Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Ghent University

The ability to rapidly and efficiently generate reliable Cre/loxP conditional transgenic mice would greatly complement global high-throughput gene targeting initiatives aimed at identifying gene function in the mouse. We report here the generation of Cre/loxP conditional ROSA26-targeted ES cells within 3-4 weeks by using Gateway cloning to build the target vectors. The cDNA of the gene of interest can be expressed either directly by the ROSA26 promoter providing a moderate level of expression or by a CAGG promoter placed in the ROSA26 locus providing higher transgene expression. Utilization of F1 hybrid ES cells with exceptional developmental potential allows the production of germ line transmitting, fully or highly ES cell-derived mice by aggregation of cells with diploid embryos. The presented streamlined procedures accelerate the examination of phenotypical consequences of transgene expression. It also provides a unique tool for comparing the biological activity of polymorphic or splice variants of a gene, or products of different genes functioning in the same or parallel pathways in an overlapping manner.

Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells.

Vascular endothelial growth factor (VEGF) and beta-catenin both act broadly in embryogenesis and adulthood, including in the skeletal and vascular systems. Increased or deregulated activity of these molecules has been linked to cancer and bone-related pathologies. By using novel mouse models to locally increase VEGF levels in the skeleton, we found that embryonic VEGF over-expression in osteo-chondroprogenitors and their progeny largely pheno-copied constitutive beta-catenin activation. Adult induction of VEGF in these cell populations dramatically increased bone mass, associated with aberrant vascularization, bone marrow fibrosis and haematological anomalies. Genetic and pharmacological interventions showed that VEGF increased bone mass through a VEGF receptor 2- and phosphatidyl inositol 3-kinase-mediated pathway inducing beta-catenin transcriptional activity in endothelial and osteoblastic cells, likely through modulation of glycogen synthase kinase 3-beta phosphorylation. These insights into the actions of VEGF in the bone and marrow environment underscore its power as pleiotropic bone anabolic agent but also warn for caution in its therapeutic use. Moreover, the finding that VEGF can modulate beta-catenin activity may have widespread physiological and clinical ramifications.

Increased skeletal VEGF enhances beta-catenin activity and results in excessively ossified bones.

Zeb2 (Sip1/Zfhx1b) &#x662F;&#x8F6C;&#x5F55; repressors &#x4EE5;&#x524D;&#x8868;&#x73B0;&#x51FA;&#x80DA;&#x80CE;&#x53D1;&#x751F;&#x4E0E;&#x80BF;&#x7624;&#x8FDB;&#x5C55;&#x8FC7;&#x7A0B;&#x8C03;&#x8282;&#x4E0A;&#x76AE;-&#x95F4;&#x8D28;&#x8F6C;&#x6362; (EMT) &#x8FDB;&#x7A0B;&#x7684;&#x950C;&#x6307; E-&#x6846;&#x7ED3;&#x5408; (&#x745E;&#x4F2F;) &#x5BB6;&#x5EAD;&#x7684;&#x6210;&#x5458;&#x3002;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#x9AD8; Zeb2 mRNA &#x8868;&#x8FBE;&#x6C34;&#x5E73;&#x5728;&#x661F;&#x548C;&#x9020;&#x8840;&#x7956;&#x7EC6;&#x80DE; (HPCs)&#xFF0C;&#x5E76;&#x5BA1;&#x67E5;&#x4E86; Zeb2 &#x51FD;&#x6570;&#x4E2D;&#x901A;&#x8FC7;&#x4F7F;&#x7528; Tie2 Cre &#x548C;&#x53D8;&#x98CE;&#x91CF;&#x7A7A;&#x8C03;&#x590D;&#x5174;&#x91CD;&#x7EC4;&#x9F20;&#x6807;&#x7EBF;&#x6761;&#x4EF6;&#x5220;&#x9664;&#x529E;&#x6CD5;&#x7684;&#x9020;&#x8840;&#x529F;&#x80FD;&#x3002;&#x7EC6;&#x80DE;&#x7684;&#x8BE6;&#x7EC6;&#x7684;&#x5206;&#x6790;&#x8868;&#x660E; Zeb2 &#x662F;&#x9020;&#x8840;&#x7FA4;&#x96C6;&#x548C; HSC &#x5F62;&#x6210;&#x4E3B;&#x52A8;&#x8109; gonadomesonephros &#x5730;&#x533A;&#x7684;&#x80DA;&#x80CE;&#xFF0C;&#x53EF;&#x6709;&#x53EF;&#x65E0;&#xFF0C;&#x800C;&#x662F;&#x6B63;&#x5E38; HSC/HPC &#x5206;&#x5316;&#x7684;&#x5FC5;&#x8981;&#x6761;&#x4EF6;&#x3002;&#x6B64;&#x5916;&#xFF0C;Zeb2 &#x7F3A;&#x661F;/HPCs &#x672A;&#x80FD;&#x6B63;&#x786E;&#x6B96;&#x6C11;&#x7684;&#x80CE;&#x809D;&#x548C;/&#x6216;&#x9AA8;&#x9AD3;&#x548C;&#x663E;&#x793A;&#x5173;&#x8054;&#x589E;&#x52A0;&#x7684; &beta;1 &#x6574;&#x5408;&#x7D20;&#x548C; Cxcr4 &#x8868;&#x8FBE;&#x589E;&#x5F3A;&#x7C98;&#x63A5;&#x6027;&#x80FD;&#x3002;&#x6B64;&#x5916;&#xFF0C;Zeb2 &#x5220;&#x9664;&#x5BFC;&#x81F4;&#x80DA;&#x80CE; &#xFF08;Tie2 Cre&#xFF09; &#x548C;&#x56E0;&#x4E3A;&#x4E25;&#x91CD;&#x7684;&#x5934;&#x90E8;&#x5927;&#x51FA;&#x8840;&#x7684;&#x56F4;&#x4EA7;&#x671F; (&#x53D8;&#x98CE;&#x91CF;&#x590D;&#x5174;&#xFF09; &#x6740;&#x4F24;&#x529B;&#x548C;&#x8840;&#x7BA1;&#x751F;&#x6210;&#x7D20;-1 &#x6C34;&#x5E73;&#x4E0B;&#x964D;&#xFF0C;&#x4EE5;&#x53CA;&#x968F;&#x540E;&#x4E0D;&#x5F53;&#x5468;&#x7EC6;&#x80DE;&#x8986;&#x76D6;&#x5934;&#x90E8;&#x8840;&#x7BA1;&#x7CFB;&#x7EDF;&#x3002;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x63ED;&#x793A;&#x91CD;&#x8981;&#x89D2;&#x8272;&#xFF0C;&#x4E3A; Zeb2 &#x5728;&#x80DA;&#x80CE;&#x9020;&#x8840;&#x529F;&#x80FD;&#xFF0C;&#x6697;&#x793A; Zeb2 &#x6210;&#x4EBA;&#x9020;&#x8840;&#x76F8;&#x5173;&#x75BE;&#x75C5;&#x4E2D;&#x7684;&#x4F5C;&#x7528;&#x3002;

EMT &#x8C03;&#x8282;&#x5668; Zeb2/Sip1 &#x5C0F;&#x9F20;&#x80DA;&#x80CE;&#x9020;&#x8840;&#x5E72;/&#x7956;&#x7EC6;&#x80DE;&#x5206;&#x5316;&#x548C;&#x52A8;&#x5458;&#x81F3;&#x5173;&#x91CD;&#x8981;&#x3002;

Zeb2 (Sip1/Zfhx1b) is a member of the zinc-finger E-box-binding (ZEB) family of transcriptional repressors previously demonstrated to regulate epithelial-to-mesenchymal transition (EMT) processes during embryogenesis and tumor progression. We found high Zeb2 mRNA expression levels in HSCs and hematopoietic progenitor cells (HPCs), and examined Zeb2 function in hematopoiesis through a conditional deletion approach using the Tie2-Cre and Vav-iCre recombination mouse lines. Detailed cellular analysis demonstrated that Zeb2 is dispensable for hematopoietic cluster and HSC formation in the aorta-gonadomesonephros region of the embryo, but is essential for normal HSC/HPC differentiation. In addition, Zeb2-deficient HSCs/HPCs fail to properly colonize the fetal liver and/or bone marrow and show enhanced adhesive properties associated with increased Î²1 integrin and Cxcr4 expression. Moreover, deletion of Zeb2 resulted in embryonic (Tie2-Cre) and perinatal (Vav-icre) lethality due to severe cephalic hemorrhaging and decreased levels of angiopoietin-1 and, subsequently, improper pericyte coverage of the cephalic vasculature. These results reveal essential roles for Zeb2 in embryonic hematopoiesis and are suggestive of a role for Zeb2 in hematopoietic-related pathologies in the adult.

The EMT regulator Zeb2/Sip1 is essential for murine embryonic hematopoietic stem/progenitor cell differentiation and mobilization.

Zeb2 (Sip1/Zfhx1b) est un membre de la famille de liaison E-bo&icirc;te (ZEB) doigts de zinc des r&eacute;presseurs transcriptionnels d&eacute;montr&eacute; ant&eacute;rieurement &agrave; r&eacute;glementer le processus de transition &eacute;pith&eacute;liale-m&eacute;senchymateuse (EMT) au cours de l&#39;embryogen&egrave;se et la progression tumorale. Nous avons trouv&eacute; haute Zeb2 mRNA niveaux d&#39;expression dans les CSH et les cellules prog&eacute;nitrices h&eacute;matopo&iuml;&eacute;tiques (HPCs) et examin&eacute; la fonction Zeb2 dans l&#39;h&eacute;matopo&iuml;&egrave;se gr&acirc;ce &agrave; une approche de suppression conditionnelle utilisant les lign&eacute;es de souris de recombinaison Tie2-Cre et Vav-iCre. Analyse cellulaire d&eacute;taill&eacute;e a montr&eacute; que Zeb2 n&#39;est pas indispensable pour le cluster h&eacute;matopo&iuml;&eacute;tique et formation HSC dans la r&eacute;gion de l&#39;aorte-gonadomesonephros de l&#39;embryon, mais qu&#39;il est essentiel de diff&eacute;renciation normale de HSC/HPC. En outre, Zeb2-deficient CSH/HPCs ne pas correctement coloniser le foie f&oelig;tal et/ou de la moelle osseuse et montrent des propri&eacute;t&eacute;s adh&eacute;sives am&eacute;lior&eacute;es associ&eacute;es &agrave; l&#39;int&eacute;grine &beta;1 accrue et expression Cxcr4. En outre, suppression de Zeb2 entra&icirc;n&eacute; embryonnaire (Tie2-Cre) et p&eacute;rinatale (Vav-icre) l&eacute;talit&eacute; due &agrave; une h&eacute;morragie c&eacute;phalique s&eacute;v&egrave;re et une diminution des taux d&#39;angiopoietin-1 et, par la suite, pericyte une mauvaise couverture de la vascularisation c&eacute;phalique. Ces r&eacute;sultats r&eacute;v&egrave;lent un r&ocirc;le essentiel pour Zeb2 dans l&#39;h&eacute;matopo&iuml;&egrave;se embryonnaire et sugg&egrave;rent l&#39;existence d&#39;un r&ocirc;le pour Zeb2 dans les pathologies li&eacute;es &agrave; h&eacute;matopo&iuml;&eacute;tique chez l&#39;adulte.

Le r&eacute;gulateur de l&#39;EMT Zeb2/Sip1 est essentiel pour la mobilisation et la diff&eacute;renciation des cellules souches/prog&eacute;nitrices h&eacute;matopo&iuml;&eacute;tiques murins embryonnaires.

Zeb2 (Sip1/Zfhx1b) ist ein Mitglied der Familie Zinkfinger E-Box-Einband (ZEB) transkriptionelle Repressoren zuvor gezeigt, um epithelial-zu-Mesenchymale Transition (EMT) Prozesse w&auml;hrend der Embryogenese und Tumor Fortschreiten zu regulieren. Wir hohe Zeb2-mRNA Ausdruck Niveaus in HSCs und h&auml;matopoetischen Vorl&auml;uferzellen (HPCs) gefunden und Zeb2-Funktion in der Blutbildung durch eine bedingte L&ouml;schung-Ansatz mit den Tie2-Cre und Vav-iCre Rekombination Maus Linien untersucht. Detaillierte Zellul&auml;re Analyse zeigte, dass Zeb2 f&uuml;r h&auml;matopoetische Cluster und HSC-Bildung in der Aorta-Gonadomesonephros-Region des Embryos entbehrlich ist, aber f&uuml;r normale HSC/HPC-Differenzierung unerl&auml;sslich. Dar&uuml;ber hinaus nicht Zeb2-defizienten HSCs/HPCs richtig besiedeln die fetale Leber und/oder Knochenmark und zeigen verbesserte Klebeeigenschaften erh&ouml;hte &beta;1-Integrin und Cxcr4 Ausdruck zugeordnet. Dar&uuml;ber hinaus L&ouml;schen von Zeb2 f&uuml;hrte zu einer embryonalen (Tie2-Cre) und Perinatale (Vav-Icre) Letalit&auml;t aufgrund schwerer zephale Blutungen und verringerte Niveaus des ANGPTL1-1 und in der Folge unsachgem&auml;&szlig;er Pericyt Berichterstattung &uuml;ber die zephale Gef&auml;&szlig;system. Diese Ergebnisse lassen sich wesentliche Rollen f&uuml;r Zeb2 in embryonalen Blutbildung und sind suggestiv einer Rolle f&uuml;r Zeb2 in h&auml;matopoetischen-bezogene Krankheiten in den Erwachsenen.

Der EMT-Regler Zeb2/Sip1 unbedingt murinen embryonalen h&auml;matopoetische Stammzellen/Stammvater Zelldifferenzierung und Mobilisierung.

Zeb2 (Sip1/Zfhx1b) &egrave; un membro della famiglia E-box-associazione (ZEB) zinc-finger di repressori trascrizionali precedentemente dimostrato che regolano i processi di transizione epitelio mesenchimale (EMT) durante la progressione del tumore e l&#39;embriogenesi. Abbiamo trovato alta Zeb2 mRNA i livelli di espressione di HSCs e cellule progenitrici ematopoietiche (HPC) ed esaminato Zeb2 funzione nell&#39;ematopoiesi attraverso un approccio di eliminazione condizionale utilizzando le linee del mouse di ricombinazione Tie2-Cre e Vav-iCre. Dettagliata analisi cellulare ha dimostrato che Zeb2 &egrave; superfluo per cluster di hematopoietic e formazione HSC aorta-gonadomesonephros della regione dell&#39;embrione, ma &egrave; essenziale per la normale differenziazione HSC/HPC. Inoltre, deficit Zeb2 HSCs/HPC non riescono a colonizzare il fegato fetale e/o del midollo osseo e mostrano una maggiore propriet&agrave; adesive associati con maggiore &beta;1 integrina e l&#39;espressione di Cxcr4 correttamente. Inoltre, eliminazione di Zeb2 provocato embrionale (Tie2-Cre) e perinatale letalit&agrave; (Vav-icre) a causa di gravi emorragie cefalica e diminuzione dei livelli di angiopoietin-1 e, successivamente, copertura Pericita improprio del sistema vascolare cefalica. Questi risultati rivelano ruoli essenziali per Zeb2 nell&#39;emopoiesi embrionale e sono suggestivi di un ruolo per Zeb2 in patologie emopoietiche nell&#39;adulto.

Il regolatore di EMT Zeb2/Sip1 &egrave; essenziale per la mobilitazione e la differenziazione delle cellule staminali/progenitrici ematopoietiche embrionale murino.

Zeb2 (Sip1/Zfhx1b) &#x4EE5;&#x524D;&#x4E0A;&#x76AE;&#x9593;&#x8449;&#x8EE2;&#x63DB; &#xFF08;EMT&#xFF09; &#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x4E2D;&#x306E;&#x80DA;&#x767A;&#x751F;&#x3068;&#x816B;&#x760D;&#x306E;&#x9032;&#x884C;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;&#x5B9F;&#x8A3C;&#x8EE2;&#x5199;&#x30EA;&#x30D7;&#x30EC;&#x30C3;&#x30B5;&#x30FC;&#x306E;&#x4E9C;&#x925B;&#x30D5;&#x30A3;&#x30F3;&#x30AC;&#x30FC; E &#x30DC;&#x30C3;&#x30AF;&#x30B9; &#x30D0;&#x30A4;&#x30F3;&#x30C7;&#x30A3;&#x30F3;&#x30B0; (ZEB) &#x5BB6;&#x65CF;&#x306E;&#x30E1;&#x30F3;&#x30D0;&#x30FC;&#x3067;&#x3059;&#x3002;&#x9AD8; Zeb2 mRNA &#x767A;&#x73FE;&#x30EC;&#x30D9;&#x30EB;&#x306F; HSCs &#x3068;&#x9020;&#x8840;&#x524D;&#x99C6;&#x7D30;&#x80DE; (HPCs&#xFF09; &#x3067;&#x767A;&#x898B;&#x3057;&#x3001;Zeb2 &#x95A2;&#x6570;&#x9020;&#x8840; Tie2 Cre &#x3068; Vav &#x74B0;&#x5883;&#x7D44;&#x63DB;&#x3048;&#x30DE;&#x30A6;&#x30B9;&#x7DDA;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x6761;&#x4EF6;&#x4ED8;&#x304D;&#x524A;&#x9664;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x3092;&#x901A;&#x3058;&#x3066;&#x691C;&#x8A0E;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x643A;&#x5E2F;&#x96FB;&#x8A71;&#x306E;&#x8A73;&#x7D30;&#x306A;&#x5206;&#x6790;&#x306F;&#x3001;Zeb2 &#x9020;&#x8840;&#x30AF;&#x30E9;&#x30B9;&#x30BF;&#x30FC;&#x3068;&#x5927;&#x52D5;&#x8108; gonadomesonephros &#x5730;&#x57DF;&#x3001;&#x80DA;&#x306E; HSC &#x306E;&#x5F62;&#x6210;&#x306B;&#x4E0D;&#x53EF;&#x6B20;&#x3067;&#x3042;&#x308B;&#x304C;&#x901A;&#x5E38;&#x306E; HSC/HPC &#x5206;&#x5316;&#x306E;&#x305F;&#x3081;&#x4E0D;&#x53EF;&#x6B20;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;Zeb2 &#x6B20;&#x640D; HSCs/HPCs &#x6B63;&#x5E38;&#x80CE;&#x5150;&#x809D;&#x81D3;&#x3084;&#x9AA8;&#x9AC4;&#x3092;&#x690D;&#x6C11;&#x5730;&#x5316;&#x3057;&#x3001;&#x5897;&#x52A0; &beta; 1 &#x30A4;&#x30F3;&#x30C6;&#x30B0;&#x30EA;&#x30F3;&#x3068; Cxcr4 &#x5F0F;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x305F;&#x62E1;&#x5F35;&#x306E;&#x63A5;&#x7740;&#x6027;&#x3092;&#x793A;&#x3059;&#x306B;&#x5931;&#x6557;&#x3057;&#x307E;&#x3059;&#x3002;&#x307E;&#x305F;&#x3001;Zeb2 &#x306E;&#x524A;&#x9664;&#x306E;&#x80DA; &#xFF08;Tie2 Cre&#xFF09; &#x3068;&#x53B3;&#x3057;&#x3044;&#x982D;&#x90E8;&#x51FA;&#x8840;&#x306B;&#x3088;&#x308B;&#x5468;&#x7523;&#x671F; (Vav &#x74B0;&#x5883;&#xFF09; &#x81F4;&#x6B7B;&#x7D50;&#x679C;&#x304A;&#x3088;&#x3073; angiopoietin 1 &#x306E;&#x30EC;&#x30D9;&#x30EB;&#x3092;&#x6E1B;&#x5C11;&#x3057;&#x3001;&#x305D;&#x306E;&#x5F8C;&#x3001;&#x4E0D;&#x9069;&#x5207;&#x306A;&#x5468;&#x76AE;&#x7D30;&#x80DE;&#x30AB;&#x30D0;&#x30EC;&#x30C3;&#x30B8;&#x982D;&#x90E8;&#x306E;&#x8840;&#x7BA1;&#x7CFB;&#x306E;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D50;&#x679C;&#x306F;&#x3001;&#x4E0D;&#x53EF;&#x6B20;&#x306A;&#x5F79;&#x5272;&#x306E; Zeb2 &#x306B;&#x304A;&#x3051;&#x308B;&#x80DA;&#x9020;&#x8840;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3001;&#x6210;&#x4EBA;&#x306E;&#x9020;&#x8840;&#x95A2;&#x9023;&#x75C5;&#x614B;&#x306B;&#x304A;&#x3051;&#x308B; Zeb2 &#x306E;&#x5F79;&#x5272;&#x306E;&#x793A;&#x5506;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

EMT &#x30EC;&#x30AE;&#x30E5;&#x30EC;&#x30FC;&#x30BF; Zeb2/Sip1 &#x30DE;&#x30A6;&#x30B9;&#x80DA;&#x9020;&#x8840;&#x5E79;/&#x524D;&#x99C6;&#x7D30;&#x80DE;&#x306E;&#x5206;&#x5316;&#x3068;&#x52D5;&#x54E1;&#x306B;&#x4E0D;&#x53EF;&#x6B20;&#x3067;&#x3059;&#x3002;

Zeb2 (Sip1/Zfhx1b) &#xC774;&#xC804;&#xC5D0; embryogenesis &#xBC0F; &#xC885;&#xC591; &#xC9C4;&#xD589; &#xC911; &#xC0C1;&#xD53C; &#xB300; &#xC5FD; &#xC804;&#xD658; (&#xC751;&#xAE09;) &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xB97C; &#xADDC;&#xC81C;&#xB97C; &#xC2DC;&#xC5F0; &#xD558;&#xB294; transcriptional repressors&#xC758; &#xC544;&#xC5F0; &#xC190;&#xAC00;&#xB77D; &#xC804;&#xC790; &#xC0C1;&#xC790; &#xBC14;&#xC778;&#xB529; (&#xC81C; &#xBC14;&#xB514;) &#xAC00;&#xC871;&#xC758; &#xC77C;&#xC6D0; &#xC774;&#xB2E4;. &#xC6B0;&#xB9AC; HSCs &#xBC0F; &#xC870; &#xD608; &#xC870;&#xC0C1; &#xC138;&#xD3EC; (HPCs) &#xC2DD; &#xC218;&#xC900; &#xB192;&#xC740; Zeb2 Mrna&#xB97C; &#xBC1C;&#xACAC; &#xD558; &#xACE0; Tie2 Cre&#xC640; Vav iCre &#xC7AC;&#xC870;&#xD569; &#xB9C8;&#xC6B0;&#xC2A4; &#xC120;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC870;&#xAC74;&#xBD80; &#xC0AD;&#xC81C; &#xBC29;&#xC2DD;&#xC744; &#xD1B5;&#xD574; &#xC870;&#xC5D0; Zeb2 &#xD568;&#xC218;&#xB97C; &#xAC80;&#xC0AC;. &#xC0C1;&#xC138;&#xD55C; &#xC138;&#xD3EC; &#xBD84;&#xC11D; &#xC2DC;&#xC5F0; zeb2&#xB294; &#xD0DC;&#xC544;&#xC758; &#xB300;&#xB3D9;&#xB9E5; gonadomesonephros &#xC9C0;&#xC5ED;&#xC5D0; HSC &#xD615;&#xC131;&#xC5D0; &#xB300; &#xD55C; &#xC870; &#xD608; &#xD074;&#xB7EC;&#xC2A4;&#xD130; &#xBD88;&#xAC00;&#xACB0; &#xD558;&#xC9C0;&#xB9CC; &#xC815;&#xC0C1;&#xC801;&#xC778; HSC/HPC &#xCC28;&#xBCC4;&#xD654; &#xC704;&#xD574; &#xD544;&#xC218;&#xC801; &#xC774;&#xB2E4;. &#xB610;&#xD55C;, Zeb2 &#xBD88;&#xCDA9;&#xBD84; &#xD55C; HSCs/HPCs &#xB418;&#xC9C0; &#xC81C;&#xB300;&#xB85C; &#xC2DD;&#xBBFC;&#xC9C0; &#xD0DC;&#xC544; &#xAC04; &#xBC0F; &#xACE8; &#xC218; &#xC99D;&#xAC00; &beta;1 integrin&#xACFC; Cxcr4 &#xC2DD;&#xACFC; &#xAD00;&#xB828; &#xB41C; &#xD5A5;&#xC0C1; &#xB41C; &#xC811;&#xCC29; &#xC18D;&#xC131;&#xC744; &#xD45C;&#xC2DC; &#xD569;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, zeb2&#xC758; &#xC0AD;&#xC81C; &#xADC0;&#xCC29;&#xB418; &#xC5C8; &#xB2E4; &#xBC30;&#xC544; (Tie2 Cre) &#xBC0F; &#xCE58; &#xC0AC; (Vav-icre) perinatal &#xC728; &#xB54C;&#xBB38;&#xC5D0; &#xC2EC;&#xD55C; &#xB450; &#xBD80; &#xCD9C;&#xD608; &#xBC0F; angiopoietin-1&#xC758; &#xAC10;&#xC18C;&#xC640;, &#xADF8; &#xD6C4;, &#xB450; &#xBD80; &#xD608;&#xAD00;&#xC758; &#xBD80;&#xC801;&#xC808; &#xD55C; pericyte &#xBC94;&#xC704;. &#xC774; &#xACB0;&#xACFC; &#xBC30;&#xC544; &#xC870;&#xC5D0; zeb2&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC218;&#xC801;&#xC778; &#xC5ED;&#xD560;&#xC744; &#xACC4;&#xC2DC; &#xD558; &#xACE0;&#xB294; &#xC131;&#xC778;&#xC5D0;&#xC11C; &#xC870; &#xD608; &#xAD00;&#xB828; pathologies&#xC5D0; zeb2&#xC5D0; &#xB300; &#xD55C; &#xC5ED;&#xD560;&#xC758; &#xC554;&#xC2DC;.

&#xAD6C;&#xAE09; &#xB300; &#xB808; &#xADE4; &#xB808;&#xC774; &#xD130; Zeb2/Sip1 murine &#xBC30;&#xC544; &#xC870; &#xD608; &#xC904;&#xAE30;/&#xBFCC;&#xB9AC; &#xC138;&#xD3EC; &#xBD84;&#xD654; &#xBC0F; &#xB3D9;&#xC6D0;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC218;&#xC801;&#xC785;&#xB2C8;&#xB2E4;.

Zeb2 (Sip1/Zfhx1b) es un miembro de la familia de E-caja-obligatorio (ZEB) de zinc-finger de represores transcripcionales demostrado previamente para regular los procesos de transici&oacute;n epitelial-a-mesenquimal (EMT) durante la embriog&eacute;nesis y progresi&oacute;n tumoral. Encontramos alta Zeb2 mRNA niveles de expresi&oacute;n en HSCs y c&eacute;lulas progenitoras hematopoy&eacute;ticas (HPCs) y examina la funci&oacute;n de Zeb2 en la hematopoyesis a trav&eacute;s de un acercamiento de borrado condicional usando las l&iacute;neas de rat&oacute;n de recombinaci&oacute;n Tie2-Cre y Vav-iCre. An&aacute;lisis celular demostr&oacute; que Zeb2 es prescindible para cluster hematopoy&eacute;tico y formaci&oacute;n de HSC en la regi&oacute;n de aorta-gonadomesonephros del embri&oacute;n, pero es esencial para la diferenciaci&oacute;n normal de HSC/HPC. Adem&aacute;s, Zeb2-deficiente HSCs/HPCs no correctamente colonizan el h&iacute;gado fetal o la m&eacute;dula &oacute;sea y Mostrar propiedades mejoradas de adhesivos asociados integrinas &beta;1 aumento y expresi&oacute;n de Cxcr4. Por otra parte, la supresi&oacute;n del Zeb2 result&oacute; en embrionaria (Tie2-Cre) y la mortalidad perinatal de (Vav-icre) debido a la severa hemorragia cef&aacute;lica y disminuci&oacute;n de los niveles de angiopoyetina-1 y, posteriormente, pericyte inadecuada cobertura de la vasculatura cef&aacute;lica. Estos resultados revelan roles esenciales para Zeb2 de hematopoyesis embrionaria y sugieren un papel para Zeb2 en patolog&iacute;as relacionadas con hematopoy&eacute;tico en el adulto.

El regulador de la EMT Zeb2/Sip1 es esencial para la movilizaci&oacute;n y la diferenciaci&oacute;n de las c&eacute;lulas madre/progenitoras hematopoy&eacute;ticas embrionarias murinas.

Classic derivation of mouse embryonic stem (ES) cells from blastocysts is inefficient, strain-dependent, and requires expert skills. Over recent years, several major improvements have greatly increased the success rate for deriving mouse ES cell lines. The first improvement was the establishment of a user-friendly and reproducible medium-alternating protocol that allows isolation of ES cells from C57BL/6 transgenic mice with efficiencies of up to 75%. A recent report describes the use of this protocol in combination with leukemia inhibitory factor and pluripotin treatment, which made it possible to obtain ES cells from F1 strains with high efficiency. We report modifications of these protocols for user-friendly and reproducible derivation of mouse ES cells with efficiencies of up to 100%. Our protocol involves a long initial incubation of primary outgrowths from blastocysts with pluripotin, which results in the formation of large spherical outgrowths. These outgrowths are morphologically distinct from classical inner cell mass (ICM) outgrowths and can be easily picked and trypsinized. Pluripotin was omitted after the first trypsinization because we found that it blocks attachment of ES cells to the feeder layer and its removal facilitated formation of ES cell colonies. The newly established ES cells exhibited normal karyotypes and generated chimeras. In summary, our user-friendly modified protocol allows formation of large spherical ICM outgrowths in a robust and reliable manner. These outgrowths gave rise to ES cell lines with success rates of up to 100%.

Efficient and user-friendly pluripotin-based derivation of mouse embryonic stem cells.

Control of cellular (de)differentiation in a temporal, cell-specific, and exchangeable manner is of paramount importance in the field of reprogramming. Here, we have generated and characterized a mouse strain that allows iPSC generation through the Cre/loxP conditional and doxycycline/rtTA-controlled inducible expression of the OSKM reprogramming factors entirely from within the ROSA26 locus. After reprogramming, these factors can be replaced by genes of interest-for example, to enhance lineage-directed differentiation-with the use of a trap-coupled RMCE reaction. We show that, similar to ESCs, Dox-controlled expression of the cardiac transcriptional regulator Mesp1 together with Wnt inhibition enhances the generation of functional cardiomyocytes upon in vitro differentiation of such RMCE-retargeted iPSCs. This ROSA26-iPSC mouse model is therefore an excellent tool for studying both cellular reprogramming and lineage-directed differentiation factors from the same locus and will greatly facilitate the identification and ease of functional characterization of the genetic/epigenetic determinants involved in these complex processes.

The ROSA26-iPSC mouse: a conditional, inducible, and exchangeable resource for studying cellular (De)differentiation.

The conditional Cre/loxP system and/or the doxycycline (Dox) inducible Tet-on/off system are widely used in mouse transgenesis but often require time consuming, inefficient cloning/screening steps and extensive mouse breeding strategies. We have therefore developed a highly efficient Gateway- and recombinase-mediated cassette exchange (RMCE)-compatible system to target conditional and/or inducible constructs to the ROSA26 locus of F1 hybrid Bl6/129 ESCs, called G4 ROSALUC ESCs. By combining the Cre/loxP system with or without the inducible Tet-on system using Gateway cloning, we can rapidly generate spatial and/or temporal controllable gain-of-function constructs that can be targeted to the RMCE-compatible ROSA26 locus of the G4 ROSALUC ESCs with efficiencies close to 100 %. These novel ESC-based technologies allow for the creation of multiple gain-of-function conditional and/or inducible transgenic ESC clones and mouse lines in a highly efficient and locus specific manner. Importantly, incorporating insulator sequences into the Dox-inducible vector system resulted in robust, stable transgene expression in undifferentiated ESCs but could not fully overcome transgene mosaicism in the differentiated state.

Efficient ROSA26-based conditional and/or inducible transgenesis using RMCE-compatible F1 hybrid mouse embryonic stem cells.

Malignant transformation of the endothelium is rare, and hemangiosarcomas comprise only 1% of all sarcomas. For this reason and due to the lack of appropriate mouse models, the genetic mechanisms of malignant endothelial transformation are poorly understood. Here, we describe a hemangiosarcoma mouse model generated by deleting p53 specifically in the endothelial and hematopoietic lineages. This strategy led to a high incidence of hemangiosarcoma, with an average latency of 25 weeks. To study the in vivo roles of autocrine or endothelial cell autonomous VEGF signaling in the initiation and/or progression of hemangiosarcomas, we genetically deleted autocrine endothelial sources of VEGF in this mouse model. We found that loss of even a single conditional VEGF allele results in substantial rescue from endothelial cell transformation. These findings highlight the important role of threshold levels of autocrine VEGF signaling in endothelial malignancies and suggest a new approach for hemangiosarcoma treatment using targeted autocrine VEGF inhibition.

Loss of autocrine endothelial-derived VEGF significantly reduces hemangiosarcoma development in conditional p53-deficient mice.

Early T-cell precursor leukaemia (ETP-ALL) is a high-risk subtype of human leukaemia that is poorly understood at the molecular level. Here we report translocations targeting the zinc finger E-box-binding transcription factor ZEB2 as a recurrent genetic lesion in immature/ETP-ALL. Using a conditional gain-of-function mouse model, we demonstrate that sustained Zeb2 expression initiates T-cell leukaemia. Moreover, Zeb2-driven mouse leukaemia exhibit some features of the human immature/ETP-ALL gene expression signature, as well as an enhanced leukaemia-initiation potential and activated Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signalling through transcriptional activation of IL7R. This study reveals ZEB2 as an oncogene in the biology of immature/ETP-ALL and paves the way towards pre-clinical studies of novel compounds for the treatment of this aggressive subtype of human T-ALL using our Zeb2-driven mouse model.

ZEB2 drives immature T-cell lymphoblastic leukaemia development via enhanced tumour-initiating potential and IL-7 receptor signalling.

Arginase-1 catalyzes the conversion of arginine to ornithine and urea, which is the final step of the urea cycle used to remove excess ammonia from the body. Arginase-1 deficiency leads to hyperargininemia in mice and man with severe lethal consequences in the former and progressive neurological impairment to varying degrees in the latter. In a tamoxifen-induced arginase-1 deficient mouse model, mice succumb to the enzyme deficiency within 2 weeks after inducing the knockout and retain <2 % enzyme in the liver. Standard clinical care regimens for arginase-1 deficiency (low-protein diet, the nitrogen-scavenging drug sodium phenylbutyrate, ornithine supplementation) either failed to extend lifespan (ornithine) or only minimally prolonged lifespan (maximum 8 days with low-protein diet and drug). A conditional, tamoxifen-inducible arginase-1 transgenic mouse strain expressing the enzyme from the Rosa26 locus modestly extended lifespan of neonatal mice, but not that of 4-week old mice, when crossed to the inducible arginase-1 knockout mouse strain. Delivery of an arginase-1/enhanced green fluorescent fusion construct by adeno-associated viral delivery (rh10 serotype with a strong cytomegalovirus-chicken β-actin hybrid promoter) rescued about 30% of male mice with lifespan prolongation to at least 6 months, extensive hepatic expression and restoration of significant enzyme activity in liver. In contrast, a vector of the AAV8 serotype driven by the thyroxine-binding globulin promoter led to weaker liver expression and did not rescue arginase-1 deficient mice to any great extent. Since the induced arginase-1 deficient mouse model displays a much more severe phenotype when compared to human arginase-1 deficiency, these studies reveal that it may be feasible with gene therapy strategies to correct the various manifestations of the disorder and they provide optimism for future clinical studies.

Strategies to rescue the consequences of inducible arginase-1 deficiency in mice.

E-cadherin-mediated cell-cell adhesion is critical for naive pluripotency of cultured mouse embryonic stem cells (mESCs). E-cadherin-depleted mESC fail to downregulate their pluripotency program and are unable to initiate lineage commitment. To further explore the roles of cell adhesion molecules during mESC differentiation, we focused on p120 catenin (p120ctn). Although one key function of p120ctn is to stabilize and regulate cadherin-mediated cell-cell adhesion, it has many additional functions, including regulation of transcription and Rho GTPase activity. Here, we investigated the role of mouse p120ctn in early embryogenesis, mESC pluripotency and early fate determination. In contrast to the E-cadherin-null phenotype, p120ctn-null mESCs remained pluripotent, but their in vitro differentiation was incomplete. In particular, they failed to form cystic embryoid bodies and showed defects in primitive endoderm formation. To pinpoint the underlying mechanism, we undertook a structure-function approach. Rescue of p120ctn-null mESCs with different p120ctn wild-type and mutant expression constructs revealed that the long N-terminal domain of p120ctn and its regulatory domain for RhoA were dispensable, whereas its armadillo domain and interaction with E-cadherin were crucial for primitive endoderm formation. We conclude that p120ctn is not only an adaptor and regulator of E-cadherin, but is also indispensable for proper lineage commitment.

Lieven Haenebalcke

Department of Biomedical Molecular Biology

Ghent University

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Lieven Haenebalcke

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biomedical Molecular Biology

Ghent University

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Department of Biomedical Molecular Biology