li>Karginov, A. V., Ding, F., Kota, P., Dokholyan, N. V., Hahn, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+allosteric+activation+of+kinases+in+living+cells.">Engineered allosteric activation of kinases in living cells.</a> Nature Biotechnology. 28 (7), 743-747 (2010).</li><li>Biechele, T. L., Moon, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+beta-catenin/TCF+transcription+with+beta-catenin/TCF+transcription-based+reporter+constructs.">Assaying beta-catenin/TCF transcription with beta-catenin/TCF transcription-based reporter constructs.</a> Methods in Molecular Biology. , 99-110 (2008).</li><li>Yi, J. 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=An+Autism-linked+mutation+disables+phosphorylation+control+of+UBE3A.">An Autism-linked mutation disables phosphorylation control of UBE3A.</a> Cell. 162 (4), 795-807 (2015).</li><li>Kuhnle, 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=Angelman+syndrome-associated+point+mutations+in+the+Zn(2+)-binding+N-terminal+(AZUL)+domain+of+UBE3A+ubiquitin+ligase+inhibit+binding+to+the+proteasome.">Angelman syndrome-associated point mutations in the Zn(2+)-binding N-terminal (AZUL) domain of UBE3A ubiquitin ligase inhibit binding to the proteasome.</a> Journal of Biological Chemistry. 293 (47), 18387-18399 (2018).</li><li>Yamamoto, Y., Huibregtse, J. M., Howley, P. 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+human+E6-AP+gene+(UBE3A)+encodes+three+potential+protein+isoforms+generated+by+differential+splicing.">The human E6-AP gene (UBE3A) encodes three potential protein isoforms generated by differential splicing.</a> Genomics. 41 (2), 263-266 (1997).</li><li>Avagliano Trezza, R., 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=Loss+of+nuclear+UBE3A+causes+electrophysiological+and+behavioral+deficits+in+mice+and+is+associated+with+Angelman+syndrome.">Loss of nuclear UBE3A causes electrophysiological and behavioral deficits in mice and is associated with Angelman syndrome.</a> Nature Neuroscience. 22 (8), 1235-1247 (2019).</li><li>Bossuyt, S. N. 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=Loss+of+nuclear+UBE3A+activity+is+the+predominant+cause+of+Angelman+syndrome+in+individuals+carrying+UBE3A+missense+mutations.">Loss of nuclear UBE3A activity is the predominant cause of Angelman syndrome in individuals carrying UBE3A missense mutations.</a> Human Molecular Genetics. 30 (6), 430-442 (2021).</li></ol>

此处描述的方案提供了一种有效且可扩展的方法来评估Ube3a变体的酶活性。使用该测定时，有几个技术细节值得仔细考虑。一个考虑因素是选择该测定中使用的Wnt报告质粒。这里描述的方案专门使用β-连环蛋白激活报告基因（BAR）21，该报告基因包含由专门设计的接头序列分隔的12个T细胞因子（TCF）响应元件的串联体，以最大限度地减少TCF结合位点的重组和丢失。文献中还...

最近的技术进步使外显子组和基因组的测序成为临床环境中的常规1，2。这导致了大量遗传变异的发现，包括数百万个错义变异，这些变异通常会改变蛋白质中的一个氨基酸。了解这些变异的功能意义仍然是一个挑战，只有一小部分（~2%）已知的错义变异具有临床解释1，3。
这个问题的一个突出例子是Ube3a，这是一种编码E3泛素连接酶的基因，该酶靶向底物蛋白进行降解4。Ube3a 位于染色体 15q11-13 内，仅由母系遗传等位基因5，6，7 表达。疾病遗传学的观察强烈表明，UBE3A酶活性不足或过度会导致明显的神经发育障碍。母体染色体 15q11-13 缺失会导致安格曼综合征 （AS）8，这是一种以严重智力障碍、运动障碍、癫痫发作、快乐举止和频繁微笑和面部畸形特征为特征的疾病8，9，10。相比之下，母体染色体15q11-13的重复或三重会导致Dup15q综合征，这是一种被认为是自闭症最普遍的综合征形式之一的异质性疾病11，12，13。此外，在Ube3a中发现了数百种错义变异，其中大多数被认为是具有不确定意义的变异（VUS），因为它们的功能和临床意义是未知的。因此，人们对开发能够实证评估Ube3a变异以确定它们是否有助于神经发育疾病的方法产生了相当大的兴趣。
UBE3A 酶属于 E3 泛素连接酶的 HECT（与 E6-AP C 末端同源）结构域家族，它们都具有同名的 HECT 结构域，其中包含接受来自 E2 酶的活化泛素并将其转移到底物蛋白所需的生化机制14。从历史上看，E3酶的表征依赖于需要纯化蛋白质集合的体外重建系统4，15，16。这种方法缓慢且费力，不适合评估大量变体的活性。在以前的工作中，UBE3A被鉴定为通过调节蛋白酶体的功能来激活HEK293T细胞中的Wnt途径，以减缓β-连环蛋白17的降解。这种洞察力允许使用 Wnt 通路报告基因作为一种有效和快速的方法来识别 Ube3a18 的功能丧失和获得变体。下面的协议详细描述了一种生成Ube3a变体的方法以及基于荧光素酶的报告基因，用于评估Ube3a变体活性的变化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA (1x), phenol red</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>1 Kb DNA ladder</td>
			<td>Lambda Biotech</td>
			<td>M108-S</td>
			<td></td>
		</tr>
		<tr>
			<td>100 bp DNA Ladder</td>
			<td>Lambda Biotech</td>
			<td>M107</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Buffer for T4 DNA Ligase with 10 mM ATP</td>
			<td>New England BioLabs</td>
			<td>B0202A</td>
			<td></td>
		</tr>
		<tr>
			<td>5x Phusion HF Reaction Buffer</td>
			<td>New England BioLabs</td>
			<td>B0518S</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic Solution</td>
			<td>Corning</td>
			<td>30004CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Black/White Isoplate-96 Black Frame White Well plate</td>
			<td>PerkinElmer</td>
			<td>6005030</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicillin Disodium Salt</td>
			<td>Midwest Scientific</td>
			<td>KCC46000-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess cell counting chamber&#160; slides</td>
			<td>Invitrogen by Thermo Fisher Scientific</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II Automated Cell Counter&#160;</td>
			<td>life technologies</td>
			<td></td>
			<td>Cell counting machine</td>
		</tr>
		<tr>
			<td>Custom DNA oligos</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxynucleotide (dNTP) Solution Mix</td>
			<td>New England BioLabs</td>
			<td>N0447S</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td>
			<td>Gibco</td>
			<td>10569044</td>
			<td>Basal medium for supporting the growth of HEK293T cell line</td>
		</tr>
		<tr>
			<td>DPBS (1x)</td>
			<td>Gibco</td>
			<td>14190-136</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega</td>
			<td>E1910</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI-HF&#160;</td>
			<td>New England BioLabs</td>
			<td>R3101S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, heat inactivated</td>
			<td>Gibco</td>
			<td>16140071</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>Fisherbrand Surface Treated Tissue Culture Dishes</td>
			<td>Fisherbrand</td>
			<td>FB012924</td>
			<td></td>
		</tr>
		<tr>
			<td>FuGENE 6 Transfection Reagent</td>
			<td>Promega</td>
			<td>E2691</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Loading Dye Purple (6x)</td>
			<td>New England BioLabs</td>
			<td>B7024A</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>High Efficiency ig 10B Chemically Competent Cells</td>
			<td>Intact Genomics</td>
			<td>1011-12</td>
			<td>E. coli DH10B cells</td>
		</tr>
		<tr>
			<td>HiSpeed Plasmid Midi Kit</td>
			<td>Qiagen</td>
			<td>12643</td>
			<td>Midi prep</td>
		</tr>
		<tr>
			<td>pCIG2 plasmid</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pGL3 BAR plasmid</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion HF DNA Polymerase</td>
			<td>New England BioLabs</td>
			<td>M0530L</td>
			<td>DNA polymerase</td>
		</tr>
		<tr>
			<td>ProFlex 3 x 32 well PCR System</td>
			<td>Applied biosystems by life technologies</td>
			<td></td>
			<td>Thermocycler</td>
		</tr>
		<tr>
			<td>pTK Renilla plasmid</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit (250)</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>Mini prep</td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit (250)</td>
			<td>Qiagen</td>
			<td>28706</td>
			<td>Gel purification</td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit (250)</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td>PCR purification</td>
		</tr>
		<tr>
			<td>rCutSmart Buffer</td>
			<td>New England BioLabs</td>
			<td>B6004S</td>
			<td></td>
		</tr>
		<tr>
			<td>SacI-HF</td>
			<td>New England BioLabs</td>
			<td>R3156S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>Synergy HTX Multi-Mode Reader</td>
			<td>BioTek</td>
			<td></td>
			<td>&#160;Plate reader runs Gen5 software v3.08 (BioTek)</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New England BioLabs</td>
			<td>M0202L</td>
			<td>Ligase</td>
		</tr>
		<tr>
			<td>TAE Buffer, Tris-Acetate-EDTA, 50x Solution, Electrophoresis</td>
			<td>Fisher Scientific</td>
			<td>BP13324</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Plate 96 wells, Flat Bottom</td>
			<td>Fisherbrand</td>
			<td>FB012931</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Ethidium Bromide Solution</td>
			<td>Invitrogen by Thermo Fisher Scientific</td>
			<td>15585011</td>
			<td></td>
		</tr>
		<tr>
			<td>XmaI</td>
			<td>New England BioLabs</td>
			<td>R0180S</td>
			<td>Restriction enzyme</td>
		</tr>
	</tbody>
</table>

a scalable, cell-based method for the functional assessment of ube3a variants

测序在医学中的使用越来越多，已经在人类基因组中发现了数百万个编码变异。这些变异中的许多发生在与神经发育障碍相关的基因中，但绝大多数变异的功能意义仍然未知。本协议描述了Ube3a变异的研究， Ube3a是一种编码与自闭症和安格曼综合征相关的E3泛素连接酶的基因。 Ube3a 的重复或三重与自闭症密切相关，而其缺失会导致Angelman综合征。因此，了解UBE3A蛋白活性变化的效价对临床结果很重要。在这里，描述了一种快速的，基于细胞的方法，该方法将 Ube3a变体与Wnt 通路报告基因配对。这种简单的测定是可扩展的，可用于确定任何 Ube3a 变体中活性变化的价和幅度。此外，该方法的便利性允许产生丰富的结构功能信息，这些信息可用于深入了解UBE3A的酶机制。

Ube3a错义变异的大规模功能筛选确定了功能丧失和获得突变的广泛前景 先前对Ube3a突变体的研究表明，Wnt反应可以作为细胞 UBE3A 蛋白活性的报告基因。扩大了这些观察结果，并进行了额外的验证实验，以研究BAR测定是否适合报告细胞中的一系列UBE3A活性。首先，用编码人WT Ube3a的不同量的质粒DNA转染HEK293T细胞。该实验表明，BAR反应随转染到细胞中的 Ube3a ?...

Watch this Scientific Journal Video about A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants at JoVE.com

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants

开发了一种简单且可扩展的方法来评估Ube3a中错义变异的功能意义， Ube3a是一种基因，其功能的丧失和获得与Angelman综合征和自闭症谱系障碍有关。

一种可扩展的、基于细胞的 Ube3a 变体功能评估方法

The increased use of sequencing in medicine has identified millions of coding variants in the human genome. Many of these variants occur in genes ...

我们的方法提供了一种快速了解Ube3a变异功能影响的方法，Ube3a是一种引起Angelman综合征的泛素连接酶，与称为Dup15q综合征的自闭症遗传形式密切相关。泛素连接酶活性的表征传统上是劳动密集型和缓慢的，并且这种方法很难并且不适合表征Ube3a鉴定的数百种变体。Angelman综合征是由母体Ube3a基因的缺失引起的，而Dup15q综合征是由母体Ube3a的重复引起的，因此推断错义变异如何改变Ube3a酶的活性非常重要，如果不对每个变异进行经验测试，这是很难做到的。演示该程序的将是我实验室的研究技术人员Jalin Stelzer。首先在HEK293T细胞中进行测定，HEK293T细胞在Dulbecco的改良鹰培养基中生长，温度为37摄氏度，在加湿的容器中含有5%的二氧化碳，如手稿中所述。在生物安全柜中，将悬浮的HEK293T细胞接种到组织培养处理的96孔平底板上，并将细胞在37度下孵育过夜。第二天，用萤火虫和雷尼拉荧光素酶报告质粒以及Ube3a质粒一式三份在生物安全柜中转染细胞。以每孔10微升的总体积进行转染。在 1.5 毫升微量离心管中为每个变体的转染创建预混液，并在室温下孵育 15 分钟。孵育后，通过敲击管轻轻搅拌转染混合物，然后将10微升转染混合物直接添加到孔中现有的生长培养基中。之后，将细胞放回培养箱，让质粒表达48小时。使用荧光显微镜，通过GFP荧光监测转染效率。超过80%的细胞应在48小时后表达GFP。小心地从转染的HEK293T细胞中吸出培养基，然后用冷PBS洗涤细胞。吸出 PBS。加入25微升非变性裂解缓冲液用于细胞裂解，并在冰上轻轻摇动孵育15分钟。然后，将 20 微升所得裂解物加入含有 100 微升萤火虫荧光素酶底物试剂的新 96 孔测定板中，并通过敲击轻轻混合板。将板加载到读板器上，并使用读数高度为1毫米，积分时间为1秒的顶部检测器测量发光。读取萤火虫荧光素酶发光后，立即向孔中加入 100 微升雷尼拉萤光素酶底物，在评估雷尼拉萤光素酶活性的同时淬灭萤火虫萤光素酶活性。通过轻轻的板搅拌混合样品并再次测量发光以评估Renilla荧光素酶活性。通过量化报告者反应并将每个变异体的萤火虫和雷尼拉值与野生型 Ube3a 标准化来比较变异蛋白的相对活性。用越来越多的编码野生型Ube3a的质粒转染的HEK293T细胞显示BAR反应的线性增加。BAR测定可识别质粒的疾病相关变异，证明了该方法的准确性和通用性。152 个 Ube3a 变体的 BAR 测定筛选显示相对于野生型 Ube3a 的良性功能丧失和功能突变增益。Ube3a位置的功能谷氨酸取代、功能脯氨酸替代丧失和另一个功能精氨酸替代丧失产生了截然不同的效果，从而强调了经验变异评估的重要性。热图显示了归一化 BAR 结果，用于在 Ube3a 中位置 588 进行全面的突变分析。白色阴影表示野生型Ube3a活性水平，蓝色阴影表示功能丧失，红色阴影表示功能突变的获得。除了我们工作的临床意义之外，Ube3a还是泛素连接酶HECT结构域家族的成员，这些家族都具有保守的HECT催化结构域。我认为我们工作的一个有趣的方面是它提供了关于Ube3a的丰富结构函数数据，这可以用来获得对这类酶的更深入的机理理解。

Research

JoVE Journal

Neuroscience

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

可伸缩流控喷油器阵列病毒完整的3 - D的大脑回路为目标

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

科研

神经科学

Functional Mapping with Simultaneous MEG and EEG

同时脑磁图和脑电图的功能映射

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the ...

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound (fTCD)

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound fTCD

儿童使用功能经颅多普勒超声评估大脑偏侧（fTCD）

There are many unanswered questions about cerebral lateralization.  In particular, it remains unclear which aspects of language and nonverbal ability are ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

高分辨率人类中脑功能磁共振成像方法

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

隔膜的神经支配膈运动神经元的功能和形态评估

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

使用高尔基Cox方法评估老年小鼠海马树突状复杂性

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

重建神经元电路：快速神经延伸和功能性神经元连接的新方法

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

小鼠蛛网膜下腔出血模型脑血管痉挛量化的容积法

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

间刺激间隔对啮齿类动物时间处理评估的力量

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

&#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

 &#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

μTongue：一个基于微流体的功能成像平台，用于在Viva的舌头

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in ...