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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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Donor plasmid design for the generation of GRdim KI rat.Upper DNA indicates the wild type sequence of the exon 3 of the rat Nr3c1 GR. In blue the residue A476. TAL 3 and TAL 6 binding sites are indicated. DP, donor plasmid sequence. Donor plasmid was synthesized with 500 pb homology arms on both ends. The pA476T point mutation is highlighted in blue. Silent mutations of the DP are shown in red bold letters and are located in the overlap of TAL 3 and Tal 6 binding sites. HaeIII site is highlighted in yellow. AluI site is not shown in this figure.
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pone-0088146-g002: Donor plasmid design for the generation of GRdim KI rat.Upper DNA indicates the wild type sequence of the exon 3 of the rat Nr3c1 GR. In blue the residue A476. TAL 3 and TAL 6 binding sites are indicated. DP, donor plasmid sequence. Donor plasmid was synthesized with 500 pb homology arms on both ends. The pA476T point mutation is highlighted in blue. Silent mutations of the DP are shown in red bold letters and are located in the overlap of TAL 3 and Tal 6 binding sites. HaeIII site is highlighted in yellow. AluI site is not shown in this figure.

Mentions: To generate GRdim KI rats, we designed a common donor plasmid for TALENs 3 and 6, bearing the pA476T mutation along with 4 silent point mutations in each TALEN binding site to prevent further nuclease activity in the targeted alleles (Fig. 2and Fig. S2 in File S1). The donor plasmid sequence had also 500 bp homology arms on 3′ and 5′ sides of the pA476T mutation, to allow homology-derived recombination as described in [25]. The donor carried two extra point mutations for rapid detection by enzyme digestion: an AluI site was removed and a HaeIII site was added close to the pA476T site (Fig. 2and Fig. S2 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)

Donor plasmid design for the generation of GRdim KI rat.Upper DNA indicates the wild type sequence of the exon 3 of the rat Nr3c1 GR. In blue the residue A476. TAL 3 and TAL 6 binding sites are indicated. DP, donor plasmid sequence. Donor plasmid was synthesized with 500 pb homology arms on both ends. The pA476T point mutation is highlighted in blue. Silent mutations of the DP are shown in red bold letters and are located in the overlap of TAL 3 and Tal 6 binding sites. HaeIII site is highlighted in yellow. AluI site is not shown in this figure.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0088146-g002: Donor plasmid design for the generation of GRdim KI rat.Upper DNA indicates the wild type sequence of the exon 3 of the rat Nr3c1 GR. In blue the residue A476. TAL 3 and TAL 6 binding sites are indicated. DP, donor plasmid sequence. Donor plasmid was synthesized with 500 pb homology arms on both ends. The pA476T point mutation is highlighted in blue. Silent mutations of the DP are shown in red bold letters and are located in the overlap of TAL 3 and Tal 6 binding sites. HaeIII site is highlighted in yellow. AluI site is not shown in this figure.
Mentions: To generate GRdim KI rats, we designed a common donor plasmid for TALENs 3 and 6, bearing the pA476T mutation along with 4 silent point mutations in each TALEN binding site to prevent further nuclease activity in the targeted alleles (Fig. 2and Fig. S2 in File S1). The donor plasmid sequence had also 500 bp homology arms on 3′ and 5′ sides of the pA476T mutation, to allow homology-derived recombination as described in [25]. The donor carried two extra point mutations for rapid detection by enzyme digestion: an AluI site was removed and a HaeIII site was added close to the pA476T site (Fig. 2and Fig. S2 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