crisprcas9-mediated-targeted-integration-vivo-using-homology-mediated

CRISPR/Cas9-mediated Targeted Integration <em>In Vivo</em> Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Chinese Academy of Sciences

Somatic nuclei can be reprogrammed into a pluripotent state by nuclear transfer, cell fusion and expression of transcription factors. However, these reprogramming processes are very inefficient, which has greatly hindered efforts to elucidate the underlying molecular mechanisms. Here, we report a new reprogramming strategy that combines the advantages of all three reprogramming methodologies into one process. We injected nuclei from cumulus cells into intact MII oocytes. Following activation, 80% of the reconstructed embryos developed to the blastocyst stage, and tetraploid (4N) embryonic stem (ES) cell lines were generated at a rate of 30% per reconstructed oocyte. We also generated triploid (3N) ES cells after injection of somatic nuclei into activated oocytes. 4N and 3N ES cells expressed pluripotent markers and differentiated into cell types of three embryonic germ layers in vivo. Moreover, all ES cells generated histocompatible, differentiated cells after being engrafted in immunocompetent B6D2F1 mice, showing that ES cells derived from this reprogramming strategy might serve as a source of genetically tailored tissues for transplantation. Thus, we have established a simple and highly efficient reprogramming procedure that provides a system for investigating the molecular mechanisms involved in somatic reprogramming.

High-efficiency somatic reprogramming induced by intact MII oocytes.

Mice generated after round spermatid injection into haploid two-cell blastomeres.

The low success rate of somatic nuclear transfer (NT) is hypothesized to be mainly due to functional defects in the trophoblast cell lineage rather than the inner cell mass (ICM); this hypothesis, however, remains to be tested directly. Here we separated the ICMs from cloned blastocysts and aggregated the cloned ICM with two fertilization-derived (FD) tetraploid (4N) embryos. We found that the full-term development of cloned ICMs was dramatically improved after the trophoblast cells in the cloned blastocysts were replaced by cells from tetraploid embryos, thus providing direct evidence that defects in trophoblast cell lineage underlie the low success rate of somatic NT.

Defects in trophoblast cell lineage account for the impaired in vivo development of cloned embryos generated by somatic nuclear transfer.

&#x7CBE;&#x5B50;&#x548C;&#x5375;&#x5B50;&#x8FDB;&#x884C;&#x53D7;&#x7CBE;&#x540E;&#x91CD;&#x65B0;&#x8BBE;&#x7F6E;&#x8C03;&#x6574;&#x7684;&#x72EC;&#x7279;&#x540E;&#x751F;&#x4FEE;&#x6539;&#x3002;&#x5728;&#x53D7;&#x7CBE;&#x5375;&#x4E2D;&#x7684;&#x7236;&#x7CFB;&#x57FA;&#x56E0;&#x7EC4;&#x7ECF;&#x5386;&#x524D;&#x7B2C;&#x4E00;&#x6B21;&#x6709;&#x4E1D;&#x5206;&#x88C2;&#x7684;&#x6D3B;&#x6027; DNA &#x53BB;&#x7532;&#x57FA;&#x3002;&#x8FD9;&#x4E2A;&#x7236;&#x4EB2;&#x8868;&#x89C2;&#x57FA;&#x56E0;&#x7EC4;&#x91CD;&#x5851;&#x673A;&#x5236;&#x4E0E;&#x751F;&#x7269;&#x610F;&#x4E49;&#x6709;&#x4ECD;&#x4E0D;&#x6E05;&#x695A;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x62A5;&#x544A;&#x5185;&#x5C0F;&#x9F20;&#x53D7;&#x7CBE;&#x5375;, 5-&#x7532;&#x57FA;&#x80DE;&#x5627;&#x5576; (5mC) &#x6C27;&#x5316;&#x53D1;&#x751F;&#x5BF9;&#x7236;&#x4EB2;&#x7684;&#x57FA;&#x56E0;&#x7EC4;&#xFF0C;5mC &#x6539; 5-hydroxymethylcytosine (5hmC)&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x6211;&#x4EEC;&#x5728;&#x8BC1;&#x660E; Tet3 &#x53CC;&#x52A0;&#x6C27;&#x9176; &#xFF08;&#x6CE8;&#x91CA; 5&#xFF09; &#x4E13;&#x95E8;&#x5728;&#x96C4;&#x6027;&#x539F;&#x6838;&#x6D53;&#x7F29;&#x3002;&#x5728; Tet3 &#x7F3A;&#x4ECE;&#x6761;&#x4EF6;&#x57FA;&#x56E0;&#x6572;&#x9664;&#x5C0F;&#x9F20;&#x7684;&#x53D7;&#x7CBE;&#x5375;&#xFF0C;&#x7236;&#x7CFB;&#x57FA;&#x56E0;&#x7EC4;&#x8F6C;&#x6362;&#x6210; 5hmC 5mC &#x672A;&#x80FD;&#x53D1;&#x751F;&#x548C; 5mC &#x6C34;&#x5E73;&#x4FDD;&#x6301;&#x4E0D;&#x53D8;&#x3002;Tet3 &#x865A;&#x8BC1;&#x4E5F;&#x963B;&#x788D;&#x4E86;&#x7236;&#x4EB2;&#x7684; Oct4 &#x548C; Nanog &#x57FA;&#x56E0;&#x7684;&#x7532;&#x57FA;&#x8FC7;&#x7A0B;&#x548C;&#x5EF6;&#x8FDF;&#x7684;&#x65E9;&#x671F;&#x80DA;&#x80CE;&#x7684;&#x7236;&#x6D3E;&#x751F; Oct4 &#x8F6C;&#x57FA;&#x56E0;&#x968F;&#x540E;&#x6FC0;&#x6D3B;&#x3002;&#x96CC;&#x6027;&#x5C0F;&#x9F20;&#x80DA;&#x82BD;&#x884C;&#x4E2D;&#x8017;&#x5C3D;&#x7684; Tet3 &#x663E;&#x793A;&#x4E25;&#x91CD;&#x964D;&#x4F4E;&#x7684;&#x751F;&#x80B2;&#x529B;&#x548C;&#x6742;&#x5408;&#x6027;&#x7A81;&#x53D8;&#x5B50;&#x5973;&#x7F3A;&#x4E4F;&#x5B55;&#x4EA7;&#x5987; Tet3 &#x906D;&#x53D7;&#x53D1;&#x75C5;&#x7387;&#x589E;&#x52A0;&#x7684;&#x53D1;&#x5C55;&#x5931;&#x8D25;&#x3002;&#x5375;&#x6BCD;&#x7EC6;&#x80DE;&#x4E5F;&#x7F3A;&#x4E4F; Tet3 &#x4F3C;&#x4E4E;&#x6709;&#x91CD;&#x65B0;&#x7F16;&#x7A0B;&#x4F53;&#x7EC6;&#x80DE;&#x7EC6;&#x80DE;&#x6838;&#x6CE8;&#x5165;&#x7684;&#x80FD;&#x529B;&#x964D;&#x4F4E;&#x3002;&#x56E0;&#x6B64;&#xFF0C;Tet3 &#x4ECB;&#x5BFC;&#x7684; DNA &#x7F9F;&#x57FA;&#x5316;&#x6240;&#x6D89;&#x53CA;&#x7684;&#x8868;&#x89C2;&#x9057;&#x4F20;&#x91CD;&#x7F16;&#x7A0B;&#x7684;&#x81EA;&#x7136;&#x53D7;&#x7CBE;&#x540E;&#x7684;&#x5408;&#x5B50;&#x7236;&#x7CFB; DNA&#xFF0C;&#x4E5F;&#x53EF;&#x80FD;&#x6709;&#x52A9;&#x4E8E;&#x4F53;&#x7EC6;&#x80DE;&#x6838;&#x91CD;&#x7F16;&#x7A0B;&#x5728;&#x52A8;&#x7269;&#x514B;&#x9686;&#x8FC7;&#x7A0B;&#x3002;

Tet3 DNA &#x53CC;&#x52A0;&#x6C27;&#x9176;&#x8868;&#x89C2;&#x9057;&#x4F20;&#x91CD;&#x7F16;&#x7A0B;&#x7684;&#x5375;&#x6BCD;&#x7EC6;&#x80DE;&#x7684;&#x4F5C;&#x7528;&#x3002;

Sperm and eggs carry distinctive epigenetic modifications that are adjusted by reprogramming after fertilization. The paternal genome in a zygote undergoes active DNA demethylation before the first mitosis. The biological significance and mechanisms of this paternal epigenome remodelling have remained unclear. Here we report that, within mouse zygotes, oxidation of 5-methylcytosine (5mC) occurs on the paternal genome, changing 5mC into 5-hydroxymethylcytosine (5hmC). Furthermore, we demonstrate that the dioxygenase Tet3 (ref. 5) is enriched specifically in the male pronucleus. In Tet3-deficient zygotes from conditional knockout mice, paternal-genome conversion of 5mC into 5hmC fails to occur and the level of 5mC remains constant. Deficiency of Tet3 also impedes the demethylation process of the paternal Oct4 and Nanog genes and delays the subsequent activation of a paternally derived Oct4 transgene in early embryos. Female mice depleted of Tet3 in the germ line show severely reduced fecundity and their heterozygous mutant offspring lacking maternal Tet3 suffer an increased incidence of developmental failure. Oocytes lacking Tet3 also seem to have a reduced ability to reprogram the injected nuclei from somatic cells. Therefore, Tet3-mediated DNA hydroxylation is involved in epigenetic reprogramming of the zygotic paternal DNA following natural fertilization and may also contribute to somatic cell nuclear reprogramming during animal cloning.

The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes.

Sperme et des &oelig;ufs portent des modifications &eacute;pig&eacute;n&eacute;tiques distinctives qui sont ajust&eacute;es par reprogrammation apr&egrave;s la f&eacute;condation. Le g&eacute;nome paternel dans un zygote subit une d&eacute;m&eacute;thylation active de l&#39;ADN avant la premi&egrave;re mitose. L&#39;importance biologique et les m&eacute;canismes de ce remodelage &eacute;pig&eacute;nome paternel sont rest&eacute;es peu claires. Nous rapportons ici que, au sein des zygotes de souris, l&#39;oxydation de la 5-m&eacute;thylcytosine (5mC) s&#39;effectue sur le g&eacute;nome paternel, changeant 5mC en 5-hydroxym&eacute;thylcytosine (5hmC). En outre, nous d&eacute;montrons que la dioxyg&eacute;nase Tet3 (R&eacute;f. 5) est enrichi, plus pr&eacute;cis&eacute;ment dans le pronucl&eacute;us m&acirc;le. Chez les zygotes d&eacute;ficients en Tet3 de souris knock-out conditionnel, conversion du g&eacute;nome paternel de 5mC dans 5hmC ne parvient pas &agrave; se produire et le niveau de 5mC reste constant. Carence en Tet3 aussi g&ecirc;ne le processus de la d&eacute;m&eacute;thylation des g&egrave;nes Oct4 et Nanog paternels et retarde l&#39;activation d&#39;un transg&egrave;ne Oct4 paternellement d&eacute;riv&eacute;e dans les embryons au stade pr&eacute;coces. Appauvri en Tet3 dans la lign&eacute;e germinale des souris femelles montrent une r&eacute;duction de la f&eacute;condit&eacute; et leur prog&eacute;niture mutant h&eacute;t&eacute;rozygote manque Tet3 maternelle subissent une augmentation de l&#39;incidence de l&#39;insuffisance du d&eacute;veloppement. Ovocytes manque aussi les Tet3 semblent avoir une diminution de la capacit&eacute; de reprogrammer l&#39;injection noyaux de cellules somatiques. Par cons&eacute;quent, hydroxylation de l&#39;ADN induite par le Tet3 est impliqu&eacute;e dans la reprogrammation &eacute;pig&eacute;n&eacute;tique de l&#39;ADN paternel zygotique apr&egrave;s la fertilisation naturelle et peut-&ecirc;tre aussi contribuer &agrave; la reprogrammation nucl&eacute;aire de cellules somatiques au cours du clonage animal.

Le r&ocirc;le de l&#39;ADN Tet3 dioxyg&eacute;nase dans la reprogrammation &eacute;pig&eacute;n&eacute;tique par ovocytes.

Sperma und Eizellen tragen markante epigenetische Ver&auml;nderungen, die angepasst sind, durch eine Anpassung nach der Befruchtung. Das v&auml;terliche Genom in einer Zygote durchl&auml;uft aktive DNA-Demethylierung vor der ersten Mitose. Die biologische Bedeutung und die Mechanismen dieser v&auml;terlichen Epigenome Umbau blieben unklar. Hier berichten wir, dass innerhalb der Maus Zygoten, Oxidation von 5-Methylcytosine (5mC) auf das v&auml;terliche Genom, auftritt 5mC in 5-Hydroxymethylcytosine (5hmC) ge&auml;ndert werden. Dar&uuml;ber hinaus zeigen wir, dass die Dioxygenase Tet3 (Ref. 5) ist speziell in der m&auml;nnlichen Pronucleus angereichert. In Tet3-defizienten Zygoten bedingte Knock-out-M&auml;use v&auml;terlichen Genoms von 5mC in 5hmC Konvertierungsfehler auftreten, und das Niveau der 5mC konstant bleibt. Ein Mangel an Tet3 auch behindert den Demethylierung Prozess der v&auml;terlichen Oct4 und Nanog Gene und Verz&ouml;gerungen die nachfolgende Aktivierung ein v&auml;terlich abgeleiteten Oct4-Transgen in fr&uuml;hen Embryonen. Weibliche M&auml;use ausgelaugt Tet3 in die Keimbahn zeigen stark reduzierte Fruchtbarkeit und deren heterozygote mutant Nachwuchs fehlt m&uuml;tterlichen Tet3 leiden eine erh&ouml;hte Inzidenz von Entwicklungsst&ouml;rungen scheitern. Eizellen fehlen Tet3 auch scheinen eine reduzierte F&auml;higkeit, die injizierten Kerne von somatischen Zellen umzuprogrammieren. Daher DNA Tet3-vermittelte Hydroxylierung epigenetischen Reprogrammierung der zygotic v&auml;terliche DNA nach nat&uuml;rlichen Befruchtung beteiligt und kann auch dazu beitragen, somatische nukleare Neuprogrammierung, w&auml;hrend das Klonen von Tieren.

Die Rolle der Tet3 DNA-Dioxygenase in epigenetischen Reprogrammierung von Eizellen.

Sperma e uova portano modifiche epigenetiche distintive che vengono regolate dalla riprogrammazione dopo la fecondazione. Il genoma paterno in uno zigote subisce la demetilazione del DNA attivo prima della prima mitosi. Il significato biologico e i meccanismi di questo rimodellamento Epigenoma paterna sono rimasti poco chiari. Qui riportiamo che, all&#39;interno di zigoti mouse, ossidazione della 5-metilcitosina (5mC) si verifica sul genoma paterno, cambiando 5mC in 5-idrossimetilcitosina (5hmC). Inoltre, dimostriamo che la diossigenasi Tet3 (Rif. 5) &egrave; arricchito in particolare il maschio pronucleus. In zigoti Tet3-carenti da topi knockout condizionale, conversione del genoma paterno di 5mC in 5hmC non riesce a verificarsi e il livello di 5mC rimane costante. Deficit di Tet3 inoltre ostacola il processo di demetilazione dei geni paterni Oct4 e Nanog e ritardano l&#39;attivazione successiva di un transgene Oct4 paterna derivato in embrioni in anticipo. Topi femminili impoveriti di Tet3 nella linea germinale Visualizza gravemente ridotta fecondit&agrave; e loro prole mutante eterozigote manca Tet3 materna subiscono un aumento dell&#39;incidenza di fallimento dello sviluppo. Gli ovociti manca anche Tet3 sembrano avere una ridotta capacit&agrave; di riprogrammare i nuclei iniettati da cellule somatiche. Pertanto, Tet3-mediato del DNA idrossilazione &egrave; coinvolto nella riprogrammazione epigenetica del DNA paterno zygotic dopo fertilizzazione naturale e pu&ograve; anche contribuire alla riprogrammazione nucleare di cellule somatiche durante la clonazione animale.

Il ruolo del DNA Tet3 diossigenasi in riprogrammazione epigenetica di ovociti.

&#x7CBE;&#x5B50;&#x3068;&#x5375;&#x53D7;&#x7CBE;&#x5F8C;&#x306E;&#x30EA;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30DF;&#x30F3;&#x30B0;&#x306B;&#x3088;&#x3063;&#x3066;&#x8ABF;&#x6574;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x72EC;&#x7279;&#x306E;&#x30A8;&#x30D4;&#x30B8;&#x30A7;&#x30CD;&#x30C6;&#x30A3;&#x30C3;&#x30AF;&#x4FEE;&#x98FE;&#x3092;&#x904B;&#x3076;&#x3002;&#x7236;&#x65B9;&#x306E;&#x30B2;&#x30CE;&#x30E0;&#x53D7;&#x7CBE;&#x5375;&#x306B;&#x6700;&#x521D;&#x306E;&#x6709;&#x7CF8;&#x5206;&#x88C2;&#x306E;&#x524D;&#x306B;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306A; DNA &#x8131;&#x30E1;&#x30C1;&#x30EB;&#x5316;&#x3092;&#x7D4C;&#x308B;&#x3002;&#x751F;&#x7269;&#x5B66;&#x7684;&#x610F;&#x7FA9;&#x3068;&#x3053;&#x306E;&#x7236;&#x65B9;&#x30A8;&#x30D4;&#x30B2;&#x30CE;&#x30E0;&#x306E;&#x30EA;&#x30E2;&#x30C7;&#x30EA;&#x30F3;&#x30B0;&#x306E;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306F;&#x660E;&#x767D;&#x3067;&#x306A;&#x304F;&#x6B8B;&#x3063;&#x305F;&#x304C;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x30DE;&#x30A6;&#x30B9;&#x53D7;&#x7CBE;&#x5375;&#x5185;&#x306B;&#x7236;&#x65B9;&#x306E;&#x30B2;&#x30CE;&#x30E0;&#x4E0A; 5-&#x30E1;&#x30C1;&#x30EB;&#x30B7;&#x30C8;&#x30B7;&#x30F3; (5mC) &#x306E;&#x9178;&#x5316;&#x304C;&#x767A;&#x751F;&#x3059;&#x308B;&#x3053;&#x3068;&#x3001;5mC (5hmC) 5-&#x30D2;&#x30C9;&#x30ED;&#x30AD;&#x30B7;&#x30E1;&#x30C1;&#x30EB;&#x30B7;&#x30C8;&#x30B7;&#x30F3;&#x306B;&#x5909;&#x66F4;&#x5831;&#x544A;&#x3057;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x6211;&#x3005; &#x306F;&#x3053;&#x3068;&#x3092;&#x793A;&#x3059;&#x30B8;&#x30AA;&#x30AD;&#x30B7;&#x30B2;&#x30CA;&#x30FC;&#x30BC; tet3 &#x304C;&#x679C;&#x305F;&#x3059; (&#x6587;&#x732E; 5&#xFF09; &#x3067;&#x7537;&#x6027;&#x306E;&#x96C4;&#x6027;&#x5177;&#x4F53;&#x7684;&#x306B;&#x6FC3;&#x7E2E;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;Tet3 &#x304C;&#x679C;&#x305F;&#x3059;&#x6B20;&#x4E4F;&#x53D7;&#x7CBE;&#x30B3;&#x30F3;&#x30C7;&#x30A3;&#x30B7;&#x30E7;&#x30CA;&#x30EB;&#x30CE;&#x30C3;&#x30AF; &#x30A2;&#x30A6;&#x30C8; &#x30DE;&#x30A6;&#x30B9;&#x304B;&#x3089;&#x767A;&#x751F;&#x3059;&#x308B; 5hmC &#x306B; 5mC &#x306E;&#x7236;&#x65B9;&#x306E;&#x30B2;&#x30CE;&#x30E0;&#x306E;&#x5909;&#x63DB;&#x304C;&#x5931;&#x6557;&#x3057;&#x305F;&#x3057;&#x3001;5mC &#x306E;&#x30EC;&#x30D9;&#x30EB;&#x3092;&#x4E00;&#x5B9A;&#x306B;&#x4FDD;&#x305F;&#x308C;&#x307E;&#x3059;&#x3002;Tet3 &#x304C;&#x679C;&#x305F;&#x3059;&#x306E;&#x6B20;&#x4E4F;&#x306F;&#x307E;&#x305F;&#x3001;&#x7236;&#x65B9;&#x306E; Oct4 &#x3068; Nanog &#x907A;&#x4F1D;&#x5B50;&#x306E;&#x8131;&#x30E1;&#x30C1;&#x30EB;&#x5316;&#x53CD;&#x5FDC;&#x904E;&#x7A0B;&#x3092;&#x59A8;&#x3052;&#x308B;&#x3001;&#x521D;&#x671F;&#x80DA;&#x306B;&#x304A;&#x3051;&#x308B;&#x7236;&#x6027;&#x6D3E;&#x751F; Oct4 transgene &#x306E;&#x5F8C;&#x7D9A;&#x306E;&#x6D3B;&#x6027;&#x5316;&#x3092;&#x9045;&#x5EF6;&#x3057;&#x307E;&#x3059;&#x3002;&#x96CC;&#x30DE;&#x30A6;&#x30B9;&#x306E;&#x751F;&#x6B96;&#x7CFB;&#x5217;&#x3067;&#x306E; tet3 &#x304C;&#x679C;&#x305F;&#x3059;&#x3092;&#x67AF;&#x6E07;&#x6DF1;&#x523B;&#x306A;&#x4F4E;&#x591A;&#x7523;&#x3068;&#x6BCD;&#x4F53; tet3 &#x304C;&#x679C;&#x305F;&#x3059;&#x6B20;&#x3051;&#x3066;&#x30D8;&#x30C6;&#x30ED;&#x5909;&#x7570;&#x5B50;&#x5B6B;&#x767A;&#x9054;&#x969C;&#x5BB3;&#x306E;&#x767A;&#x751F;&#x7387;&#x306E;&#x5897;&#x52A0;&#x306B;&#x82E6;&#x3057;&#x307F;&#x307E;&#x3059;&#x3002;Tet3 &#x304C;&#x679C;&#x305F;&#x3059;&#x3082;&#x6B20;&#x3051;&#x3066;&#x3044;&#x308B;&#x5375;&#x6BCD;&#x7D30;&#x80DE;&#x306F;&#x4F53;&#x7D30;&#x80DE;&#x304B;&#x3089;&#x6CE8;&#x5165;&#x3055;&#x308C;&#x305F;&#x6838;&#x518D;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30E0;&#x3059;&#x308B;&#x80FD;&#x529B;&#x304C;&#x4F4E;&#x4E0B;&#x3092;&#x6301;&#x3063;&#x3066;&#x3044;&#x308B;&#x3088;&#x3046;&#x3067;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;tet3 &#x304C;&#x679C;&#x305F;&#x3059;&#x4EF2;&#x4ECB;&#x3055;&#x308C;&#x305F; DNA &#x6C34;&#x9178;&#x5316;&#x30A8;&#x30D4;&#x30B8;&#x30A7;&#x30CD;&#x30C6;&#x30A3;&#x30C3;&#x30AF;&#x306A;&#x81EA;&#x7136;&#x53D7;&#x7CBE;&#x30B3;&#x30B3;&#x30E4;&#x30B7;&#x306E;&#x7236;&#x65B9;&#x306E; DNA &#x306E;&#x30EA;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30DF;&#x30F3;&#x30B0;&#x3001;&#x4F53;&#x7D30;&#x80DE;&#x6838;&#x306E;&#x4E2D;&#x306B;&#x30AF;&#x30ED;&#x30FC;&#x30F3;&#x52D5;&#x7269;&#x30EA;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30DF;&#x30F3;&#x30B0;&#x306B;&#x307E;&#x305F;&#x8CA2;&#x732E;&#x3059;&#x308B;&#x304B;&#x3082;&#x3057;&#x308C;&#x306A;&#x3044;&#x3002;

&#x5375;&#x6BCD;&#x7D30;&#x80DE;&#x306B;&#x3088;&#x308B;&#x30A8;&#x30D4;&#x30B8;&#x30A7;&#x30CD;&#x30C6;&#x30A3;&#x30C3;&#x30AF;&#x306A;&#x30EA;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30DF;&#x30F3;&#x30B0;&#x306B; tet3 &#x304C;&#x679C;&#x305F;&#x3059; DNA &#x30B8;&#x30AA;&#x30AD;&#x30B7;&#x30B2;&#x30CA;&#x30FC;&#x30BC;&#x306E;&#x5F79;&#x5272;&#x3002;

&#xC815;&#xC790;&#xC640; &#xC218;&#xC815; &#xD6C4; &#xC7AC;&#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D; &#xD558; &#xC5EC; &#xC870;&#xC815; &#xD558;&#xB294; &#xB3C5;&#xD2B9;&#xD55C; epigenetic &#xC218;&#xC815; &#xC218;&#xD589; &#xD569;&#xB2C8;&#xB2E4;. &#xC811;&#xD569; &#xC790;&#xC5D0;&#xC11C; &#xC544;&#xBC84;&#xC9C0;&#xC758; &#xAC8C;&#xB188; &#xCCAB; &#xBC88;&#xC9F8; &#xC720;&#xC0AC; &#xBD84;&#xC5F4; &#xD558;&#xAE30; &#xC804;&#xC5D0; &#xD65C;&#xC131; DNA demethylation&#xB97C; &#xACAA; &#xC2B5;. &#xC0DD;&#xBB3C;&#xD559; &#xC758;&#xBBF8; &#xBC0F;&#xC774; &#xC544;&#xBC84;&#xC9C0; epigenome &#xAC1C;&#xC7A5;&#xC758; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC740; &#xBD88;&#xBD84;&#xBA85; &#xB0A8;&#xC544; &#xC788;&#xB2E4;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC; &#xBCF4;&#xACE0;&#xB294;, &#xB9C8;&#xC6B0;&#xC2A4; zygotes&#xC5D0;&#xC11C; 5-methylcytosine (5mC)&#xC758; &#xC0B0;&#xD654; &#xC544;&#xBC84;&#xC9C0;&#xC758; &#xAC8C;&#xB188;&#xC5D0;&#xC11C; &#xBC1C;&#xC0DD; 5mC 5-hydroxymethylcytosine (5hmC)&#xB85C; &#xBCC0;&#xACBD; &#xD569;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, &#xC6B0;&#xB9AC;&#xB294; &#xBCF4;&#xC5EC; dioxygenase Tet3 (5 &#xCC38;&#xACE0;) &#xD2B9;&#xD788; &#xB0A8;&#xC131; pronucleus&#xC5D0;&#xC11C;&#xC5D0;&#xC11C; &#xD48D;&#xC131; &#xD558; &#xAC8C;. &#xC870;&#xAC74;&#xBD80; &#xB179;&#xC544;&#xC6C3; &#xC950;&#xC5D0;&#xC11C; Tet3 &#xBD88;&#xCDA9;&#xBD84; &#xD55C; zygotes, 5hmc&#xB85C; 5mC &#xC544;&#xBC84;&#xC9C0;&#xC758; &#xAC8C;&#xB188; &#xBCC0;&#xD658; &#xBC1C;&#xC0DD;&#xC744; &#xC2E4;&#xD328; &#xD558; &#xACE0; 5mC &#xC218;&#xC900;&#xC744; &#xC77C;&#xC815; &#xD558; &#xAC8C; &#xC720;&#xC9C0;. Tet3&#xC758; &#xACB0;&#xD54D;&#xB3C4; &#xC544;&#xBC84;&#xC9C0; Oct4 &#xBC0F; Nanog &#xC720;&#xC804;&#xC790;&#xC758; demethylation &#xACFC;&#xC815;&#xC744; &#xBC29;&#xD574; &#xD558; &#xACE0; &#xCD08;&#xAE30; &#xBC30;&#xC544;&#xC5D0; paternally &#xD30C;&#xC0DD;&#xB41C; Oct4 transgene&#xC758; &#xD6C4;&#xC18D; &#xD65C;&#xC131;&#xD654;&#xB97C; &#xC9C0;&#xC5F0;. &#xC5EC;&#xC131; &#xC950; &#xC138;&#xADE0; &#xB77C;&#xC778;&#xC5D0;&#xC11C; tet3&#xC758; &#xACE0;&#xAC08; &#xC2EC;&#xAC01; &#xD558; &#xAC8C; &#xAC10;&#xC18C; fecundity &#xB098;&#xD0C0;&#xB098;&#xACE0; &#xBAA8;&#xC131; Tet3 &#xBD80;&#xC871; heterozygous &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xC790;&#xC190;&#xC774; &#xADF8;&#xB4E4;&#xC758; &#xACE0;&#xD1B5;&#xC744; &#xBC1C;&#xB2EC; &#xC7A5;&#xC560;&#xC758; &#xBC1C;&#xC0DD;&#xB960;&#xC774; &#xC99D;&#xAC00; &#xD569;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C; Tet3 &#xBD80;&#xC871; oocytes &#xCCB4;&#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xC8FC;&#xC785; &#xB41C; &#xD575; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC744; &#xAC10;&#xC18C; &#xB2A5;&#xB825; &#xAC83; &#xAC19;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, Tet3-&#xC911;&#xC7AC; DNA hydroxylation &#xC790;&#xC5F0; &#xC218;&#xC815;&#xC5D0; &#xB530;&#xB77C; zygotic &#xC544;&#xBC84;&#xC9C0;&#xC758; DNA&#xC758; epigenetic &#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D;&#xC5D0; &#xCC38;&#xC5EC; &#xD558; &#xACE0; &#xB610;&#xD55C; &#xCCB4;&#xC138;&#xD3EC; &#xD575; &#xB3D9;&#xBB3C; &#xBCF5;&#xC81C; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D;&#xC5D0; &#xAE30;&#xC5EC;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

Tet3 DNA dioxygenase oocytes &#xC5EC; epigenetic &#xC7AC;&#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D;&#xC5D0;&#xC11C; &#xC5ED;&#xD560;.

Espermatozoides y &oacute;vulos llevan distintivas modificaciones epigen&eacute;ticas que se ajustan por reprogramaci&oacute;n despu&eacute;s de la fertilizaci&oacute;n. El genoma paterno en un cigoto sufre desmetilaci&oacute;n activa del ADN antes de la primera mitosis. La importancia biol&oacute;gica y mecanismos de esta remodelaci&oacute;n del epigenoma paternal han permanecido claros. Aqu&iacute; divulgamos que, dentro de cigotos de rat&oacute;n, oxidaci&oacute;n de la 5-METILCITOSINA (5mC) se produce en el genoma paterno, cambiando 5mC en 5-hidroximetilcitosina (5hmC). Adem&aacute;s, demostramos que el dioxygenase Tet3 (Ref. 5) se enriquece espec&iacute;ficamente en el pron&uacute;cleo masculino. En cigotos Tet3 deficientes de ratones knockout condicional, conversi&oacute;n de genoma paterno de 5mC en 5hmC no se produce y se mantiene constante el nivel de 5mC. Deficiencia de Tet3 tambi&eacute;n impide el proceso de la desmetilaci&oacute;n de los genes paternos de Oct4 y Nanog y retrasa la activaci&oacute;n subsiguiente de un transgen de Oct4 paternal derivado en embriones tempranos. Ratones hembras empobrecidos de Tet3 en la l&iacute;nea germinal muestran severamente reducida fecundidad y su descendencia mutante heterozig&oacute;tico carecer Tet3 materna sufre una mayor incidencia de fracaso del desarrollo. Ovocitos carecer Tet3 tambi&eacute;n parecen tener una capacidad reducida para reprogramar los n&uacute;cleos inyectado de c&eacute;lulas som&aacute;ticas. Por lo tanto, hidroxilaci&oacute;n mediada por Tet3 ADN participa en reprogramaci&oacute;n epigen&eacute;tica del ADN paterno cig&oacute;ticos tras fecundaci&oacute;n natural y tambi&eacute;n puede contribuir a la reprogramaci&oacute;n nuclear de c&eacute;lulas som&aacute;ticas durante la clonaci&oacute;n animal.

El papel del ADN Tet3 dioxygenase en reprogramaci&oacute;n epigen&eacute;tica de ovocitos.

Haploid cells are amenable for genetic analysis. Recent success in the derivation of mouse haploid embryonic stem cells (haESCs) via parthenogenesis has enabled genetic screening in mammalian cells. However, successful generation of live animals from these haESCs, which is needed to extend the genetic analysis to the organism level, has not been achieved. Here, we report the derivation of haESCs from androgenetic blastocysts. These cells, designated as AG-haESCs, partially maintain paternal imprints, express classical ESC pluripotency markers, and contribute to various tissues, including the germline, upon injection into diploid blastocysts. Strikingly, live mice can be obtained upon injection of AG-haESCs into MII oocytes, and these mice bear haESC-carried genetic traits and develop into fertile adults. Furthermore, gene targeting via homologous recombination is feasible in the AG-haESCs. Our results demonstrate that AG-haESCs can be used as a genetically tractable fertilization agent for the production of live animals via injection into oocytes.

Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells.

Mammalian oocytes undergo a cortical reaction after fertilization, releasing cortical granules and other proteins into the perivitelline space and inhibiting polyspermy. Few studies have evaluated the biological functions and properties of these proteins.

Secretome profile of mouse oocytes after activation using mass spectrum.

Identification of the function of all genes in the mammalian genome is critical in understanding basic mechanisms of biology. However, the diploidy of mammalian somatic cells has greatly hindered efforts to elucidate the gene function in numerous biological processes by mutagenesis-based genetic approaches. Recently, mouse haploid embryonic stem (haES) cells have been successfully isolated from parthenogenetic and androgenetic embryos, providing an ideal tool for genetic analyses. In these studies, mouse haES cells have already shown that they could be used in cell-based forward or reverse genetic screenings and in generating gene-targeting via homologous recombination. In particular, haES cells from androgenetic embryos can be employed as novel, renewable form of fertilization agent for yielding live-born mice via injection into oocytes, thus showing the possibility that genetic analysis can be extended from cellular level to organism level.

Haploid embryonic stem cells: an ideal tool for mammalian genetic analyses.

Recent success in the derivation of haploid embryonic stem cells (haESCs) from mouse via parthenogenesis and androgenesis has enabled genetic screening in mammalian cells and generation of gene-modified animals. However, whether haESCs can be derived from primates remains unknown. Here, we report the derivation of haESCs from parthenogenetic blastocysts of Macaca fascicularis monkeys. These cells, termed as PG-haESCs, are pluripotent and can differentiate to cells of three embryonic germ layers in vitro or in vivo. Interestingly, the haploidy of one monkey PG-haESC line (MPH1) is more stable compared with that of the other one (MPH2), as shown by the existence of haploid cells for more than 140 days without fluorescence-activated cell sorting (FACS) enrichment of haploid cells. Importantly, transgenic monkey PG-haESC lines can be generated by lentivirus- and piggyBac transposon-mediated gene transfer. Moreover, genetic screening is feasible in monkey PG-haESCs. Our results demonstrate that PG-haESCs can be generated from monkeys, providing an ideal tool for genetic analyses in primates.

Generation of haploid embryonic stem cells from Macaca fascicularis monkey parthenotes.

Regulation of ubiquitin-proteasome system (UPS), which controls the turnover of short-lived proteins in eukaryotic cells, is critical in maintaining cellular proteostasis. Here we show that USP14, a major deubiquitinating enzyme that regulates the UPS, is a substrate of Akt, a serine/threonine-specific protein kinase critical in mediating intracellular signaling transducer for growth factors. We report that Akt-mediated phosphorylation of USP14 at Ser432, which normally blocks its catalytic site in the inactive conformation, activates its deubiquitinating activity in vitro and in cells. We also demonstrate that phosphorylation of USP14 is critical for Akt to regulate proteasome activity and consequently global protein degradation. Since Akt can be activated by a wide range of growth factors and is under negative control by phosphoinosotide phosphatase PTEN, we suggest that regulation of UPS by Akt-mediated phosphorylation of USP14 may provide a common mechanism for growth factors to control global proteostasis and for promoting tumorigenesis in PTEN-negative cancer cells.

Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system.

Targeted integration of transgenes can be achieved by strategies based on homologous recombination (HR), microhomology-mediated end joining (MMEJ) or non-homologous end joining (NHEJ). The more generally used HR is inefficient for achieving gene integration in animal embryos and tissues, because it occurs only during cell division, although MMEJ and NHEJ can elevate the efficiency in some systems. Here we devise a homology-mediated end joining (HMEJ)-based strategy, using CRISPR/Cas9-mediated cleavage of both transgene donor vector that contains guide RNA target sites and ∼800 bp of homology arms, and the targeted genome. We found no significant improvement of the targeting efficiency by the HMEJ-based method in either mouse embryonic stem cells or the neuroblastoma cell line, N2a, compared to the HR-based method. However, the HMEJ-based method yielded a higher knock-in efficiency in HEK293T cells, primary astrocytes and neurons. More importantly, this approach achieved transgene integration in mouse and monkey embryos, as well as in hepatocytes and neurons in vivo, with an efficiency much greater than HR-, NHEJ- and MMEJ-based strategies. Thus, the HMEJ-based strategy may be useful for a variety of applications, including gene editing to generate animal models and for targeted gene therapies.

Homology-mediated end joining-based targeted integration using CRISPR/Cas9.

Precisely targeted genome editing is highly desired for clinical applications. However, the widely used homology-directed repair (HDR)-based genome editing strategies remain inefficient for certain in vivo applications. We here demonstrate a microhomology-mediated end-joining (MMEJ)-based strategy for precisely targeted gene integration in transfected neurons and hepatocytes in vivo with efficiencies up to 20%, much higher (up to 10 fold) than HDR-based strategy in adult mouse tissues. As a proof of concept of its therapeutic potential, we demonstrate the efficacy of MMEJ-based strategy in correction of Fah mutation and rescue of Fahliver failure mice, offering an efficient approach for precisely targeted gene therapies.

CRISPR/Cas9 - Mediated Precise Targeted Integration In Vivo Using a Double Cut Donor with Short Homology Arms.

The CRISPR/Cas9 system is an efficient gene-editing method, but the majority of gene-edited animals showed mosaicism, with editing occurring only in a portion of cells. Here we show that single gene or multiple genes can be completely knocked out in mouse and monkey embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs (spaced 10-200 bp apart) that target only a single key exon of each gene. Phenotypic analysis of F0 mice following targeted deletion of eight genes on the Y chromosome individually demonstrated the robustness of this approach in generating knockout mice. Importantly, this approach delivers complete gene knockout at high efficiencies (100% on Arntl and 91% on Prrt2) in monkey embryos. Finally, we could generate a complete Prrt2 knockout monkey in a single step, demonstrating the usefulness of this approach in rapidly establishing gene-edited monkey models.

One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs.

Stimulation of TNFR1 by TNFα can promote three distinct alternative mechanisms of cell death: necroptosis, RIPK1-independent and -dependent apoptosis. How cells decide which way to die is unclear. Here, we report that TNFα-induced phosphorylation of RIPK1 in the intermediate domain by TAK1 plays a key role in regulating this critical decision. Using phospho-Ser321 as a marker, we show that the transient phosphorylation of RIPK1 intermediate domain induced by TNFα leads to RIPK1-independent apoptosis when NF-κB activation is inhibited by cycloheximide. On the other hand, blocking Ser321 phosphorylation promotes RIPK1 activation and its interaction with FADD to mediate RIPK1-dependent apoptosis (RDA). Finally, sustained phosphorylation of RIPK1 intermediate domain at multiple sites by TAK1 promotes its interaction with RIPK3 and necroptosis. Thus, absent, transient and sustained levels of TAK1-mediated RIPK1 phosphorylation may represent distinct states in TNF-RSC to dictate the activation of three alternative cell death mechanisms, RDA, RIPK1-independent apoptosis and necroptosis.TNFα can promote three distinct mechanisms of cell death: necroptosis, RIPK1-independent and dependent apoptosis. Here the authors show that TNFα-induced phosphorylation of RIPK1 in the intermediate domain by TAK1 plays a key role in regulating this decision.

Regulation of RIPK1 activation by TAK1-mediated phosphorylation dictates apoptosis and necroptosis.

The CRISPR/Cas9 system has become an efficient gene editing method for generating cells carrying precise gene mutations, including the rearrangement and deletion of chromosomal segments. However, whether an entire chromosome could be eliminated by this technology is still unknown.

CRISPR/Cas9-mediated targeted chromosome elimination.

Generation of knock-in cynomolgus monkey via CRISPR/Cas9 editing.

Despite rapid progresses in the genome-editing field, in vivo simultaneous overexpression of multiple genes remains challenging. We generated a transgenic mouse using an improved dCas9 system that enables simultaneous and precise in vivo transcriptional activation of multiple genes and long noncoding RNAs in the nervous system. As proof of concept, we were able to use targeted activation of endogenous neurogenic genes in these transgenic mice to directly and efficiently convert astrocytes into functional neurons in vivo. This system provides a flexible and rapid screening platform for studying complex gene networks and gain-of-function phenotypes in the mammalian brain.

Article	Year
High-efficiency somatic reprogramming induced by intact MII oocytes. Cell research\| PubMed ID: 20603641	2010
Mice generated after round spermatid injection into haploid two-cell blastomeres. Cell research\| PubMed ID: 21423273	2011
Defects in trophoblast cell lineage account for the impaired in vivo development of cloned embryos generated by somatic nuclear transfer. Cell stem cell\| PubMed ID: 21474101	2011
The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature\| PubMed ID: 21892189	2011
Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells. Cell\| PubMed ID: 22541431	2012
Secretome profile of mouse oocytes after activation using mass spectrum. Journal of assisted reproduction and genetics\| PubMed ID: 22573034	2012
Haploid embryonic stem cells: an ideal tool for mammalian genetic analyses. Protein & cell\| PubMed ID: 23055337	2012
Generation of haploid embryonic stem cells from Macaca fascicularis monkey parthenotes. Cell research\| PubMed ID: 23856644	2013
Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system. eLife\| PubMed ID: 26523394	2015
Homology-mediated end joining-based targeted integration using CRISPR/Cas9. Cell research\| PubMed ID: 28524166	2017
CRISPR/Cas9 - Mediated Precise Targeted Integration In Vivo Using a Double Cut Donor with Short Homology Arms. EBioMedicine\| PubMed ID: 28527830	2017
One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs. Cell research\| PubMed ID: 28585534	2017
Regulation of RIPK1 activation by TAK1-mediated phosphorylation dictates apoptosis and necroptosis. Nature communications\| PubMed ID: 28842570	2017
CRISPR/Cas9-mediated targeted chromosome elimination. Genome biology\| PubMed ID: 29178945	2017
Generation of knock-in cynomolgus monkey via CRISPR/Cas9 editing. Cell research\| PubMed ID: 29327726	2018
In vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR-dCas9-activator transgenic mice. Nature neuroscience\| PubMed ID: 29335603	2018

Linyu Shi

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