enrichment-purging-human-embryonic-stem-cells-detection-cell-surface

Enrichment and Purging of Human Embryonic Stem Cells by Detection of Cell Surface Antigens Using the Monoclonal Antibodies TG30 and GCTM-2

Human embryonic stem cells (hESC) can self-renew indefinitely in vitro, and with the appropriate cues can be induced to differentiate into potentially all ...

CSIRO

&#x810A;&#x67F1;&#x548C;&#x5EF6;&#x9AD3;&#x808C;&#x840E;&#x7F29;&#x75C7; &#xFF08;SBMA&#xFF09; &#x662F;&#x6210;&#x4EBA;&#x53D1;&#x75C5;&#x8FD0;&#x52A8;&#x795E;&#x7ECF;&#x5143;&#x75BE;&#x75C5;&#xFF0C;&#x9020;&#x6210;&#x7684;&#x6269;&#x5927;&#x7684;&#x5916;&#x663E;&#x5B50; 1 &#x7684;&#x96C4;&#x6FC0;&#x7D20;&#x53D7;&#x4F53; (AR) &#x57FA;&#x56E0;&#x7684;&#x4E09;&#x6838;&#x82F7;&#x9178;&#x91CD;&#x590D; &#xFF08;TNR&#xFF09;&#x3002;&#x6B64;&#x969C;&#x788D;&#x53D8;&#x6027;&#x7684;&#x8FD0;&#x52A8;&#x53CA;&#x611F;&#x89C9;&#x795E;&#x7ECF;&#x5143;&#x3001; &#x8FD1;&#x7AEF;&#x808C;&#x8089;&#x840E;&#x7F29;&#x3001; &#x548C;&#x5185;&#x5206;&#x6CCC;&#x7684;&#x5F02;&#x5E38;&#xFF0C;&#x5982;&#x7537;&#x6027;&#x4E73;&#x623F;&#x53D1;&#x80B2;&#x75C7;&#x7684;&#x7279;&#x70B9;&#x662F;&#xFF0C;&#x548C;&#x964D;&#x4F4E;&#x751F;&#x80B2;&#x7387;&#x3002;&#x6211;&#x4EEC;&#x63CF;&#x8FF0;&#x53D1;&#x5C55;&#x8F6C;&#x57FA;&#x56E0;&#x6A21;&#x578B;&#x7684; SBMA &#x8868;&#x793A;&#x4EBA;&#x7C7B; AR (hAR) cDNA &#x643A;&#x5E26; 65 &#xFF08;AR(65)) &#x6216; 120 CAG &#x91CD;&#x590D;&#x5E8F;&#x5217; &#xFF08;AR(120))&#xFF0C;&#x4E0E;&#x5DE8;&#x7EC6;&#x80DE;&#x75C5;&#x6BD2;&#x542F;&#x52A8;&#x5B50;&#x9A71;&#x52A8;&#x7684;&#x666E;&#x904D;&#x8868;&#x8FBE;&#x3002;&#x643A;&#x5E26; AR(120) &#x663E;&#x793A;&#x8F6C;&#x57FA;&#x56E0;&#x7684;&#x884C;&#x4E3A;&#x548C;&#x8FD0;&#x52A8;&#x529F;&#x80FD;&#x969C;&#x788D;&#x7684;&#x5C0F;&#x9F20;&#xFF0C;&#x5C0F;&#x9F20;&#x540C;&#x65F6;&#x643A;&#x5E26; 65 CAG &#x91CD;&#x590D;&#x663E;&#x793A;&#x8F7B;&#x5EA6;&#x7684;&#x8868;&#x578B;&#x3002;&#x9010;&#x6B65;&#x7684;&#x808C;&#x8089;&#x65E0;&#x529B;&#x548C;&#x840E;&#x7F29; AR(120) &#x5C0F;&#x9F20;&#x4E2D;&#xFF0C;&#x88AB;&#x53D1;&#x73B0;&#x548C;&#x810A;&#x9AD3; &alpha; &#x8FD0;&#x52A8;&#x795E;&#x7ECF;&#x5143;&#x7684;&#x635F;&#x5931;&#x6709;&#x5173;&#x8054;&#x3002;&#x6CA1;&#x6709;&#x4EFB;&#x4F55;&#x5176;&#x4ED6;&#x7684;&#x5927;&#x8111;&#x7ED3;&#x6784;&#x4E2D;&#x795E;&#x7ECF;&#x53D8;&#x6027;&#x7684;&#x8BC1;&#x636E;&#x3002;&#x8FD0;&#x52A8;&#x529F;&#x80FD;&#x969C;&#x788D;&#x6709;&#x4EBA;&#x6307;&#x51FA;&#x8FD9;&#x4E24;&#x4E2A;&#x7537;&#x6027;&#x548C;&#x96CC;&#x6027;&#x52A8;&#x7269;&#xFF0C;&#x663E;&#x793A;&#x51FA;&#x5728; SBMA polyglutamine &#x91CD;&#x590D;&#x6269;&#x5F20;&#x5BFC;&#x81F4;&#x5360;&#x4E3B;&#x5BFC;&#x5730;&#x4F4D;&#x7684;&#x589E;&#x76CA;&#x7684;&#x51FD;&#x6570;&#x7A81;&#x53D8;&#x4E2D;&#x4EBA;&#x624D;&#x57F9;&#x8BAD;&#x4E2D;&#x5FC3;&#x3002;&#x96C4;&#x6027;&#x5C0F;&#x9F20;&#x7CBE;&#x5B50;&#x751F;&#x4EA7;&#x7B26;&#x5408;&#x4EBA;&#x7C7B;&#x60A3;&#x8005;&#x6240;&#x62A5;&#x544A;&#x7684;&#x777E;&#x4E38;&#x7F3A;&#x9677;&#x4E2D;&#x663E;&#x793A;&#x9010;&#x6B65;&#x51CF;&#x5C11;&#x3002;&#x8FD9;&#x4E9B;&#x5C0F;&#x9F20;&#x8868;&#x793A;&#x7B2C;&#x4E00;&#x79CD;&#x6A21;&#x5F0F;&#x6765;&#x91CD;&#x73B0; SBMA&#xFF0C;&#x4F7F;&#x4ED6;&#x4EEC;&#x6210;&#x4E3A;&#x6709;&#x7528;&#x8D44;&#x6E90;&#x8868;&#x5F81;&#x75C5;&#x60C5;&#x53D1;&#x5C55;&#x548C;&#x6D4B;&#x8BD5; polyglutamine &#x548C;&#x8FD0;&#x52A8;&#x795E;&#x7ECF;&#x5143;&#x75BE;&#x75C5;&#x7684;&#x6CBB;&#x7597;&#x7B56;&#x7565;&#x7684;&#x4E3B;&#x8981;&#x7279;&#x70B9;&#x3002;

&#x810A;&#x67F1;&#x548C;&#x5EF6;&#x9AD3;&#x808C;&#x840E;&#x7F29;&#x75C7;&#x7684;&#x5C0F;&#x9F20;&#x6A21;&#x578B;&#x3002;

Spinal and bulbar muscular atrophy (SBMA) is an adult-onset motor neuron disease, caused by the expansion of a trinucleotide repeat (TNR) in exon 1 of the androgen receptor (AR) gene. This disorder is characterized by degeneration of motor and sensory neurons, proximal muscular atrophy, and endocrine abnormalities, such as gynecomastia and reduced fertility. We describe the development of a transgenic model of SBMA expressing a full-length human AR (hAR) cDNA carrying 65 (AR(65)) or 120 CAG repeats (AR(120)), with widespread expression driven by the cytomegalovirus promoter. Mice carrying the AR(120) transgene displayed behavioral and motor dysfunction, while mice carrying 65 CAG repeats showed a mild phenotype. Progressive muscle weakness and atrophy was observed in AR(120) mice and was associated with the loss of alpha-motor neurons in the spinal cord. There was no evidence of neurodegeneration in other brain structures. Motor dysfunction was observed in both male and female animals, showing that in SBMA the polyglutamine repeat expansion causes a dominant gain-of-function mutation in the AR. The male mice displayed a progressive reduction in sperm production consistent with testis defects reported in human patients. These mice represent the first model to reproduce the key features of SBMA, making them a useful resource for characterizing disease progression, and for testing therapeutic strategies for both polyglutamine and motor neuron diseases.

A mouse model of spinal and bulbar muscular atrophy.

Atrophie musculaire spinale et bulbaire (SBMA) est une maladie du motoneurone d&#39;&acirc;ge adulte, caus&eacute;e par l&#39;expansion d&#39;une r&eacute;p&eacute;tition du trinucl&eacute;otide (TNR) dans l&#39;exon 1 du g&egrave;ne du r&eacute;cepteur (AR) androg&egrave;ne. Ce trouble se caract&eacute;rise par une d&eacute;g&eacute;n&eacute;rescence des neurones moteurs et sensoriels, atrophie musculaire proximale et anomalies endocriniennes, comme la gyn&eacute;comastie et r&eacute;duit la fertilit&eacute;. Les auteurs d&eacute;crivent le d&eacute;veloppement d&#39;un mod&egrave;le transg&eacute;nique de SBMA exprimant un ADNc humain complet de AR (hAR) transportant 65 (AR(65)) ou r&eacute;p&eacute;titions CAG 120 (AR(120)), avec une expression tr&egrave;s r&eacute;pandue par le promoteur de cytom&eacute;galovirus. Souris porteuses de la dysfonction motrice et comportementale du transg&egrave;ne affich&eacute;e AR(120), tandis que les souris transportant 65 CAG r&eacute;p&egrave;te montrent un ph&eacute;notype doux. Une atrophie et une faiblesse musculaire progressive a &eacute;t&eacute; observ&eacute;e chez les souris AR(120) et a &eacute;t&eacute; associ&eacute;es &agrave; la perte des alpha-motoneurones dans la moelle &eacute;pini&egrave;re. Rien n&#39;indiquait de la neurod&eacute;g&eacute;n&eacute;rescence dans d&#39;autres structures c&eacute;r&eacute;brales. Dysfonction motrice a &eacute;t&eacute; observ&eacute;e chez les m&acirc;les et femelles, montrant que dans SBMA la polyglutamine r&eacute;p&eacute;ter expansion provoque une mutation dominante de gain de fonction dans l&#39;EI. Les souris m&acirc;les affichent une r&eacute;duction progressive dans la production de sperme conforme aux testicules d&eacute;fauts signal&eacute;s chez des patients humains. Ces souris repr&eacute;sentent le premier mod&egrave;le &agrave; reproduire les caract&eacute;ristiques principales de SBMA, rendant une ressource utile pour caract&eacute;riser l&#39;&eacute;volution de la maladie et pour tester des strat&eacute;gies th&eacute;rapeutiques pour les maladies polyglutamine et de neurones moteurs.

Un mod&egrave;le murin d&#39;amyotrophie spinale et bulbaire.

Ein Maus-Modell der spinalen und bulb&#xE4;ren Muskelatrophie.

Atrofia muscolare spinale e bulbare (SBMA) &egrave; una malattia del motoneurone ad esordio adulto, causata dall&#39;espansione di una ripetizione di trinucleotide (TNR) nell&#39;esone 1 del gene androgeno recettore (AR). Questa malattia &egrave; caratterizzata da degenerazione dei neuroni motori e sensoriali, atrofia muscolare e anomalie endocrine, quali ginecomastia e ridotta fertilit&agrave;. Descriviamo lo sviluppo di modelli transgenici di SBMA esprimendo un full-length cDNA AR (hAR) umano portando 65 (AR(65)) o 120 ripetizioni CAG (AR(120)), con espressione diffusa guidato dal promotore citomegalovirus. Topi portatori il transgene visualizzato AR(120) comportamentale e motore disfunzione, mentre topi portando 65 CAG ripetizioni hanno mostrato un fenotipo pi&ugrave; lieve. Atrofia e debolezza muscolare progressiva &egrave; stata osservata nei topi AR(120) ed &egrave; state associate con la perdita dei motoneuroni alfa del midollo spinale. Non non c&#39;era nessuna prova di neurodegenerazione in altre strutture cerebrali. Motore disfunzione &egrave; stata osservata in entrambi maschio e femminili animali, mostrando che in SBMA delle poliglutamine ripetere espansione provoca una mutazione dominante di guadagno di funzione in AR. I topi maschi visualizzata una progressiva riduzione nella produzione di spermatozoi coerente con testicolo difetti segnalati in pazienti umani. Questi topi rappresentano il primo modello di riprodurre le caratteristiche chiave di SBMA, che li rende una risorsa utile per caratterizzare la progressione della malattia e per testare strategie terapeutiche per malattie delle poliglutamine sia motoneuroni.

Un modello murino di atrofia muscolare spinale e bulbare.

&#x810A;&#x9AC4;&#x3001;&#x5EF6;&#x9AC4;&#x7B4B;&#x840E;&#x7E2E;&#x75C7; (SBMA) &#x30C8;&#x30EA;&#x30D7;&#x30EC;&#x30C3;&#x30C8;&#x30EA;&#x30D4;&#x30FC;&#x30C8; &#x30EA;&#x30D4;&#x30FC;&#x30C8; &#xFF08;TNR&#xFF09; &#x306E; exon 1 &#x30A2;&#x30F3;&#x30C9;&#x30ED;&#x30B2;&#x30F3;&#x53D7;&#x5BB9;&#x4F53; (AR) &#x907A;&#x4F1D;&#x5B50;&#x306E;&#x81A8;&#x5F35;&#x306B;&#x3088;&#x3063;&#x3066;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3055;&#x308C;&#x305F;&#x6210;&#x4EBA;&#x767A;&#x75C7;&#x904B;&#x52D5;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x75C5;&#x3067;&#x3059;&#x3002;&#x3053;&#x306E;&#x969C;&#x5BB3;&#x306F;&#x30E2;&#x30FC;&#x30BF;&#x30FC;&#x305D;&#x3057;&#x3066;&#x611F;&#x899A;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x3001;&#x8FD1;&#x4F4D;&#x7B4B;&#x840E;&#x7E2E;&#x3001;&#x5973;&#x6027;&#x5316;&#x4E73;&#x623F;&#x306A;&#x3069;&#x306E;&#x5185;&#x5206;&#x6CCC;&#x7570;&#x5E38;&#x306E;&#x5909;&#x6027;&#x306B;&#x3088;&#x3063;&#x3066;&#x7279;&#x5FB4;&#x3065;&#x3051;&#x3089;&#x308C;&#x308B;&#x3057;&#x3001;&#x4E0D;&#x598A;&#x6CBB;&#x7642;&#x3092;&#x524A;&#x6E1B;&#x3057;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x304C;&#x904B;&#x3076; 65 (AR(65)) &#x307E;&#x305F;&#x306F; 120 CAG &#x30EA;&#x30D4;&#x30FC;&#x30C8; (AR(120)), &#x30B5;&#x30A4;&#x30C8;&#x30E1;&#x30AC;&#x30ED; &#x30A6;&#x30A4;&#x30EB;&#x30B9; &#x30D7;&#x30ED;&#x30E2;&#x30FC;&#x30BF;&#x30FC;&#x306B;&#x3088;&#x308B;&#x5E83;&#x7BC4;&#x306A;&#x5F0F;&#x3002; &#x30D5;&#x30EB;&#x30EC;&#x30F3;&#x30B0;&#x30B9;&#x4EBA;&#x9593; AR (hAR) cDNA &#x3092;&#x8868;&#x73FE;&#x3059;&#x308B; SBMA &#x306E;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30B8;&#x30A7;&#x30CB;&#x30C3;&#x30AF; &#x30E2;&#x30C7;&#x30EB;&#x306E;&#x958B;&#x767A;&#x3092;&#x8A18;&#x8FF0;&#x3057;&#x307E;&#x3059;&#x3002;AR(120) &#x8868;&#x793A; transgene &#x884C;&#x52D5;&#x3068;&#x904B;&#x52D5;&#x6A5F;&#x80FD;&#x969C;&#x5BB3;&#x3092;&#x904B;&#x3076;&#x30DE;&#x30A6;&#x30B9; &#x30DE;&#x30A6;&#x30B9;&#x306A;&#x304C;&#x3089;&#x7A4F;&#x3084;&#x304B;&#x306A;&#x8868;&#x73FE;&#x578B;&#x3092;&#x793A;&#x3057;&#x305F; 65 CAG &#x3092;&#x904B;&#x3076;&#x7E70;&#x308A;&#x8FD4;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x9032;&#x884C;&#x6027;&#x7B4B;&#x8131;&#x529B;&#x3068;&#x840E;&#x7E2E; AR(120) &#x30DE;&#x30A6;&#x30B9;&#x3067;&#x8A8D;&#x3081;&#x3089;&#x308C;&#x810A;&#x9AC4;&#x306E; &alpha; &#x904B;&#x52D5;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x640D;&#x5931;&#x3068;&#x95A2;&#x9023;&#x3057;&#x3066;&#x3044;&#x305F;&#x3002;&#x4ED6;&#x306E;&#x8133;&#x306E;&#x69CB;&#x9020;&#x3067;&#x306E;&#x795E;&#x7D4C;&#x5909;&#x6027;&#x306E;&#x8A3C;&#x62E0;&#x306F;&#x306A;&#x304B;&#x3063;&#x305F;&#x3002;&#x4E21;&#x65B9;&#x306E;&#x7537;&#x6027;&#x306E;&#x904B;&#x52D5;&#x6A5F;&#x80FD;&#x969C;&#x5BB3;&#x304C;&#x89B3;&#x5BDF;&#x3055;&#x308C;&#x3001;&#x62E1;&#x5F35; AR &#x3067;&#x306E;&#x652F;&#x914D;&#x7684;&#x306A;&#x6A5F;&#x80FD;&#x7372;&#x5F97;&#x5909;&#x7570;&#x306E;&#x539F;&#x56E0;&#x3092; SBMA&#x3001;&#x30DD;&#x30EA;&#x30B0;&#x30EB;&#x30BF;&#x30DF;&#x30F3;&#x793A;&#x3059;&#x5973;&#x6027;&#x306E;&#x52D5;&#x7269;&#x3092;&#x7E70;&#x308A;&#x8FD4;&#x3057;&#x307E;&#x3059;&#x96C4;&#x30DE;&#x30A6;&#x30B9;&#x7CBE;&#x5B50;&#x751F;&#x7523;&#x4E00;&#x8CAB;&#x6027;&#x306E;&#x3042;&#x308B;&#x4EBA;&#x9593;&#x306E;&#x60A3;&#x8005;&#x3067;&#x5831;&#x544A;&#x7CBE;&#x5DE3;&#x6B20;&#x9665;&#x3092;&#x6709;&#x3059;&#x308B;&#x306E;&#x30D7;&#x30ED;&#x30B0;&#x30EC;&#x30C3;&#x30B7;&#x30D6;&#x524A;&#x6E1B;&#x3092;&#x8868;&#x793A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x30DE;&#x30A6;&#x30B9;&#x306F;&#x305D;&#x308C;&#x3089;&#x306F;&#x75C5;&#x6C17;&#x306E;&#x9032;&#x884C;&#x3092;&#x7279;&#x5FB4;&#x4ED8;&#x3051;&#x308B;&#x305F;&#x3081;&#x3068;&#x30DD;&#x30EA;&#x30B0;&#x30EB;&#x30BF;&#x30DF;&#x30F3;&#x3068;&#x904B;&#x52D5;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x75BE;&#x60A3;&#x306E;&#x6CBB;&#x7642;&#x6226;&#x7565;&#x3092;&#x30C6;&#x30B9;&#x30C8;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x6709;&#x7528;&#x306A;&#x30EA;&#x30BD;&#x30FC;&#x30B9;&#x3092;&#x4F5C;&#x308B; SBMA &#x306E;&#x4E3B;&#x8981;&#x306A;&#x6A5F;&#x80FD;&#x3092;&#x518D;&#x73FE;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x6700;&#x521D;&#x306E;&#x30E2;&#x30C7;&#x30EB;&#x3092;&#x8868;&#x3057;&#x307E;&#x3059;&#x3002;

&#x810A;&#x9AC4;&#x3001;&#x5EF6;&#x9AC4;&#x6027;&#x7B4B;&#x840E;&#x7E2E;&#x75C7;&#x306E;&#x30DE;&#x30A6;&#x30B9;&#x30E2;&#x30C7;&#x30EB;

&#xCC99;&#xCD94; &#xBC0F; &#xC548;&#xAD6C; &#xADFC;&#xC721; &#xC704;&#xCD95; (SBMA) 1 &#xC548; &#xB4DC;&#xB85C; &#xAC90; &#xC218;&#xC6A9; &#xCCB4; (AR) &#xC720;&#xC804;&#xC790;&#xC758; exon&#xC5D0; trinucleotide &#xBC18;&#xBCF5; (TNR)&#xC758; &#xD655;&#xC7A5;&#xC73C;&#xB85C; &#xC778; &#xD55C; &#xC131;&#xC778;-&#xBC1C;&#xBCD1; &#xBAA8;&#xD130; &#xC2E0;&#xACBD; &#xC9C8;&#xBCD1; &#xC774;&#xB2E4;. &#xC774; &#xC7A5;&#xC560; &#xBAA8;&#xD130;&#xC640; &#xAC10;&#xAC01; &#xC2E0;&#xACBD;, &#xADFC; &#xC704; &#xADFC;&#xC721; &#xC704;&#xCD95;, &#xC5EC; &#xC131;&#xD615; &#xC720; &#xBC29; &#xB4F1; &#xB0B4; &#xBD84; &#xBE44; &#xC885;&#xC591;&#xC758; &#xBCC0;&#xC131;&#xC5D0; &#xC758;&#xD574; &#xD2B9;&#xC9D5; &#xBC0F; &#xBE44; &#xC625;&#xC744; &#xAC10;&#xC18C;. &#xC6B0;&#xB9AC;&#xAC00; &#xB4E4;&#xACE0; 65 (AR(65)) &#xB610;&#xB294; 120 CAG &#xBC18;&#xBCF5; (AR(120)), &#xC138;&#xD3EC; &#xBC1C;&#xAE30;&#xC778;&#xC5D0; &#xD798;&#xC785;&#xC5B4; &#xAD11;&#xBC94;&#xC704; &#xD558; &#xAC8C; &#xC2DD; &#xC804;&#xCCB4; &#xAE38;&#xC774; &#xC778;&#xAC04;&#xC758; &#xC544;&#xCE78;&#xC18C; (hAR) Cdna&#xB97C; &#xD45C;&#xD604; SBMA&#xC758; &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615; &#xBAA8;&#xB378;&#xC758; &#xAC1C;&#xBC1C;&#xC5D0; &#xC124;&#xBA85;. AR(120) &#xD45C;&#xC2DC; transgene &#xD589;&#xB3D9; &#xBC0F; &#xBAA8;&#xD130; &#xC7A5;&#xC560;&#xB97C; &#xB4E4;&#xACE0; &#xB9C8;&#xC6B0;&#xC2A4;, &#xC950; &#xD558;&#xBA74;&#xC11C; &#xB4E4;&#xACE0; 65 CAG &#xBC18;&#xBCF5; &#xAC00;&#xBCBC;&#xC6B4; &#xD45C;&#xD604; &#xD615;&#xC744; &#xBCF4;&#xC600;&#xB2E4;. &#xC9C4;&#xBCF4;&#xC801;&#xC778; &#xADFC;&#xC721; &#xC57D;&#xD654;&#xC640; &#xC704;&#xCD95; AR(120) &#xB9C8;&#xC6B0;&#xC2A4;&#xC5D0;&#xC11C; &#xAD00;&#xCC30; &#xB418;&#xC5C8;&#xB2E4; &#xCC99;&#xC218;&#xC5D0;&#xC11C; &#xC54C;&#xD30C; &#xBAA8;&#xD130; &#xB274;&#xB7F0;&#xC758; &#xC190;&#xC2E4;&#xC5D0; &#xC5F0;&#xACB0;. &#xB2E4;&#xB978; &#xB1CC; &#xAD6C;&#xC870;&#xC5D0;&#xC11C; neurodegeneration&#xC758; &#xC99D;&#xAC70;&#xAC00; &#xD588;&#xB2E4;. &#xBAA8;&#xD130; &#xBD80;&#xC804; &#xBAA8;&#xB450; &#xB0A8;&#xC131;&#xC5D0;&#xC11C; &#xAD00;&#xCC30; &#xB418;&#xC5C8;&#xB2E4; &#xACE0; SBMA&#xB294; polyglutamine&#xC5D0;&#xC11C; &#xBCF4;&#xC5EC;&#xC8FC;&#xB294; &#xC5EC;&#xC131; &#xB3D9;&#xBB3C; &#xBC18;&#xBCF5; &#xD655;&#xC7A5; &#xD569;&#xB2C8;&#xB2E4; &#xC544;&#xCE78;&#xC18C;&#xC5D0;&#xC11C; &#xC9C0;&#xBC30;&#xC801;&#xC778; &#xD568;&#xC218; &#xC774;&#xB4DD; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xBC1C;&#xC0DD; &#xB0A8;&#xC131; &#xC0DD;&#xC950; &#xACE0;&#xD658; &#xACB0;&#xD568; &#xC778;&#xAC04;&#xC758; &#xD658;&#xC790;&#xC5D0;&#xC11C; &#xBCF4;&#xACE0;&#xC640; &#xC77C;&#xCE58;&#xC758; &#xC815;&#xC790; &#xC0DD;&#xC0B0;&#xC5D0; &#xC9C4;&#xBCF4;&#xC801;&#xC778; &#xAC10;&#xC18C; &#xD45C;&#xC2DC;. &#xC774;&#xB7EC;&#xD55C; &#xC950; &#xADF8;&#xB4E4; &#xC9C8;&#xBCD1;&#xC758; &#xC9C4;&#xD589;&#xC744; &#xD2B9;&#xC131;&#xD654; &#xD558; &#xACE0; &#xD14C;&#xC2A4;&#xD2B8; polyglutamine &#xBC0F; &#xBAA8;&#xD130; &#xC2E0;&#xACBD; &#xC9C8;&#xD658;&#xC5D0; &#xB300; &#xD55C; &#xCE58;&#xB8CC; &#xC804;&#xB7B5;&#xC5D0; &#xB300; &#xD55C; &#xC720;&#xC6A9;&#xD55C; &#xB9AC;&#xC18C;&#xC2A4;&#xB97C; &#xB9CC;&#xB4DC;&#xB294; SBMA&#xC758; &#xC8FC;&#xC694; &#xD2B9;&#xC9D5;&#xC744; &#xC7AC;&#xD604; &#xD558;&#xB294; &#xCCAB; &#xBC88;&#xC9F8; &#xBAA8;&#xB378;&#xC744; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;.

&#xCC99;&#xCD94; &#xBC0F; &#xC548;&#xAD6C; &#xADFC;&#xC721; &#xC704;&#xCD95; &#xC99D;&#xC758; &#xB9C8;&#xC6B0;&#xC2A4; &#xBAA8;&#xB378;.

Atrofia muscular espinal y bulbar (SBMA) es una enfermedad de la motoneurona del adulto-inicio, causada por la expansi&oacute;n de una repetici&oacute;n del Trinucle&oacute;tido (TNR) en el ex&oacute;n 1 del gene del receptor (AR) de andr&oacute;genos. Este trastorno se caracteriza por la degeneraci&oacute;n de las neuronas motoras y sensoriales, atrofia muscular proximal y anormalidades endocrinas, como ginecomastia y reduce la fertilidad. Describimos el desarrollo de un modelo transg&eacute;nico de SBMA expresando un cDNA de AR (hAR) humano integral que lleva 65 (AR(65)) o 120 repeticiones de CAG (AR(120)), con la expresi&oacute;n generalizada, impulsado por el promotor del citomegalovirus. Ratones portando la disfunci&oacute;n conductual y motor de AR(120) transgen muestra, mientras que los ratones que lleva 65 CAG repite demostr&oacute; un fenotipo suave. Atrofia y debilidad muscular progresiva fue observado en ratones AR(120) y fue asociadas a la p&eacute;rdida de alfa-motoneuronas de la m&eacute;dula espinal. No hubo pruebas de neurodegeneraci&oacute;n en otras estructuras cerebrales. Disfunci&oacute;n motora se observ&oacute; en ambos hombre y animales femeninos, mostrando que en SBMA el poliglutaminas repetici&oacute;n dilataci&oacute;n provoca una mutaci&oacute;n dominante de ganancia de funci&oacute;n en la AR. Los ratones machos muestran una reducci&oacute;n progresiva en la producci&oacute;n de espermatozoides consistente con los defectos de test&iacute;culo divulgados en pacientes humanos. Estos ratones representan el primer modelo para reproducir las caracter&iacute;sticas clave de SBMA, convirti&eacute;ndolos en un recurso &uacute;til para caracterizar la progresi&oacute;n de la enfermedad y probar estrategias terap&eacute;uticas para enfermedades de la neurona motora tanto poliglutaminas.

Un modelo de rat&oacute;n de la atrofia muscular espinal bulbar.

Vectors used for gene targeting experiments usually consist of a selectable marker flanked by two regions of homology to the targeted gene. In a homologous recombination event, the selectable marker replaces an essential element of the target gene rendering it inactive. Other applications of gene targeting technology include gene replacement (knockins) and conditional vectors which allow for the generation of inducible or tissue-specific gene-targeting events. The assembly of gene-targeting vectors is generally a laborious process requiring considerable technical skill. The procedures presented here report the application of transposons as tools for the construction of targeting vectors. Two mini-Mu transposons were sequentially inserted by in vitro transposition at each side of the region targeted for deletion. One such transposon carries an antibiotic resistance marker suitable for selection in mammalian cells. A deletion is then generated between the two transposons either by LoxP-induced recombination or by restriction digestion followed by ligation. This deletion removes part of both transposons plus the targeted region in between, leaving a transposon carrying the selectable marker flanked by two arms which are homologous to the targeted gene. Targeting vectors constructed using these transposons were electroporated into embryonic stem cells and shown to be effective in gene-targeting events.

Transposon-mediated generation of targeting vectors for the production of gene knockouts.

Previous reports and the current study have found that germ cell precursor cells appear in embryoid bodies (EBs) formed from mouse embryonic stem cells as identified by positive expression of specific germ cell markers such as Oct-3/4, Mvh, c-kit, Stella, and DAZL. We hypothesized that if exposed to appropriate growth factors, the germ cell precursor cells within the EBs would differentiate into gametes. The source for growth factors used in the present study is conditioned medium collected from testicular cell cultures prepared from the testes of newborn males. Testes at this stage of development contain most growth factors required for the transformation of germ stem cells into differentiated gametes. When EBs were cultured in the conditioned medium, they developed into ovarian structures, which contained putative oocytes. The oocytes were surrounded by one to two layers of flattened cells and did not have a visible zona pellucida. However, oocyte-specific markers such as Fig-alpha and ZP3 were found expressed by the ovarian structures. The production of oocytes using this method is repeatable and reliable and may be applicable to other mammalian species, including the human.

Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes.

Surface marker expression forms the basis for characterization and isolation of human embryonic stem cells (hESCs). Currently, there are few well-defined protein epitopes that definitively mark hESCs. Here we combine immunotranscriptional profiling of hESC lines with membrane-polysome translation state array analysis (TSAA) to determine the full set of genes encoding potential hESC surface marker proteins. Three independently isolated hESC lines (HES2, H9, and MEL1) grown under feeder and feeder-free conditions were sorted into subpopulations by fluorescence-activated cell sorting based on coimmunoreactivity to the hESC surface markers GCTM-2 and CD9. Colony-forming assays confirmed that cells displaying high coimmunoreactivity to GCTM-2 and CD9 constitute an enriched subpopulation displaying multiple stem cell properties. Following microarray profiling, 820 genes were identified that were common to the GCTM-2(high)/CD9(high) stem cell-like subpopulation. Membrane-polysome TSAA analysis of hESCs identified 1,492 mRNAs encoding actively translated plasma membrane and secreted proteins. Combining these data sets, 88 genes encode proteins that mark the pluripotent subpopulation, of which only four had been previously reported. Cell surface immunoreactivity was confirmed for two of these markers: TACSTD1/EPCAM and CDH3/P-Cadherin, with antibodies for EPCAM able to enrich for pluripotent hESCs. This comprehensive listing of both hESCs and spontaneous differentiation-associated transcripts and survey of translated membrane-bound and secreted proteins provides a valuable resource for future study into the role of the extracellular environment in both the maintenance of pluripotency and directed differentiation.

Identification of human embryonic stem cell surface markers by combined membrane-polysome translation state array analysis and immunotranscriptional profiling.

This unit describes a useful approach to preparing highly reproducible samples of human embryonic stem cell (hESC) total RNA suitable for transcriptional profiling from heterogeneous mixtures of cells containing undifferentiated hESC and differentiated cell types. In this unit, fluorescence-activated cell sorting (FACS) is used to sub-fractionate hESC populations on the basis of their levels of co-expression of two previously published hESC surface markers, CD9(TG30) and GCTM-2. This sub-fractionation allows for the separation of undifferentiated hESC (CD9hi, GCTM-2hi) from the early stages in hESC differentiation (CD9neg or low, GCTM-2neg or low).

Preparation of defined human embryonic stem cell populations for transcriptional profiling.

Recent RNA-sequencing studies have shown remarkable complexity in the mammalian transcriptome. The ultimate impact of this complexity on the predicted proteomic output is less well defined. We have undertaken strand-specific RNA sequencing of multiple cellular RNA fractions (>20 Gb) to uncover the transcriptional complexity of human embryonic stem cells (hESCs). We have shown that human embryonic stem (ES) cells display a high degree of transcriptional diversity, with more than half of active genes generating RNAs that differ from conventional gene models. We found evidence that more than 1000 genes express long 5' and/or extended 3'UTRs, which was confirmed by "virtual Northern" analysis. Exhaustive sequencing of the membrane-polysome and cytosolic/untranslated fractions of hESCs was used to identify RNAs encoding peptides destined for secretion and the extracellular space and to demonstrate preferential selection of transcription complexity for translation in vitro. The impact of this newly defined complexity on known gene-centric network models such as the Plurinet and the cell surface signaling machinery in human ES cells revealed a significant expansion of known transcript isoforms at play, many predicting possible alternative functions based on sequence alterations within key functional domains.

Article	Year
A mouse model of spinal and bulbar muscular atrophy. Human molecular genetics\| PubMed ID: 12189162	2002
Transposon-mediated generation of targeting vectors for the production of gene knockouts. Nucleic acids research\| PubMed ID: 15699181	2005
Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes. Stem cells (Dayton, Ohio)\| PubMed ID: 16109761	2006
Identification of human embryonic stem cell surface markers by combined membrane-polysome translation state array analysis and immunotranscriptional profiling. Stem cells (Dayton, Ohio)\| PubMed ID: 19650036	2009
Preparation of defined human embryonic stem cell populations for transcriptional profiling. Current protocols in stem cell biology\| PubMed ID: 20575147	2010
Deep-transcriptome and ribonome sequencing redefines the molecular networks of pluripotency and the extracellular space in human embryonic stem cells. Genome research\| PubMed ID: 22042643	2011

Hun Chy

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