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Generation of TALEN-mediated GRdim knock-in rats by homologous recombination.

Ponce de León V, Mérillat AM, Tesson L, Anegón I, Hummler E - PLoS ONE (2014)

Bottom Line: We assembled TALENs and designed a linear donor insert targeting a pA476T mutation in the rat Glucocorticoid Receptor (Nr3c1) namely GR(dim), that prevents receptor homodimerization in the mouse.TALEN mRNA and linear double-stranded donor were microinjected into rat one-cell embryos.Overall, we observed targeted genomic modifications in 17% of the offspring, indicating high TALEN cutting efficiency in rat zygotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland.

ABSTRACT
Transcription Activator-Like Effector Nucleases (TALEN) are potential tools for precise genome engineering of laboratory animals. We report the first targeted genomic integration in the rat using TALENs (Transcription Activator-Like Effector Nucleases) by homology-derived recombination (HDR). We assembled TALENs and designed a linear donor insert targeting a pA476T mutation in the rat Glucocorticoid Receptor (Nr3c1) namely GR(dim), that prevents receptor homodimerization in the mouse. TALEN mRNA and linear double-stranded donor were microinjected into rat one-cell embryos. Overall, we observed targeted genomic modifications in 17% of the offspring, indicating high TALEN cutting efficiency in rat zygotes.

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Related in: MedlinePlus

Detection of random donor integration in rat founders.Representative Southern blot analysis of founder rat genomic DNA following HincII digestion of genomic DNA and hybridization with exon 3-derived probe. Indicated is the 3.6 kb endogenous exon 3-containing genomic fragment and additional random integrations in founder 5.4 (knockout) and 8.1 (wildtype) rats. Rats 5.5 and 6.1 are knockouts as well but presented no off-target donor integration.
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pone-0088146-g005: Detection of random donor integration in rat founders.Representative Southern blot analysis of founder rat genomic DNA following HincII digestion of genomic DNA and hybridization with exon 3-derived probe. Indicated is the 3.6 kb endogenous exon 3-containing genomic fragment and additional random integrations in founder 5.4 (knockout) and 8.1 (wildtype) rats. Rats 5.5 and 6.1 are knockouts as well but presented no off-target donor integration.

Mentions: To check whether our linearized donor was integrated elsewhere into the genome, we performed a Southern blot analysis of all 58 founders using a hybridization probe around exon 3 and searched for additional integration events to the 3.6 kb HincII endogenous fragment (Fig. 5). In total, 7 live born pups out of 58 (12%) exhibited an off-target integration of the donor sequence. 5 out of 48 (10%) wild type founders and 2 out of 10 (20%) KO/KI founders harbored randomly one or several copies of the donor insert.


Generation of TALEN-mediated GRdim knock-in rats by homologous recombination.

Ponce de León V, Mérillat AM, Tesson L, Anegón I, Hummler E - PLoS ONE (2014)

Detection of random donor integration in rat founders.Representative Southern blot analysis of founder rat genomic DNA following HincII digestion of genomic DNA and hybridization with exon 3-derived probe. Indicated is the 3.6 kb endogenous exon 3-containing genomic fragment and additional random integrations in founder 5.4 (knockout) and 8.1 (wildtype) rats. Rats 5.5 and 6.1 are knockouts as well but presented no off-target donor integration.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921256&req=5

pone-0088146-g005: Detection of random donor integration in rat founders.Representative Southern blot analysis of founder rat genomic DNA following HincII digestion of genomic DNA and hybridization with exon 3-derived probe. Indicated is the 3.6 kb endogenous exon 3-containing genomic fragment and additional random integrations in founder 5.4 (knockout) and 8.1 (wildtype) rats. Rats 5.5 and 6.1 are knockouts as well but presented no off-target donor integration.
Mentions: To check whether our linearized donor was integrated elsewhere into the genome, we performed a Southern blot analysis of all 58 founders using a hybridization probe around exon 3 and searched for additional integration events to the 3.6 kb HincII endogenous fragment (Fig. 5). In total, 7 live born pups out of 58 (12%) exhibited an off-target integration of the donor sequence. 5 out of 48 (10%) wild type founders and 2 out of 10 (20%) KO/KI founders harbored randomly one or several copies of the donor insert.

Bottom Line: We assembled TALENs and designed a linear donor insert targeting a pA476T mutation in the rat Glucocorticoid Receptor (Nr3c1) namely GR(dim), that prevents receptor homodimerization in the mouse.TALEN mRNA and linear double-stranded donor were microinjected into rat one-cell embryos.Overall, we observed targeted genomic modifications in 17% of the offspring, indicating high TALEN cutting efficiency in rat zygotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland.

ABSTRACT
Transcription Activator-Like Effector Nucleases (TALEN) are potential tools for precise genome engineering of laboratory animals. We report the first targeted genomic integration in the rat using TALENs (Transcription Activator-Like Effector Nucleases) by homology-derived recombination (HDR). We assembled TALENs and designed a linear donor insert targeting a pA476T mutation in the rat Glucocorticoid Receptor (Nr3c1) namely GR(dim), that prevents receptor homodimerization in the mouse. TALEN mRNA and linear double-stranded donor were microinjected into rat one-cell embryos. Overall, we observed targeted genomic modifications in 17% of the offspring, indicating high TALEN cutting efficiency in rat zygotes.

Show MeSH
Related in: MedlinePlus