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High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases.

Qiu Z, Liu M, Chen Z, Shao Y, Pan H, Wei G, Yu C, Zhang L, Li X, Wang P, Fan HY, Du B, Liu B, Liu M, Li D - Nucleic Acids Res. (2013)

Bottom Line: Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism.One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation.In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.

ABSTRACT
Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ∼40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background.

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Somatic mutations induced by TALENs in mice. (a) Frequencies and sequences of TALEN-induced mutations. The binding sites of the TALEN half-site are shown in bold. A dash represents a single base deletion. Insertions or substitutions are indicated in red letters. Microhomology that was likely used by NHEJ is underlined. The length and frequency of indels are labeled to the right of each sequence. (b) TALENs induce a wide range of indel length. The percentage is from the number of clones of different indel lengths divided by the number of total clones sequenced.
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gkt258-F3: Somatic mutations induced by TALENs in mice. (a) Frequencies and sequences of TALEN-induced mutations. The binding sites of the TALEN half-site are shown in bold. A dash represents a single base deletion. Insertions or substitutions are indicated in red letters. Microhomology that was likely used by NHEJ is underlined. The length and frequency of indels are labeled to the right of each sequence. (b) TALENs induce a wide range of indel length. The percentage is from the number of clones of different indel lengths divided by the number of total clones sequenced.

Mentions: To determine the mutations generated in individual founders, PCR products were TA cloned, and at least six colonies for each founder mouse were sequenced. All the mutations identified by sequencing are listed in Figure 3a and Supplementary Figure S1. Similar to other species, some of the founders were biallelically modified. For instance, founders 1, 4, 6, 10, 12, 13 of the Rprm gene had the same biallelic non-frame-shifting 3-bp deletions. Founders 2, 3, 7, 12 of the Gpr55 gene had a combination of frame-shifting and non-frame-shifting deletions (Figure 3a). Also, there are mosaic founders where more than two allele types have been identified from a single mouse, such as founder 8 of the Lepr gene in the FVB/N background, founders 3 and 7 of the Rprm gene and founder 19 of Fbxo6 gene (Figure 3a). However, biallelic mutations were not observed in the founders of other genes tested in this study. In line with the indels induced by TALEN in zebrafish (31), our TALENs generated indels with a broad range of lengths in mice, from 1 to 173 bp, with an average length of 15.4 bp (n = 61 independent indels in total) (Figure 3b). In accord with the deletions induced by ZFNs in mice (18), the deletions generated by TALENs contain a microhomology of 1–4 bp at the deletion boundary (Figure 3a). The statistic of the injections and mutation rates is shown in Table 1.Figure 3.


High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases.

Qiu Z, Liu M, Chen Z, Shao Y, Pan H, Wei G, Yu C, Zhang L, Li X, Wang P, Fan HY, Du B, Liu B, Liu M, Li D - Nucleic Acids Res. (2013)

Somatic mutations induced by TALENs in mice. (a) Frequencies and sequences of TALEN-induced mutations. The binding sites of the TALEN half-site are shown in bold. A dash represents a single base deletion. Insertions or substitutions are indicated in red letters. Microhomology that was likely used by NHEJ is underlined. The length and frequency of indels are labeled to the right of each sequence. (b) TALENs induce a wide range of indel length. The percentage is from the number of clones of different indel lengths divided by the number of total clones sequenced.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675477&req=5

gkt258-F3: Somatic mutations induced by TALENs in mice. (a) Frequencies and sequences of TALEN-induced mutations. The binding sites of the TALEN half-site are shown in bold. A dash represents a single base deletion. Insertions or substitutions are indicated in red letters. Microhomology that was likely used by NHEJ is underlined. The length and frequency of indels are labeled to the right of each sequence. (b) TALENs induce a wide range of indel length. The percentage is from the number of clones of different indel lengths divided by the number of total clones sequenced.
Mentions: To determine the mutations generated in individual founders, PCR products were TA cloned, and at least six colonies for each founder mouse were sequenced. All the mutations identified by sequencing are listed in Figure 3a and Supplementary Figure S1. Similar to other species, some of the founders were biallelically modified. For instance, founders 1, 4, 6, 10, 12, 13 of the Rprm gene had the same biallelic non-frame-shifting 3-bp deletions. Founders 2, 3, 7, 12 of the Gpr55 gene had a combination of frame-shifting and non-frame-shifting deletions (Figure 3a). Also, there are mosaic founders where more than two allele types have been identified from a single mouse, such as founder 8 of the Lepr gene in the FVB/N background, founders 3 and 7 of the Rprm gene and founder 19 of Fbxo6 gene (Figure 3a). However, biallelic mutations were not observed in the founders of other genes tested in this study. In line with the indels induced by TALEN in zebrafish (31), our TALENs generated indels with a broad range of lengths in mice, from 1 to 173 bp, with an average length of 15.4 bp (n = 61 independent indels in total) (Figure 3b). In accord with the deletions induced by ZFNs in mice (18), the deletions generated by TALENs contain a microhomology of 1–4 bp at the deletion boundary (Figure 3a). The statistic of the injections and mutation rates is shown in Table 1.Figure 3.

Bottom Line: Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism.One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation.In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.

ABSTRACT
Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ∼40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background.

Show MeSH
Related in: MedlinePlus