Genética reversa III. Mutagénesis dirigida.

Presentación del tema: "Genética reversa III. Mutagénesis dirigida."— Transcripción de la presentación:

1 Genética reversa III. Mutagénesis dirigida.
Gene targeting mediante recombinación homóloga El DNA transformante es llevado hasta el gen mediante secuencias homólogas Knockout por inserción Mutación por conversión génica Ventajas: El investigador elige el gen que quiere modificar El investigador tiene un control total sobre la forma en la que modifica el gen (mutación puntual, cambio de expresión KO….)

2 Genética reversa III. Mutagénesis dirigida.
¿Por qué no se usa siempre? La eficacia del proceso depende de la ratio HR/IR Partners and pathways: repairing a double-strand break (2000). James E. Haber. TIG 16 (6) DNA double strand break repair in mammalian cells (2000). Peter Karran. Current Opinion in Genetics & Development, 10:144–150 Genetic aspects of targeted insertion mutagenesis in yeasts (2004) U. Klinner *, B. Schäfer. FEMS Microbiology Reviews 28: 201–223

3 Genética reversa III. Mutagénesis dirigida.
HR/IR.

4 Genética reversa III. Mutagénesis dirigida.
Las proteínas implicadas en HR e IR se encuentran conservadas desde levaduras a humanos. El método preferente de reparación es, sin embargo, específico Finalmente, la eficacia de gene targeting depende de la utilización de HR en el organismo. Gene targeting in Physcomitrella patens (2001). Didier G Schaefer. Current Opinion in Plant Biology 2001, 4:143–150. Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Mario R. Capecchi. NATURE REVIEWS. GENETICS VOLUME 6. JUNE 2005, 507 From sequence to phenotype: Reverse Genetics in Drosophila melanogaster. Melissa D. Adams and Jeff J. Sekelsky. NATURE REVIEWS. GENETICS. VOLUME 3. MARCH

5 Genética reversa III. Mutagénesis dirigida.

6 Genética reversa III. Mutagénesis dirigida. Ratón
Figure 4 | Positive–negative selection. A selection protocol used to enrich for embryonic stem (ES) cell lines that contain a targeted disruption of any chosen gene (here, gene X), regardless of its function or expression in ES cells30. a | The targeting vector contains an insertion of a neomycin resistance (neor) gene in an exon of gene X and a linked herpes virus thymidine kinase (HSV-tk) gene at one end. The vector is shown pairing with a chromosomal copy of gene X. Homologous recombination between the targeting vector and the cognate chromosomal gene results in the disruption of one genomic copy of gene X and the loss of the vector HSV-tk gene. Cells in which this event has occurred will be X+/–, neor+ and HSV-tk–, and will be resistant to both G418 and FIAU. b | Most frequently, the targeting vector will be integrated into the host cell genome at a random site, through non-homologous recombination. Because non-homologous insertion of exogenous DNA into the host cell chromosome occurs through the ends of the linearized targeting vector, the HSV-tk gene will remain linked to the neor gene. Cells derived from this type of recombination event will be X+/+, neor+ and HSV-tk+ and therefore resistant to G418 but killed by FIAU (a drug that kills cells containing a functional HSV-tk gene, but which is not toxic to cells with only the cellular tk gene). ES cells that contain the desired targeting event survive the selection procedure, but cells that contain the much more common random integration of the targeting vector do not.

7 Genética reversa III. Mutagénesis dirigida. Ratón
Figure 4 | Positive–negative selection. A selection protocol used to enrich for embryonic stem (ES) cell lines that contain a targeted disruption of any chosen gene (here, gene X), regardless of its function or expression in ES cells30. a | The targeting vector contains an insertion of a neomycin resistance (neor) gene in an exon of gene X and a linked herpes virus thymidine kinase (HSV-tk) gene at one end. The vector is shown pairing with a chromosomal copy of gene X. Homologous recombination between the targeting vector and the cognate chromosomal gene results in the disruption of one genomic copy of gene X and the loss of the vector HSV-tk gene. Cells in which this event has occurred will be X+/–, neor+ and HSV-tk–, and will be resistant to both G418 and FIAU. b | Most frequently, the targeting vector will be integrated into the host cell genome at a random site, through non-homologous recombination. Because non-homologous insertion of exogenous DNA into the host cell chromosome occurs through the ends of the linearized targeting vector, the HSV-tk gene will remain linked to the neor gene. Cells derived from this type of recombination event will be X+/+, neor+ and HSV-tk+ and therefore resistant to G418 but killed by FIAU (a drug that kills cells containing a functional HSV-tk gene, but which is not toxic to cells with only the cellular tk gene). ES cells that contain the desired targeting event survive the selection procedure, but cells that contain the much more common random integration of the targeting vector do not.

8 Genética reversa III. Mutagénesis dirigida. Ratón
Figure 5 | Generation of mouse germline chimaeras from embryonic stem cells that contain the desired targeted mutation. a | The first step involves the isolation of a clonal embryonic stem (ES) cell line that contains the desired mutation. Positive–negative selection (see also FIG. 4) can be used to enrich for ES cell lines that contain the desired modified gene. b | The second step is to use these ES cells to generate chimeric mice that are able to transmit the mutant gene to their progeny. This is accomplished by injecting ES cells that contain the desired targeted mutation into a recipient pre-implantation mouse embryo, a blastocyst. These embryos are then surgically transferred to a recipient pseudopregnant foster mother to allow the embryos to come to term. To facilitate isolation of the desired progeny, the ES cells and recipient blastocysts are derived from mice with distinguishable coat-colour alleles (that is, ES cells from agouti brown mice and blastocysts from black mice). The extent of the contribution of ES cells to the formation of the chimeric mouse can be evaluated by visual assessment of coat-colour chimerism. ES cell contribution to the germline can be evaluated by observing the coat colour of the progeny that is derived by breeding the chimeric mouse with black mice.

9 Genética reversa III. Mutagénesis dirigida. Ratón
¿Análisis genético? Figure 5 | Generation of mouse germline chimaeras from embryonic stem cells that contain the desired targeted mutation. a | The first step involves the isolation of a clonal embryonic stem (ES) cell line that contains the desired mutation. Positive–negative selection (see also FIG. 4) can be used to enrich for ES cell lines that contain the desired modified gene. b | The second step is to use these ES cells to generate chimeric mice that are able to transmit the mutant gene to their progeny. This is accomplished by injecting ES cells that contain the desired targeted mutation into a recipient pre-implantation mouse embryo, a blastocyst. These embryos are then surgically transferred to a recipient pseudopregnant foster mother to allow the embryos to come to term. To facilitate isolation of the desired progeny, the ES cells and recipient blastocysts are derived from mice with distinguishable coat-colour alleles (that is, ES cells from agouti brown mice and blastocysts from black mice). The extent of the contribution of ES cells to the formation of the chimeric mouse can be evaluated by visual assessment of coat-colour chimerism. ES cell contribution to the germline can be evaluated by observing the coat colour of the progeny that is derived by breeding the chimeric mouse with black mice.

10 Genética reversa III. Mutagénesis dirigida.
Análisis Genético Nº de copias de la construcción? Modificación del gen diana? Ligamiento fenotipo alteración del gen diana? Varias líneas

11 Genética reversa III. Mutagénesis dirigida condicional, CRE-loxP targeting

12 Genética reversa III. Mutagénesis dirigida.
Generation of mice with a novel conditional null allele of the Sox9 gene. Sook Peng Yap, Xing Xing • Petra Kraus, V. Sivakamasundari, Hsiao Yun Chan, Thomas Lufkin. Biotechnol Lett (2011) 33:1551–1558

13 Genética reversa III. Mutagénesis dirigida.
Sox9 es un factor de transcripción esencial en el desarrollo del cartílago y regulador clave del desarrollo de testículos. También participa en el desarrollo de otros tejidos: glia, cresta neural, corazon, pancreas….. Mutaciones en Sox-9 son responsables del desarrollo de displasia campomélica, una enfermedad autosómica dominante (OMIM #114290) La enfermedad está caracterizada por huesos arqueados, deformaciones en pelvis, defectos craneofaciales, reversión sexual, malformaciones en riñón, corazón, ………….

14 Genética reversa III. Mutagénesis dirigida.
La creación de un modelo en ratón no es fácil: los ratones Sox9+/- mueren al poco de nacer, mientras que la deficiencia total en Sox9 da lugar a letalidad embriónica temprana. El problema podría ser resuelto mediante la generación de un alelo mutante condicional de Sox9. La inactivación del gen en un tejido o situación determinados permitiría el análisis de la función del gen en esas situaciones.

15 Genética reversa III. Mutagénesis dirigida.
Para el desarrollo de un KO condicional se han empleado los sistemas de recombinación FLP-FRT y CRE-LOXP The Sox9 conditional knock-out targeting vector was generated by flanking the Sox9 exon 1 with loxP sites, and the neomycin cassette flanked by FRT sites was inserted immediately after the upstream loxP site. The linearized targeting vector was electroporated into V6.4 mouse ES cells and selected with G418 (200–400 lg/ml) for 8 days. El vector linearizado fue introducido por electroporación en células madre embrionarias de ratón. Las células transformadas se seleccionaron durante 8 días con G418 (200–400 ug/ml). ¿Selección negativa?

16 Genética reversa III. Mutagénesis dirigida.
Las colonias G418r se utilizaron para micro-inyectar embriones en estadio 6-8 células. Finalmente se obtuvieron 28 quimeras Sox9+/floxFRTNeo, procedentes de 2 clones ES. The individual G418-resistant ES cell colonies were screened by Southern blotting for targeted homologous recombination in the Sox9 locus with the indicated probes (Fig. 1). The Sox9+/floxFRTNeo ES cell clones were microinjected into 2–8 cell stage mouse embryos isolated from C57BL/6 J mice (Kraus et al. 2010). Sox9+/floxFRTNeo chimeras were crossed to wild type C57BL6/6 J or CD1 mice to generate heterozygous mice. We have generated six and 22 high percentage Sox9+/floxFRTNeo chimeras for the two selected independent euploid clones #35 and #84, respectively.

17 Genética reversa III. Mutagénesis dirigida.
El cassette Neo fue eliminado por recombinación mediada por FLPe. Las quimeras se cruzarón con ratones FLPe-deleter lo cual produjo una descendencia Sox9+/flox:WT 1:1. La ausencia del cassette Neo fue determinada mediante Southern blot genómico. El alelo Sox9flox parece perfectamente funcional al menos en Het y de hecho obtienen ratones homocigotos Sox9flox/flox de fenotipo WT Initially, after genotyping 85 (clone 35) and 126 (clone 84) offspring at weaning age from mating the two independent lines of Sox9+/floxFRTNeo chimeras to C57BL/6 J females, no heterozygous mice were recovered……………prior to the removal of the selection cassette, we have generated a Sox9 null allele, owing to the presence of a poly(A) tail in the neomycin cassette, preceding the Sox9 translation start codon. To remove the FRT-flanked Neo STOP cassette through FLPe/FRT recombination the male Sox9+/floxFRTNeo chimeras were crossed with FLPe–deleter mice (Farley et al. 2000), and the removal of the FRT-flanked Neo STOP cassette was confirmed by Southern blotting (Fig. 1b). This cross yielded viable Sox9+/flox mice and WT mice in the expected Mendelian ratio. With EcoRI and XhoI double digestion and Probe 1, the Sox9floxFRTNeo and Sox9flox alleles were detected at 3.8 and 2.4 kb respectively, confirming the removal of the neomycin cassette mediated by FLPe/FRT recombination. Hence, by removing the selection cassette, we could rescue the Sox9 null phenotype, with the floxed Sox9 allele being a fully functional Sox9 allele after FLPe/FRT recombination.

18 Genética reversa III. Mutagénesis dirigida.
Para activar la mutación, los ratones Sox9flox/flox se cruzaron con ratones Col2a1-Cre que expresan fuertemente la recombinasa Cre en el cartílago del esqueleto axial To test for the conditional inactivation of Sox9 in vivo, these newly established Sox9 conditional knockout mice, Sox9flox/flox were mated to Col2a1-Cre mice (Ovchinnikov et al. 2000), which were previously demonstrated to achieve tissue-specific gene expression in chondrocytes (Akiyama et al. 2002). We confirmed this Cre-activity in our hands by crossing the Col2a1-Cre mice with the Cre-tester mice (Soriano 1999). The presence of Col2a-Cre activity was observed by the detection of beta-galactosidase activity upon X-gal staining. Strong X-gal staining was observed in the cartilage of the axial skeleton of E11.0 double transgenic embryos, reflecting the expression domains of Sox9 and Col2a1at E11.5 (Fig. 2). Tail-tip PCR genotyping of flox, Cre transgene, and floxdel alleles were performed using genomic DNA with the indicated bands detected El genotipado se lleva a cabo mediante PCR con ensayos diseñados para detectar Lox-P antes y después de la recombinación

19 Genética reversa III. Mutagénesis dirigida.
El cruzamiento inicial Sox9flox/flox por Col2a1-Cre produjo doble heterocigotos Sox9+/floxdel; Col2a1-Cre que ya mostraban cierto fenotipo Estos heterocigotos se cruzaron con ratones Sox9flox/flox para dar lugar a embriones Sox9floxdel/floxdel; Col2a1-Cre (en cartílago) que fueron utilizados en análisis histológicos Fig. 3 Generation of Sox9floxdel/floxdel; Col2a1-Cre mice. Gross appearance of a 4-week old Sox9?/floxdel; Col2a1-Cre mice and b E18.5 Sox9floxdel/floxdel; Col2a1-Cre embryos To test for the conditional inactivation of Sox9 in vivo, these newly established Sox9 conditional knockout mice, Sox9flox/flox were mated to Col2a1-Cre mice (Ovchinnikov et al. 2000)

20 Genética reversa III. Mutagénesis dirigida.
La inactivación de Sox9 en el esqueleto axial determina osificación prematura en heterocigotos y malformaciones severas en homocigosis. Fig. 5 Tissue specific Sox9 dose dependency. Alizarin red and Alcian blue staining of bone and cartilage tissue respectively in E17.5 littermates indicates dosage sensitive premature ossification of the vertebrae after tissue specific Sox9 inactivation. Lumbar vertebrae of Sox9?/flox are unaffected a while loss of only one copy of Sox9 causes premature ossification of the vertebrae bodies of Sox9?/floxdel; Col2a1-Cre littermates (b) and severe premature ossification and malformation in Sox9floxdel/floxdel; Col2a1-Cre littermates (c). Tissues without Col2a1-Cre activity remain unaffected and will continue to express Sox9 as shown by RNA-ISH of the hindgut with probes for Col2a1 (g, i) and Sox9 Exon1 (h, j) on sections of Sox9?/? (g, h) and Sox9floxdel/floxdel (i, j) E13.5 embryos

21 Genética reversa III. Otro ejemplo
A Global In Vivo Drosophila RNAi Screen Identifies NOT3 as a Conserved Regulator of Heart Function Cell 141, 142–153, April 2, 2010 La función Not 3 en mamíferos es desconocida, los autores deciden crear un ratón KO por recombinación homóloga. Figure 5. not3+/ Mice Exhibit Reduced Heart Contractility, Ex Vivo Function, and Histone Modifications that Can Be Rescued by Treatment with HDAC Inhibitors (A) Gene targeting strategy. Exons 2 to 9 of the not3 gene (official symbol cnot3) were replaced with a PGK-Neo cassette by homologous recombination in A9 ESCs. The wild-type allele, targeting vector, mutant allele, and PGK-Neo and DTH selection cassettes are shown. Blue boxes indicate exons.

22 Genética reversa III. Otro ejemplo
Los ratones heterocigotos para el KO muestran una expresión menor del gen y defectos cardiacos agravados por la edad. Figure 5. not3+/ Mice Exhibit Reduced Heart Contractility, Ex Vivo Function, and Histone Modifications that Can Be Rescued by Treat ment with HDAC Inhibitors. (B) Real-time PCR analyses for not3 mRNA expression in 3-month-old wild-type and not3+/– hearts. Values were normalized to gapdh mRNA expression. n = 6 mice per group. (C) not3+/– mice display a significant reduction in percent fractional shortening at 4 months of age, which became more pronounced with age. n = 6–8 mice per group. Fractional shortening was determined by echocardiography.

23 Genética reversa III. Otro ejemplo
Los defectos no se deben a la ausencia de función Not3 en otros tejidos, en experimentos “ex vivo” los corazones muestran defectos medidos de distintas formas. Figure 5. not3+/ Mice Exhibit Reduced Heart Contractility, Ex Vivo Function, and Histone Modifications that Can Be Rescued by Treatment with HDAC Inhibitors (F) Impaired contractile response of ex vivo not3+/– hearts to electrical field stimulation (EFS) compared with littermate not3+/+ hearts. Representative data for left ventricular pressure (LVP) at 20 V stimulation are shown.

24 Genética reversa III. Otro ejemplo
ETC…. Sobre caracterización fenotípica del heterocigoto not3+/not3-, sometido a diferentes estreses…. Y en humanos? es posible la genética reversa? En una revisión de proyectos de GWAS, los autores encuentran una asociación significativa entre un polimorfismo en el promotor de Not3 y un fenotipo cardiaco: repolarización cardiaca anormal, medida como alteraciones en el intervalo QT, que predispone a muerte súbita. Figure 7. not3 Is a Conserved Regulator of Heart Function (F) Regional visualization of the association signal between common variants in the NOT3 region and the adjusted QT interval (QTc). SNP rs36643 in the 50 region of NOT3 (969 bp from the transcription start and 924 bp from the TATA box) showed a significant regional association (p = ). (G) Association between the T allele of SNP rs36643 and a prolongation of QTc. * p < from linear regression with inverse variance weighting using an additive genetic model. Data are derived from a meta-analysis of genomewide association scans in several populations (Pfeufer et al., 2009).