ES2627552T3 - Edición de genoma en ratas usando nucleasas con dedos de cinc - Google Patents
Edición de genoma en ratas usando nucleasas con dedos de cinc Download PDFInfo
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- ES2627552T3 ES2627552T3 ES09830727.5T ES09830727T ES2627552T3 ES 2627552 T3 ES2627552 T3 ES 2627552T3 ES 09830727 T ES09830727 T ES 09830727T ES 2627552 T3 ES2627552 T3 ES 2627552T3
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
Un método in vitro de modificación de un gen IgM endógeno en una célula de rata, comprendiendo el método: introducir, en la célula de rata, uno o más ARNm que codifican primera y segunda nucleasas con dedos de cinc (ZFN) que se unen a sitios diana en el gen IgM endógeno en condiciones tales que las ZFN escindan el uno o más genes celulares endógenos tal que el gen IgM endógeno se modifique, en el que la primera ZFN comprende una ZFP que tiene las regiones de hélice de reconocimiento como se muestra en las ZFP designadas 17747, 17759, 17756, 17767 y 17764 como se expone en una única fila de la Tabla 6.
Description
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por ejemplo, por co-inmunoprecipitación, ensayos de dos híbridos o complementación, tanto genética como bioquímica. Véase, por ejemplo, Fields et al., (1989) Nature 340:245-246; patente de EE.UU. N.º 5.585.245 y documento PCT WO 98/44350.
Nucleasas con dedos de cinc
En el presente documento se describen nucleasas con dedos de cinc (ZFN) que pueden usarse para la edición genómica (por ejemplo, escisión, alteración, inactivación y/o mutación aleatoria) de uno o más genes de rata. Las ZFN comprenden una proteína de dedos de cinc (ZFP) y un dominio (de escisión) de nucleasa (por ejemplo, mediodominio de escisión).
A. Proteínas de dedos de cinc
Los dominios de unión de dedos de cinc pueden manipularse para unirse a una secuencia de elección. Véanse, por ejemplo, Beerli et al., (2002) Nature Biotechnol. 20:135-141; Pabo et al., (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al., (2001) Nature Biotechnol. 19:656-660; Segal et al., (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al., (2000) Curr. Opin. Struct. Biol. 10:411-416. Un dominio de unión de dedos de cinc manipulado puede tener una especificidad de unión novedosa, en comparación con una proteína de dedos de cinc que se produce naturalmente. Los métodos de manipulación incluyen, pero no se limitan a, diseño racional y diversos tipos de selección. El diseño racional incluye, por ejemplo, usar bases de datos que comprenden secuencias de triplete (o cuadruplete) de nucleótidos y secuencias de aminoácidos de dedos de cinc individuales, en las que cada secuencia de nucleótidos de triplete o cuadruplete está asociada con una o más secuencias de aminoácidos de dedos de cinc que se unen a la secuencia de triplete o cuadruplete particular. Véanse, por ejemplo, las patentes de EE.UU. del mismo solicitante
6.453.242 y 6.534.261, incorporadas por referencia en el presente documento en sus totalidades.
Métodos de selección a modo de ejemplo, que incluyen presentación en fagos y sistemas de dos híbridos, se desvelan en las patentes de EE.UU. 5.789.538; 5.925.523; 6.007.988; 6.013.453; 6.410.248; 6.140.466; 6.200.759; y 6.242.568; además de los documentos WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 y GB 2.338.237. Además, se ha descrito el potenciamiento de la especificidad de unión por dominios de unión de dedos de cinc, por ejemplo, en el documento WO 02/077227 del mismo solicitante.
Selección de sitios diana; ZFP y métodos de diseño y construcción de proteínas de fusión (y polinucleótidos que codifican las mismas) son conocidos para aquellos expertos en la materia y se describen en detalle en las publicaciones de solicitud de patente de EE.UU. N.º 20050064474 y 20060188987, incorporadas por referencia en sus totalidad en el presente documento.
Además, como se ha desvelado en estas y otras referencias, los dominios de dedos de cinc y/o las proteínas de dedos de cinc multi-dedos pueden unirse juntas usando cualquier secuencia conectora adecuada, que incluye, por ejemplo, conectores de 5 o más aminoácidos de longitud (por ejemplo, TGEKP (SEQ ID NO:1), TGGQRP (SEQ ID NO:2), TGQKP (SEQ ID NO:3) y/o TGSQKP (SEQ ID NO:4)). Véanse, por tanto, las patentes de EE.UU. N.º 6.479.626; 6.903.185; y 7.153.949 para secuencias conectoras a modo de ejemplo de 6 o más aminoácidos de longitud. Las proteínas descritas en el presente documento pueden incluir cualquier combinación de conectores adecuados entre los dedos de cinc individuales de la proteína.
Como se describe más adelante, un dominio de unión de cuatro, cinco o seis dedos está fusionado con un mediodominio de escisión, tal como, por ejemplo, el dominio de escisión de una endonucleasa de restricción de tipo IIS tal como Fok I. Se usan uno o más pares de tales fusiones de medio-dominios de dedo de cinc/nucleasa para la escisión dirigida, como se desvela, por ejemplo, en la publicación de patente de EE.UU. N.º 20050064474.
Para la escisión dirigida, los bordes próximos de los sitios de unión pueden separarse 5 o más pares de nucleótidos, y cada una de las proteínas de fusión puede unirse a una hebra opuesta del ADN diana. Todas las combinaciones en parejas 1 pueden usarse para la escisión dirigida de un gen de rata. Siguiendo la presente divulgación, las ZFN pueden dirigirse a cualquier secuencia en el genoma de rata.
En algunas realizaciones, el dominio de unión de ADN es un dominio manipulado de un efector TAL derivado del patógeno de planta Xanthomonas (véanse Boch et al, (2009) Science 29 Oct 2009 (10.1126/science, 117881) y Moscou y Bogdanove, (2009) Science 29 Oct 2009 (10.1126/science, 1178817).
B. Dominios de escisión
Las ZFN también comprenden una nucleasa (dominio de escisión, medio-dominio de escisión). La porción del dominio de escisión de las proteínas de fusión desveladas en el presente documento puede obtenerse de cualquier endonucleasa o exonucleasa. Endonucleasas a modo de ejemplo de las que puede derivarse un dominio de escisión incluyen, pero no se limitan a, endonucleasas de restricción y endonucleasas de asentamiento. Véase, por ejemplo, 2002-2003 Catalogue, New England Biolabs, Beverly, MA; y Belfort et al., (1997) Nucleic Acids Res. 25:3379-3388. Se conocen enzimas adicionales que escinden ADN (por ejemplo, nucleasa S1; nucleasa de judías mungo; DNasa I pancreática; nucleasa microcócica; endonucleasa HO de levadura; véase también Linn et al., (eds.) Nucleases, Cold
13
- Nombre de ZFN
- F1 F2 F3 F4
- 10360
- RSDNLAR (SEQ ID NO:103) RSDHLTT (SEQ ID NO:104) RSDNLSQ (SEQ ID NO:105) ASNDRKK (SEQ ID NO:106)
- 10362
- RSDHLSE (SEQ ID NO:87) RSAALAR (SEQ ID NO:107) RSDHLSE (SEQ ID NO:87) RNQHRIT (SEQ ID NO:108)
- 10361
- RSDNLAR (SEQ ID NO:103) RSDHLTT (SEQ ID NO:104) RSDNLSE (SEQ ID NO:43) DSRSRIN (SEQ ID NO:109)
- 10363
- DRSHLSR (SEQ ID NO:110) RSDDLTR (SEQ ID NO:16) RSDHLSR (SEQ ID NO:44) DRSHLAR (SEQ ID NO:12)
Tabla 2: Dianas de ZFN específicas de p53 de rata
- Nombre de ZFN
- Sitio diana (5' a 3')
- 10356
- aaGCGGAAGGGGCGggccatagcccggg (SEQ ID NO:111)
- 10358
- caGGACGTGCGGAAtgcgttaagggaat (SEQ ID NO:112)
- 10359
- caGGACGTGCGGAAtgcgttaagggaat (SEQ ID NO:112)
- 10357
- aaGCGGAAGGGGCGggccatagcccggg (SEQ ID NO:111)
- 10360
- ctTCCCAGTGGGAGgtgacagaaccctg (SEQ ID NO:113)
- 10362
- acCGGCGGGTGCGGgcggactgcactta (SEQ ID NO:114)
- 10361
- ctTCCCAGTGGGAGgtgacagaaccctg (SEQ ID NO:113)
- 10363
- ccGGCGGGtGCGGGCggactgcacttag (SEQ ID NO:115)
Se transfectaron plásmidos que codificaban ZFN en células C6 de rata. Para determinar la actividad de ZFN en el
5 locus p53, se realizaron ensayos de desapareamiento de CEL-I esencialmente según las instrucciones del fabricante (Trangenomic SURVEYOR™). Se recogieron las células y se preparó ADN cromosómico usando un kit Quickextract™ según indicaciones del fabricante (Epicentre®). Se amplificó por PCR la región apropiada del locus p53 usando ADN polimerasa Accuprime™ High-fidelity (Invitrogen). Se calentaron reacciones de PCR a 94 ºC, y se enfriaron gradualmente hasta temperatura ambiente. Se mezclaron aproximadamente 200 ng del ADN hibridado con
10 0,33 µl de enzima CEL-I y se incubaron durante 20 minutos a 42 ºC. Los productos de reacción se analizaron por electroforesis en gel de poliacrilamida en 1X tampón Tris-borato-EDTA.
Los resultados se muestran en la Figura 1 donde se probaron en combinación diversos pares de ZFN específicas de p53 descritas en las Tablas 1 y 2. El porcentaje de desapareamientos, una medida de la actividad de NHEJ, se muestra debajo de cada carril. Los resultados indican que estas ZFN son activas contra este locus de rata.
15 Se diseñaron ZFN dirigidas a GFP y se incorporaron en plásmidos esencialmente como se describe en Urnov et al. (2005) Nature 435(7042):646-651. Se cribaron pares de ZFN para la actividad en un sistema cromosómico basado en levadura como se describe en U.S. N.º de serie 12/284.887, titulada "Rapid in vivo Identification of Biologically Active Nucleases". Brevemente, se transformaron ZFN inducibles por galactosa en una cepa de levadura que contiene un indicador de hibridación monocatenaria (ySSA) integrado, que consistió en la secuencia de eGFP
20 completa insertada entre dos segmentos de solapamiento del gen MEL1 conducido por el promotor PGK. La expresión de las ZFN se indujo durante 6 horas, luego se reprimió durante 18 horas, tiempo después del cual se usó un ensayo colorimétrico estándar para cuantificar la cantidad de proteína MEL1 en el sobrenadante.
Las hélices de reconocimiento para diseños de dedos de cinc de GFP representativos se muestran a continuación en la Tabla 3.
25
17
Tabla 3: Diseños de dedos de cinc de GFP
- Nombre de ZFN
- F1 F2 F3 F4 F5 F6
- 16833 "33"
- RSAHLSR (SEQ ID NO:5) TSANLSR (SEQ ID NO:6) RSDNLSV (SEQ ID NO:7) DRSNLTR (SEQ ID NO:8)
- 16834 "34"
- RSDTLSQ (SEQ ID NO:9) QRDBRIK (SEQ ID NO:10) DRSNLSR (SEQ ID NO:11) DRSHLAR (SEQ ID NO: 12) DRSNLTR (SEQ ID NO:8)
- 16855 "55"
- RSDHLSA (SEQ ID NO: 13) DSSTRKT (SEQ ID NO: 14) TSGSLSR (SEQ ID NO:15) RSDDLTR (SEQ ID NO: 16) TSANLSR (SEQ ID NO:6)
- 16856 "56"
- RSDNLST (SEQ ID NO:17) DSSSRIK (SEQ ID NO:18) RSAVLSE (SEQ ID NO:19) TNSNRIT (SEQ ID NO:20) RSAHLSR (SEQ ID NO:5) QSGNLAR (SEQ ID NO:21)
- 16859 "59"
- TSGSLSR (SEQ ID NO:15) QSGSLTR (SEQ ID NO:22) TSGSLSR (SEQ ID NO:15) QSSDLRR (SEQ ID NO:23) RSDALSR (SEQ ID NO:24) TSGSLTR (SEQ ID NO:25)
- 16860 "60"
- RSANLSV (SEQ ID NO:30) DRANLSR (SEQ ID NO:29) DRSDLSR (SEQ ID NO:28) RSDSLSV (SEQ ID NO:27) DSSARKK (SEQ ID NO:26)
Se muestran sitios diana de los diseños de dedos de cinc de GFP a continuación en la Tabla 4. Los nucleótidos en el sitio diana que se ponen en contacto por las hélices de reconocimiento de ZFP se indican en letras mayúsculas; nucleótidos que no se ponen en contacto se indican en minúscula.
Tabla 4: Sitios diana de dedos de cinc de GFP
- Nombre de ZFN
- Sitio diana (5' a 3')
- 16833
- GACCAGGATGGG (SEQ ID NO:31)
- 16834
- GACGGCGACgTAAACG (SEQ ID NO:32)
- 16855
- GATGCGGTTcACCAGG (SEQ ID NO:33)
- 16856
- GAAGGGCATCGAcTTCAAG (SEQ ID NO:34)
- 16859
- GTTGTGGCTGTTGTAGTT (SEQ ID NO:35)
- 16860
- ATCATGGCCGACAAG (SEQ ID NO:36)
Se transfectaron construcciones de expresión activas de ZFN dirigidas a GFP en células C6 de rata que contenían una construcción de expresión de GFP.
10 Como se muestra en la FIG. 2, todos los pares de ZFN probados escindieron el gen GFP en las células diana.
Ejemplo 2: Las ZFN inducen la rotura dirigida en ratas transgénicas (ejemplo comparativo)
ZFN específicas de GFP como se describe en el Ejemplo 1 también se introdujeron por inyección pronuclear (PNI) o inyección citoplásmica (de ARNm de ZFN) a concentraciones variables en embriones unicelulares obtenidos de ratas transgénicas que expresan GFP descritos en Michalkiewicz et al. (2007) J. Amer. Phys. Society 293:H881-H894.
15 Véase la Fig. 3.
Los embriones inyectados se cultivaron durante 2-3 días hasta que alcanzaron el estadio de 2-4 células. Algunos de los embriones de 2-4 células se transfirieron entonces a hembras pseudo-preñadas. Se extrajo ADN de tanto embriones cultivados como embriones transferidos y se evaluó la escisión del gen GFP.
Los resultados del diferente modo de inyección y la concentración de ZFN inyectada en los embriones inyectados 20 usando el par de ZFN 16859/16860 se muestran en la Tabla 5 a continuación.
18
sin la adición de ADN genómico de rata no mutante, sugiriendo que ninguna de las ratas tiene una mutación homocigótica.
Se clonaron productos de PCR GJC153F/GJC154R de las ratas positivas y se secuenciaron. Una descripción de los alelos mutados está en la Tabla 8.
Tabla 8
- Rata
- Alelo Cifra % de NHEJ aprox. Notas
- 6
- No mutante 8 49
- 6
- Δ9 2 deleción en marco de DEN
- 7
- No mutante 5 31
- 7
- Δ5 1 fuera de marco
- 7
- Δ13 1 fuera de marco
- 7
- Δ15 3 deleción en marco de SDENL
- 7
- Δ18 1 deleción en marco de DENLA
- 7
- Δ39 1 del. en marco de SCESPLSDENLVA
- 8
- No mutante 7 25
- 8
- Δ3, mut. de 7b pb 3 deleción en marco de D, E->P
- 8
- Δ23 2 fuera de marco
- 19
- No mutante 7 70
- 19
- Δ64 17 deleción más grande, fuera de marco
- 46
- No mutante 9 47
- 46
- Δ5 2 fuera de marco
- Cifra se refiere al número de veces que se aisló una secuencia particular. % de NHEJ es el porcentaje aproximado de cromosomas modificados en el ADN de la cola
La secuenciación del locus de IgM en estas ratas confirmó los resultados del ensayo de nucleasas Surveyor™. Todas las deleciones solapan los sitios de unión de ZFN. El espectro de deleciones pequeñas observado aquí es típico de mutación mediada por NHEJ. Las ratas 7 y 8 tienen más de un alelo mutado y son, por tanto, mosaicos
10 para la mutación de IgM. Aunque la secuenciación de ratas 6, 19 y 46 solo dio un alelo mutado, puede ser mosaico para la modificación de IgM en otros tejidos.
Así, las ZFN modificaron satisfactoriamente el locus de IgM de rata endógeno.
Para determinar si el propio plásmido de ZFN se integró en el genoma de rata, se desarrolló un ensayo basado en PCR para probar la integración de plásmidos de ZFN. Brevemente, se mezclaron ADN genómico de rata y el 15 plásmido de ZFN para imitar una frecuencia de inserción de plásmido de una vez por genoma. El realizar 35 ciclos de amplificación por PCR de esta mezcla con un oligonucleótido en el promotor CAG (5'-GCT AAC CAT GTT CAT GCC TTC-3') (SEQ ID NO:49) y otro oligonucleótido en la región 2A del plásmido (5'-CAT CCT AGG GCC GGG ATT CTC-3') (SEQ ID NO:50) dio una banda de 1338 pb (Figura 7, carril 3). Cuando se analizó ADN genómico de ratas no mutantes y las cinco modificadas con ZFN, no fue detectable producto de PCR; que indica que la inserción del
20 plásmido en el genoma de rata no es un evento de alta frecuencia.
Además, la rata N.º 19 modificada en IgM se analizó adicionalmente por ensayo de CEL-I y secuenciación. Como se muestra en las Figuras 7A y 7B, las ZFN de IgM produjeron una deleción de 64 pares de bases en esta rata en el locus IgM.
Finalmente, también se evaluó la escisión de ZFN en sitios inespecíficos. Se usó un algoritmo informático para
25 predecir la localización de los sitios inespecíficos más probables (Doyon et al (2008) Nature Biotechnology 26(6):702-708). Se ensayaron todos los sitios inespecíficos probables para la modificación de ZFN usando el ensayo
21
de nucleasas Surveyor™ como se ha descrito anteriormente. Los resultados de este análisis se muestran en la Tabla 9 y las Figs. 9A-C.
Tabla 9
- Sitio
- Puntuación Secuencia Mm Gen PCR Frag. A Frag. B Acierto
- 1
- 8,18E-17 8 320 221 99 No
- 2
- 2,90E-18 9 325 222 103 No
- 3
- 1,67E-18 9 379 239 40 No
- 4
- 7,75E-19 8 322 218 104 No
- 5
- 6,44E-19 9 Pde4d 396 200 196 No
- 6
- 1,49E-19 8 LOC499 913 342 200 142 No
- 7
- 1,14E-19 9 567 317 250 No
- 8
- 1,07E-19 9 Actn1 354 255 99 No
- Mm: desapareamientos con respecto al sitio diana previsto Frag. A, B: tamaños esperados de los productos de escisión por nucleasa Surveyor™ Acierto: Ratas que muestran productos de escisión por nucleasa Surveyor™ correctos
5 Como se muestra, ningún sitio inespecífico probado mostró evidencia de modificación. Como se muestra, ningún sitio inespecífico probado mostró evidencia de modificación. El análisis de secuenciación de la rata N.º 19 positiva para CEL-I mostrado en la Figura 9A y cinco de sus crías mostradas en la Figura 11 en el Sitio 1 revelaron que la señal positiva para CEL-I era debida a un SNP cerca del posible sitio inespecífico. El desapareamiento se produce debido a que las ratas son heterocigóticas para este SNP que también se encontró en ratas no tratadas (datos no
10 mostrados). Aunque está presente en el 50 % de los cromosomas en animales positivos para CEL-I, el SNP es malamente reconocido por la enzima CEL-I, produciendo productos de escisión de intensidad inesperadamente más baja.
B. Rab38 (ejemplo comparativo)
También se diseñaron ZFN para dirigir el locus Rab38 endógeno en ratas, particularmente el exón 1 del gen Rab38 15 de rata. Diseños de dedos de cinc de Rab38 a modo de ejemplo se muestran en la Tabla 8 a continuación.
Tabla 10: Diseños de dedos de cinc de Rab38
- Nombre de ZFN
- F1 F2 F3 F4 F5 F6
- 18160
- DRSNLSS (SEQ ID NO:81) RSHSLLR (SEQ ID NO:82) RSDSLSA (SEQ ID NO:38) TSGSLTR (SEQ ID NO:25) QSGNLAR (SEQ ID NO:21) QSGHLSR (SEQ ID NO:83)
- 18181
- TSGHLSR (SEQ ID NO:52) HKWQRNK (SEQ ID NO:84) DRSVLRR (SEQ ID NO:85) DSSTRKK (SEQ ID NO:86) RSDHLSE (SEQ ID NO:87) DKSNRKK (SEQ ID NO:88)
22
- Nombre de ZFN
- F1 F2 F3 F4 F5 F6
- 16897
- RSDTLSE (SEQ ID NO:89) QKRNRTK (SEQ ID NO:90) RSDSLSA (SEQ ID NO:38) TSGSLTR (SEQ ID NO:25) QSGNLAR (SEQ ID NO:21) QSGHLSR (SEQ ID NO:83)
- 16898
- RSDHLSK (SEQ ID NO:91) HNDSRTN (SEQ ID NO:92) DRSDLSR (SEQ ID NO:28) RSDHLSE (SEQ ID NO:87) DKSNRKK (SEQ ID NO:88) N/A
- 18173
- RSDYLPR (SEQ ID NO:93) QSNDLNS (SEQ ID NO:94) DRSDLSR (SEQ ID NO:28) RSDHLSE (SEQ ID NO:87) DKSNRKK (SEQ ID NO:88) N/A
- 18174
- RSDYLPR (SEQ ID NO:93) QRVTRDA (SEQ ID NO:95) DRSDLSR (SEQ ID NO:28) RSDHLSE (SEQ ID NO:87) DKSNRKK (SEQ ID NO:88) N/A
- 18175
- HSNARKT (SEQ ID NO:96) ASKTRTN (SEQ ID NO:97) DRSDLSR (SEQ ID NO:28) RSDHLSE (SEQ ID NO:87) DKSNRKK (SEQ ID NO:88) N/A
- 18161
- RSHSLLR (SEQ ID NO:82) RSDSLSA (SEQ ID NO:38) TSGSLTR (SEQ ID NO:25) QSGNLAR (SEQ ID NO:21) QSGHLSR (SEQ ID NO:83) N/A
- 18183
- RSHSLLR (SEQ ID NO:82) RSDYLPR (SEQ ID NO:93) DRSVLRR (SEQ ID NO:85) DSSTRKK (SEQ ID NO:86) RSDHLSE (SEQ ID NO:87) DKSNRKK (SEQ ID NO:88)
Tabla 11: Sitios diana de dedos de cinc de Rab38
Se muestran sitios diana de los diseños de dedos de cinc dirigidos a Rab38 de rata a continuación en la Tabla 11. Los nucleótidos en el sitio diana que se ponen en contacto por las hélices de reconocimiento de ZFP se indican en letras mayúsculas; los nucleótidos que no se ponen en contacto se indican en minúscula.
- Nombre de ZFN
- Sitio diana (5' a 3')
- 18161
- gaGGAGAAGTTTTGGTGCACgtagcgct (SEQ ID NO:98)
- 18181
- acTACCGGGCCACCATTGGTgtggactt (SEQ ID NO:99)
- 16897
- gaGGAGAAGTTTTGgTGCACGtagcgct (SEQ ID NO:98)
- 16898
- acTACCGGGCCacCATTGGtgtggactt (SEQ ID NO:99)
- 18173
- acTACCGGGCCaCCATTGgtgtggactt (SEQ ID NO:99)
- 18174
- acTACCGGGCCaCCATTGgtgtggactt (SEQ ID NO:99)
- 18175
- acTACCGGGCCACCATTggtgtggactt (SEQ ID NO:99)
- 18160
- gaGGAGAAGTTTTGGTGcacgtagcgct (SEQ ID NO:98)
- 18183
- acTACCGGGCCACCaTTGGTGtggactt (SEQ ID NO:99)
Todas las ZFN dirigidas a Rab38 contuvieron las mutaciones EL/KK Fok I como se describe en la publicación de patente de EE.UU. N.º 2008/0131962. La expresión de ZFN se condujo por tanto el promotor CAG como del CMV. Se transfectaron ZFN (1 µg de cada una) en 200.000 células C6 mediante nucleofección de Amaxa usando la
10 disolución SF y el nucleofactor de 96 pocillos Amaxa Shuttle. La escisión se ensayó con la nucleasa Surveyor™ de CEL-I como se describe, por ejemplo, en las publicaciones de patente de EE.UU. N.º 20080015164; 20080131962 y 20080159996.
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CN102625655A (zh) | 2012-08-01 |
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SG172760A1 (en) | 2011-08-29 |
US20190062789A9 (en) | 2019-02-28 |
JP2012510812A (ja) | 2012-05-17 |
EP2352369A4 (en) | 2012-05-02 |
CN102625655B (zh) | 2016-07-06 |
KR20110101175A (ko) | 2011-09-15 |
JP5681114B2 (ja) | 2015-03-04 |
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