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

Detta arbete stöddes av en Simons Foundation Bridge to Independence Award (SFARI Award #387972; JJY), ett NARSAD Young Investigator Award från Brain and Behavior Research Foundation (J.J.Y.), ett forskningsstipendium från Alfred P. Sloan Foundation (J.J.Y.) och forskningsbidrag från Angelman Syndrome Foundation (J.J.Y.), Whitehall Foundation (J.J.Y.) och NIMH (R01MH122786; J.J.Y.).

1. Mutagenesis kloning för att generera Ube3a varianter
<ol><li>Klona alla Ube3a-varianter i pCIG2-plasmiden (figur 1A), en bicistronisk vektor som innehåller en kyckling-β-aktinpromotor och en intern ribosominmatningsplats (IRES) för EGFP-uttryck19. Ube3a-konstruktioner i full längd innehåller en N-terminal Myc-tag-sekvens och klonas till pCIG2 med hjälp av en 5'SacI-plats och en 3' XmaI-plats. Dessutom används na...

<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>

Protokollet som beskrivs här ger en effektiv och skalbar metod för att bedöma den enzymatiska aktiviteten hos Ube3a-varianter. Det finns flera tekniska detaljer som motiverar noggrant övervägande när du använder denna analys. Ett övervägande är valet av Wnt-reporterplasmider som används i denna analys. Protokollet som beskrivs här använder specifikt β-catenin-aktiverad reporter (BAR)21, en reporter som innehåller en sammanfogare av 12 T-cellfaktorsvarselement (TCF) separera...

De senaste tekniska framstegen har gjort sekvenseringen av exomer och genom rutin i kliniska miljöer 1,2. Detta har lett till upptäckten av ett stort antal genetiska varianter, inklusive miljontals missense-varianter som vanligtvis förändrar en aminosyra i ett protein. Att förstå den funktionella betydelsen av dessa varianter är fortfarande en utmaning, och endast en liten bråkdel (~ 2%) av de kända missense-varianterna har en klinisk tolkning 1,3.
Ett framträdande exempel på detta problem är Ube3a, en gen som kodar för en E3 ubiquitin-ligas som riktar sig mot substratproteiner för nedbrytning4. Ube3a finns inom kromosom 15q11-13 och uttrycks uteslutande från den moderligt ärvda allelen 5,6,7. Observationer från sjukdomsgenetik tyder starkt på att otillräcklig eller överdriven aktivitet hos UBE3A-enzymet orsakar distinkta neuroutvecklingsstörningar. Radering av moderns kromosom 15q11-13 orsakar Angelmans syndrom (AS)8, en störning som kännetecknas av allvarlig intellektuell funktionsnedsättning, motoriska funktionsnedsättningar, anfall, ett lyckligt uppträdande med frekvent leende och dysmorfa ansiktsdrag 8,9,10. Däremot orsakar duplicering eller tredubbling av moderns kromosom 15q11-13 Dup15q syndrom, ett heterogent tillstånd som erkänns som en av de vanligaste syndromiska formerna av autism11,12,13. Dessutom finns det hundratals missense-varianter identifierade i Ube3a, varav de flesta betraktas som varianter av osäker betydelse (VUS) eftersom deras funktionella och kliniska betydelse är okänd. Det finns således ett stort intresse för att utveckla metoder som empiriskt kan bedöma Ube3a-varianter för att avgöra om de bidrar till utvecklingsneurologisk sjukdom.
UBE3A-enzymet tillhör HECT-domänfamiljen (homolog till E6-AP C-terminus) av E3-ubiquitin-ligaser som alla har den eponymous HECT-domänen, som innehåller det biokemiska maskineriet som är nödvändigt för att acceptera aktiverat ubiquitin från E2-enzymer och överföra det till substratproteiner14. Historiskt sett har karakteriseringen av E3-enzymer förlitat sig på rekonstituerade in vitro-system som kräver en ensemble av renade proteiner 4,15,16. Sådana metoder är långsamma och arbetsintensiva och inte mottagliga för att bedöma aktiviteten hos ett stort antal varianter. I tidigare arbete identifierades UBE3A för att aktivera Wnt-vägen i HEK293T-celler genom att modulera proteasomens funktion för att bromsa nedbrytningen av β-catenin-17. Denna insikt gör det möjligt att använda Wnt-vägreportrar som en effektiv och snabb metod för att identifiera både förlust- och förstärkningsvarianter av Ube3a18. Protokollet nedan beskriver i detalj en metod för att generera Ube3a-varianter samt en luciferasbaserad reporter för att bedöma förändringar i aktiviteten hos Ube3a-varianter.

<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

Den ökade användningen av sekvensering inom medicin har identifierat miljontals kodande varianter i det mänskliga genomet. Många av dessa varianter förekommer i gener associerade med neurodevelopmental störningar, men den funktionella betydelsen av de allra flesta varianter är fortfarande okänd. Det nuvarande protokollet beskriver studien av varianter för Ube3a, en gen som kodar för en E3 ubiquitin-ligas kopplad till både autism och Angelmans syndrom. Duplicering eller tredubbling av Ube3a är starkt kopplad till autism, medan dess radering orsakar Angelmans syndrom. Således är det viktigt för kliniska resultat att förstå valensen av förändringar i UBE3A-proteinaktivitet. Här beskrivs en snabb, cellbaserad metod som parar ihop Ube3a-varianter med en Wnt-vägreporter. Denna enkla analys är skalbar och kan användas för att bestämma valensen och storleken på aktivitetsförändringar i alla Ube3a-varianter . Dessutom möjliggör anläggningen för denna metod generering av en mängd strukturfunktionsinformation, som kan användas för att få djupa insikter i de enzymatiska mekanismerna för UBE3A.

Storskalig funktionell screening av Ube3a-missense-varianter identifierar ett brett landskap av förlust- och gain-of-function-mutationer Tidigare arbete med Ube3a-mutanter föreslog att Wnt-svaret kan fungera som en reporter av cellulär UBE3A-proteinaktivitet. Dessa observationer utökades och ytterligare valideringsexperiment utfördes för att undersöka om BAR-analysen är lämplig för att rapportera en rad UBE3A-aktiviteter i cellen. Först transfekterades HEK293T-celler med v...

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

En enkel och skalbar metod utvecklades för att bedöma den funktionella betydelsen av missensevarianter i Ube3a, en gen vars förlust och funktionsförstärkning är kopplad till både Angelmans syndrom och autismspektrumstörning.

En skalbar, cellbaserad metod för funktionsbedömning av Ube3a-varianter

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

Vår metod ger ett snabbt sätt att förstå den funktionella effekten av varianter i Ube3a, en ubiquitinligas som orsakar Angelmans syndrom och är starkt kopplad till en genetisk form av autism som kallas Dup15q-syndrom. Karakteriseringen av ubiquitin-ligasaktivitet är traditionellt arbetsintensiv och långsam, och sådana metoder är svåra och inte riktigt mottagliga för att karakterisera de hundratals varianter som identifierats för Ube3a. Angelmans syndrom orsakas av raderingen av moderns Ube3a-gen, medan Dup15q-syndromet orsakas av duplicering av moderns Ube3a, så det är viktigt att härleda hur missense-varianter förändrade aktiviteten hos Ube3a-enzymet, och detta är något som är svårt att göra utan att empiriskt testa varje variant.Demonstrerar proceduren kommer att vara Jalin Stelzer, en forskningstekniker i mitt laboratorium. Börja med att utföra analyserna i HEK293T-celler, odlade i Dulbeccos modifierade Eagle Medium vid 37 grader Celsius, med 5% koldioxid i en fuktad behållare, som beskrivs i manuskriptet. I ett biosäkerhetsskåp plåtar du de upphängda HEK293T-cellerna på en vävnadsodlingsbehandlad 96 väl plattbottnad platta och inkuberar cellerna över natten vid 37 grader.Den andra dagen transfekterar cellerna i ett biosäkerhetsskåp med Firefly och Renilla luciferase reporter plasmider och Ube3a plasmider i tre exemplar. Utför transfektionerna i en total volym på 10 mikroliter per brunn. Skapa en masterblandning för transfekterna för varje variant i ett 1,5 ml mikrocentrifugrör och inkubera vid rumstemperatur i 15 minuter.Efter inkubationen, agitera försiktigt transfektionsblandningen genom att knacka på röret och tillsätt sedan 10 mikroliter av transfektionsblandningen direkt till det befintliga tillväxtmediet i brunnarna. Därefter återför cellerna till inkubatorn och låt plasmiderna uttrycka i 48 timmar. Använd ett fluorescensmikroskop för att övervaka transfektionseffektiviteten med GFP-fluorescens.Mer än 80% av cellerna ska uttrycka GFP efter 48 timmar. Aspirera försiktigt odlingsmediet från transfekterade HEK293T-celler och tvätta sedan cellerna med kall PBS. Aspirera PBS.Tillsätt 25 mikroliter icke-denaturerande lysbuffert för celllys och inkubera i 15 minuter på is med mild gungning. Tillsätt sedan 20 mikroliter av det resulterande lysatet i en ny 96-brunns analysplatta innehållande 100 mikroliter av ett Firefly-luciferassubstratreagens och blanda plattan försiktigt genom att knacka. Ladda ner plattan till en plattläsare och mät luminescensen med hjälp av en toppdetektor med en läshöjd på en millimeter och en integrationstid på en sekund.Omedelbart efter att ha läst Firefly luciferas-luminescensen, tillsätt 100 mikroliter Renilla luciferas-substrat till brunnarna, vilket släcker Firefly-luciferasaktiviteten medan du bedömer Renilla luciferasaktivitet. Blanda proverna genom försiktig plattomrörning och mät luminescensen igen för att bedöma Renilla luciferasaktiviteten. Jämför den relativa aktiviteten hos variantproteiner genom att kvantifiera reportersvaren och normalisera Firefly- och Renilla-värdet för varje variant mot den vilda typen Ube3a.HEK293T-celler transfekterade med ökande mängder plasmid som kodar för Ube3a av vild typ visar en linjär ökning av BAR-svaret. BAR-analysen identifierar sjukdomskopplade varianter av plasmiderna, vilket visar noggrannheten och generaliteten hos denna metod. BAR-analysskärm för 152 Ube3a-varianter visar godartad förlust av funktion och förstärkning av funktionsmutationer i förhållande till Ube3a av vild typ.Förstärkningen av funktionsglutamatsubstitution, förlust av funktion prolinsubstitution och en annan förlust av funktion argininsubstitution vid position 588 av Ube3a gav drastiskt olika effekter, vilket understryker vikten av empirisk variantbedömning. Värmediagrammet visar normaliserade BAR-resultat för omfattande mutationsanalys vid position 588 i Ube3a. Den vita skuggningen representerar Ube3a-aktivitetsnivåer av vild typ, den blå skuggningen indikerar funktionsförlust och den röda skuggningen indikerar förstärkning av funktionsmutationer.Utöver de kliniska konsekvenserna av vårt arbete är Ube3a medlem i HECT-domänfamiljen av ubiquitin-ligaser, som alla har den bevarade HECT-katalytiska domänen. Och jag tror att en av de fascinerande aspekterna av vårt arbete är att det ger rika strukturfunktionsdata om Ube3a, och detta kan användas för att få en djupare mekanistisk förståelse för denna klass av enzymer.

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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

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

Skalbar fluidic Injektor arrayer för Viral Inriktning av intakt 3-D Brain Kretsar

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

Functional Mapping with Simultaneous MEG and EEG

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the processing of simple sensory stimuli. We will use somatosensory stimuli to locate the hand somatosensory areas, auditory stimuli to locate the auditory cortices, visual stimuli in four quadrants of the visual field to locate the early visual areas. These type of experiments are used for functional mapping in epileptic and brain tumor patients to locate eloquent cortices. In basic neuroscience similar experimental protocols are used to study the orchestration of cortical activity. The acquisition protocol includes quality assurance procedures, subject preparation for the combined MEG/EEG study, and acquisition of evoked-response data with somatosensory, auditory, and visual stimuli. We also demonstrate analysis of the data using the equivalent current dipole model and cortically-constrained minimum-norm estimates. Anatomical MRI data are employed in the analysis for visualization and for deriving boundaries of tissue boundaries for forward modeling and cortical location and orientation constraints for the minimum-norm estimates.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to map brain areas involved in the processing of simple sensory stimuli.

Funktionell kartläggning med samtidig MEG och EEG

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)

There are many unanswered questions about cerebral lateralization. In particular, it remains unclear which aspects of language and nonverbal ability are lateralized, whether there are any disadvantages associated with atypical patterns of cerebral lateralization, and whether cerebral lateralization develops with age. In the past, researchers interested in these questions tended to use handedness as a proxy measure for cerebral lateralization, but this is unsatisfactory because handedness is only a weak and indirect indicator of laterality of cognitive functions1. Other methods, such as fMRI, are expensive for large-scale studies, and not always feasible with children2.
Here we will describe the use of functional transcranial Doppler ultrasound (fTCD) as a cost-effective, non-invasive and reliable method for assessing cerebral lateralization. The procedure involves measuring blood flow in the middle cerebral artery via an ultrasound probe placed just in front of the ear. Our work builds on work by Rune Aaslid, who co-introduced TCD in 1982, and Stefan Knecht, Michael Deppe and their colleagues at the University of M&uuml;nster, who pioneered the use of simultaneous measurements of left- and right middle cerebral artery blood flow, and devised a method of correcting for heart beat activity. This made it possible to see a clear increase in left-sided blood flow during language generation, with lateralization agreeing well with that obtained using other methods3.
The middle cerebral artery has a very wide vascular territory (see Figure 1) and the method does not provide useful information about localization within a hemisphere. Our experience suggests it is particularly sensitive to tasks that involve explicit or implicit speech production. The 'gold standard' task is a word generation task (e.g. think of as many words as you can that begin with the letter 'B') 4, but this is not suitable for young children and others with limited literacy skills. Compared with other brain imaging methods, fTCD is relatively unaffected by movement artefacts from speaking, and so we are able to get a reliable result from tasks that involve describing pictures aloud5,6. Accordingly, we have developed a child-friendly task that involves looking at video-clips that tell a story, and then describing what was seen.

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

Functional transcranial Doppler sonography (fTCD) is a simple and non-invasive ultrasound technique which can be used to assess the lateralization of cognitive functions, especially language, and is suitable for use with children.

Bedömning av Cerebral Lateralization hos barn med hjälp av funktionell Transkraniell Doppler ultraljud (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

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 measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Högupplösta functional magnetic resonance imaging metoder för Human mitthjärnan

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

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

Funktionell och morfologisk Bedömning av Membran Innervation av Phrenic motorneuroner

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

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Bedömning av hippocampal dendritisk komplexitet hos åldrade möss med användning av Golgi-Cox-metoden

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

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 accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

Rewiring Neuronal Circuits: En ny metod för snabb Neurit förlängning och funktionell Neuronal anslutning

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

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

En volymetrisk metod för kvantifiering av Cerebral Vasospasm i en murin modell av subarachnoidalblödning

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

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Kraften i Interstimulus intervall för bedömning av Temporal bearbetning i gnagare

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

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in the taste sensory organ. By integrating microfluidics into the intravital tongue imaging window, the &#181;Tongue provides reliable functional images of taste cells in vivo under controlled exposure to multiple tastants. In this paper, a detailed step-by-step procedure to utilize the &#181;Tongue system is presented. There are five subsections: preparing of tastant solutions, setting up of a microfluidic module, sample mounting, acquiring functional image data, and data analysis. Some tips and techniques to solve the practical issues that may arise when using the &#181;Tongue are also presented.

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

The article introduces the &#181;Tongue (microfluidics-on-a-tongue) device for functional taste cell imaging in vivo by integrating microfluidics into an intravital imaging window on the tongue.

μTongue: En mikrofluidikbaserad funktionell bildplattform för tungan i Vivo

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