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Tema 8. Genética reversa III.
Mutagénesis dirigida.
Mutagenesis dirigida. Recombinación homóloga, vectores.
Análisis genético.
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
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). 259-264.
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
Genética reversa III. Mutagénesis dirigida.
HR/IR.
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.
Genética reversa III. Mutagénesis dirigida.
Genética reversa III. Mutagénesis dirigida.
Vectores: Insertion
Genética reversa III. Mutagénesis dirigida.
Vectores:
Genética reversa III. Mutagénesis dirigida.
Vectores: replacement
Genética reversa III. Mutagénesis dirigida.
Vectores:
Genética
reversa
III.
Mutagénesis
dirigida.
Genética reversa III. Mutagénesis dirigida.
Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf-1 gene
Mario Mikula et al., EMBO Journal, Vol. 20, No. 8 pp. 1952-1962, 2001
An efficient method to successively introduce transgenes into a given genomic locus in the
mouse. Zhao R, Fahs SA, Weiler H, Duncan SA. BMC Dev Biol. 2001;1(1):10.
Genome engineering via homologous recombination in mouse embryonic stem (ES) cells: an
amazingly versatile tool for the study of mammalian biology. C. BABINET and M. COHENTANNOUDJI An. Acad. Bras. C. vol.73 no.3, 2001
Electroporation and selection of ES cell clones
E14.1 ES cells grown on -irradiated embryonic fibroblasts were
electroporated (260 V, 500 µF) with the AscI-linearized targeting vector
and selected with G418 (0.2 mg/ml). Homologous recombinants were
obtained with a frequency of 1 in 35, as detected by nested PCR and
Southern analysis. Positive clones were electroporated with a plasmid
expressing the Cre recombinase. Cre expression led to deletion of either
the exon 3 or the Neo/TK cassette, or both. The latter two were enriched
by negative selection with gancyclovir.
Construction of the targeting vector
A genomic DNA clone containing a portion of Raf-1 was isolated from a
129/Sv mouse genomic fix library. An 8.5 kb 5'-XbaI–BglII-3' fragment
containing exon 3 and surrounding sequences was used to assemble the
targeting construct in pBSIISK–. loxP sites were inserted as doublestranded oligonucleotides in the HindIII site 3' of exon 3 and in the
BamHI site 5' of exon 3. A Neo/TK cassette containing an upstream loxP
site was excised from plasmid pGH1 and cloned into an XbaI–HindIII
site contained in the loxP site 5' of exon 3.
Genética reversa III. Mutagénesis
dirigida.
Generation of chimeras
Two clones showing deletion between the most distant loxP sites were
used to generate chimeras. C57BL/6 blastocyst stage embryos were
injected with Raf-1+/– ES cells and then transferred to pseudopregnant
B6CBAF1 mice for further development. Chimeric mice were mated to
C57BL/6 and 129/Sv animals, and agouti offspring were genotyped.
Germline transmission of the knock-out allele was detected by either
Southern blot or PCR analysis of tail DNA.
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