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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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Phenotypes of mice with biallelic indels of Lepr gene generated by TALEN-induced somatic mutations. (a) an 8-week-old Lepr8 (right) and its wild-type littermate (left) were anesthetized and photographed. (b) Body weight of an 8-week-old Lepr8 and its wild-type littermate. (c) Lepr8 and its wild-type littermate were fasted overnight (14–16 h) and injected IP with glucose (2 mg/g body weight) and whole-blood glucose levels were monitored for 3 h. (d) Lepr8 and its wild-type littermate were fasted for up to 4 h, and injected ip with insulin (1.5 U/kg), and whole-blood glucose levels were monitored for 1 h. (e) Comparison of H&E-stained WAT of Lepr8 (upper) and its wild-type littermate (lower). Dramatic cellular hypertrophy is observed in WAT. Scale bars: 100 μm. (f) Oil Red O staining of liver sections shows marked increase of lipid accumulation in Lepr8 (upper) relative to its wild-type littermate (lower).
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gkt258-F4: Phenotypes of mice with biallelic indels of Lepr gene generated by TALEN-induced somatic mutations. (a) an 8-week-old Lepr8 (right) and its wild-type littermate (left) were anesthetized and photographed. (b) Body weight of an 8-week-old Lepr8 and its wild-type littermate. (c) Lepr8 and its wild-type littermate were fasted overnight (14–16 h) and injected IP with glucose (2 mg/g body weight) and whole-blood glucose levels were monitored for 3 h. (d) Lepr8 and its wild-type littermate were fasted for up to 4 h, and injected ip with insulin (1.5 U/kg), and whole-blood glucose levels were monitored for 1 h. (e) Comparison of H&E-stained WAT of Lepr8 (upper) and its wild-type littermate (lower). Dramatic cellular hypertrophy is observed in WAT. Scale bars: 100 μm. (f) Oil Red O staining of liver sections shows marked increase of lipid accumulation in Lepr8 (upper) relative to its wild-type littermate (lower).

Mentions: One of our targeted genes, Lepr, encodes a receptor for Leptin, which has been shown to regulate food intake, body weight and glucose homeostasis and also has the potential for treating diabetes (32). The Lepr mutant db/db mice demonstrate obesity by the time they are 3–4 weeks old and have been widely used as the mouse model for type 2 diabetes (33–35). Biallelic mutation of the Lepr gene in mice will not cause embryonic lethality, and the obesity phenotype makes them easy to be distinguished, which is why we chose the Lepr gene as the target to test whether TALENs can generate somatic mutations with a similar phenotype. Our biallelically modified Lepr founder 8 (Lepr8) in the FVB/N strain, with different frame-shifting deletions, exhibited an obese phenotype by 4 weeks of age (Figure 4a). The body weight was approximately double the weight (44 versus 23 g) of wild-type littermates (Figure 4b). The glucose homeostasis of Lepr8 was severely impaired. In glucose tolerance tests, Lepr8 showed a marked higher fasting glucose level and glucose intolerance compared with its wild-type littermate (Figure 4c). During insulin tolerance testing, Lepr8 was unresponsive to insulin administration (Figure 4d), indicating resistance to insulin treatment. Moreover, examination of white adipose tissue (WAT) from Lepr8 revealed a considerable cellular hypertrophy compared with wild-type control (Figure 4e). In addition, Oil-Red-O staining of frozen sections from Lepr8 liver showed strong signals indicating lipids, whereas the counterpart section from the littermate control was barely stained, indicating a marked increase of lipid accumulation in the liver of Lepr8 (Figure 4f). These results suggested that the somatic mutations induced by TALEN effectively impaired target gene function.Figure 4.


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)

Phenotypes of mice with biallelic indels of Lepr gene generated by TALEN-induced somatic mutations. (a) an 8-week-old Lepr8 (right) and its wild-type littermate (left) were anesthetized and photographed. (b) Body weight of an 8-week-old Lepr8 and its wild-type littermate. (c) Lepr8 and its wild-type littermate were fasted overnight (14–16 h) and injected IP with glucose (2 mg/g body weight) and whole-blood glucose levels were monitored for 3 h. (d) Lepr8 and its wild-type littermate were fasted for up to 4 h, and injected ip with insulin (1.5 U/kg), and whole-blood glucose levels were monitored for 1 h. (e) Comparison of H&E-stained WAT of Lepr8 (upper) and its wild-type littermate (lower). Dramatic cellular hypertrophy is observed in WAT. Scale bars: 100 μm. (f) Oil Red O staining of liver sections shows marked increase of lipid accumulation in Lepr8 (upper) relative to its wild-type littermate (lower).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt258-F4: Phenotypes of mice with biallelic indels of Lepr gene generated by TALEN-induced somatic mutations. (a) an 8-week-old Lepr8 (right) and its wild-type littermate (left) were anesthetized and photographed. (b) Body weight of an 8-week-old Lepr8 and its wild-type littermate. (c) Lepr8 and its wild-type littermate were fasted overnight (14–16 h) and injected IP with glucose (2 mg/g body weight) and whole-blood glucose levels were monitored for 3 h. (d) Lepr8 and its wild-type littermate were fasted for up to 4 h, and injected ip with insulin (1.5 U/kg), and whole-blood glucose levels were monitored for 1 h. (e) Comparison of H&E-stained WAT of Lepr8 (upper) and its wild-type littermate (lower). Dramatic cellular hypertrophy is observed in WAT. Scale bars: 100 μm. (f) Oil Red O staining of liver sections shows marked increase of lipid accumulation in Lepr8 (upper) relative to its wild-type littermate (lower).
Mentions: One of our targeted genes, Lepr, encodes a receptor for Leptin, which has been shown to regulate food intake, body weight and glucose homeostasis and also has the potential for treating diabetes (32). The Lepr mutant db/db mice demonstrate obesity by the time they are 3–4 weeks old and have been widely used as the mouse model for type 2 diabetes (33–35). Biallelic mutation of the Lepr gene in mice will not cause embryonic lethality, and the obesity phenotype makes them easy to be distinguished, which is why we chose the Lepr gene as the target to test whether TALENs can generate somatic mutations with a similar phenotype. Our biallelically modified Lepr founder 8 (Lepr8) in the FVB/N strain, with different frame-shifting deletions, exhibited an obese phenotype by 4 weeks of age (Figure 4a). The body weight was approximately double the weight (44 versus 23 g) of wild-type littermates (Figure 4b). The glucose homeostasis of Lepr8 was severely impaired. In glucose tolerance tests, Lepr8 showed a marked higher fasting glucose level and glucose intolerance compared with its wild-type littermate (Figure 4c). During insulin tolerance testing, Lepr8 was unresponsive to insulin administration (Figure 4d), indicating resistance to insulin treatment. Moreover, examination of white adipose tissue (WAT) from Lepr8 revealed a considerable cellular hypertrophy compared with wild-type control (Figure 4e). In addition, Oil-Red-O staining of frozen sections from Lepr8 liver showed strong signals indicating lipids, whereas the counterpart section from the littermate control was barely stained, indicating a marked increase of lipid accumulation in the liver of Lepr8 (Figure 4f). These results suggested that the somatic mutations induced by TALEN effectively impaired target gene function.Figure 4.

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