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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

Founder KI female 3.4 genotyping from subcloned PCR amplicons of tail biopsies.Wt: Wild type; DP: donor plasmid. Point mutations in the DP are indicated in red bold letters. The pA476T mutation is highlighted in blue.
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pone-0088146-g003: Founder KI female 3.4 genotyping from subcloned PCR amplicons of tail biopsies.Wt: Wild type; DP: donor plasmid. Point mutations in the DP are indicated in red bold letters. The pA476T mutation is highlighted in blue.

Mentions: To reveal homologous recombination events located in the correct locus, we amplified the genomic DNA of the founders with the forward primer designed outside of the homology region and reverse primer inside of the homology region (“outside-in”) and digested them with AluI and HaeIII enzymes. Primers used are described in Table S2 in File S1. Gel digestion of the Nr3c1 exon 3 revealed one KI female founder (namely 3.4) from the first injection series of higher TALEN mRNA out of 225 zygotes (KI founder gel digestion in Fig. S3 in File S1), and thus representing 1.7% of all live born pups. Sequencing of the Nr3c1 exon 3 revealed that the 3.4 rat also presented a full donor DNA integration by homology-derived recombination (HDR). In addition to the KI allele, rat 3.4 also had a wild type and a 7 bp depleted alleles of the Nr3c1 gene (Fig. 3and Fig. S4 in File S1).


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)

Founder KI female 3.4 genotyping from subcloned PCR amplicons of tail biopsies.Wt: Wild type; DP: donor plasmid. Point mutations in the DP are indicated in red bold letters. The pA476T mutation is highlighted in blue.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0088146-g003: Founder KI female 3.4 genotyping from subcloned PCR amplicons of tail biopsies.Wt: Wild type; DP: donor plasmid. Point mutations in the DP are indicated in red bold letters. The pA476T mutation is highlighted in blue.
Mentions: To reveal homologous recombination events located in the correct locus, we amplified the genomic DNA of the founders with the forward primer designed outside of the homology region and reverse primer inside of the homology region (“outside-in”) and digested them with AluI and HaeIII enzymes. Primers used are described in Table S2 in File S1. Gel digestion of the Nr3c1 exon 3 revealed one KI female founder (namely 3.4) from the first injection series of higher TALEN mRNA out of 225 zygotes (KI founder gel digestion in Fig. S3 in File S1), and thus representing 1.7% of all live born pups. Sequencing of the Nr3c1 exon 3 revealed that the 3.4 rat also presented a full donor DNA integration by homology-derived recombination (HDR). In addition to the KI allele, rat 3.4 also had a wild type and a 7 bp depleted alleles of the Nr3c1 gene (Fig. 3and Fig. S4 in File S1).

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