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Generation of knockout rabbits using transcription activator-like effector nucleases.

Wang Y, Fan N, Song J, Zhong J, Guo X, Tian W, Zhang Q, Cui F, Li L, Newsome PN, Frampton J, Esteban MA, Lai L - Cell Regen (Lond) (2014)

Bottom Line: Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research.They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci.This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Regenerative Biology of the Chinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, 510530 China.

ABSTRACT
Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

No MeSH data available.


Related in: MedlinePlus

In vitro and in vivo testing of TALENs. A. Schematic depiction of the SSA test as explained in step 4 of the procedure. B. Flow cytometry analysis of HEK 293 T cells transfected with a GFP “dead” reporter shows increased GFP activity with co-transfection of a specific pair of TALEN plasmids compared to the control. C. T7 endonuclease I test result. Lanes 1 and 2 are negative controls, 3 and 5 are samples A and B before treatment, respectively, 4 and 6 are samples A and B after treatment, respectively. White arrows indicate the expected fragments after cleavage by T7 endonuclease I; bp stands for base pairs.
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Fig3: In vitro and in vivo testing of TALENs. A. Schematic depiction of the SSA test as explained in step 4 of the procedure. B. Flow cytometry analysis of HEK 293 T cells transfected with a GFP “dead” reporter shows increased GFP activity with co-transfection of a specific pair of TALEN plasmids compared to the control. C. T7 endonuclease I test result. Lanes 1 and 2 are negative controls, 3 and 5 are samples A and B before treatment, respectively, 4 and 6 are samples A and B after treatment, respectively. White arrows indicate the expected fragments after cleavage by T7 endonuclease I; bp stands for base pairs.

Mentions: Construct a “dead” reporter plasmid by inserting the DNA sequence targeted by the TALENs’ EBEs and the spacer sequence (~48 base pairs) into the GFP (green fluorescent protein) coding sequence (Figure 3A). A similar readout can be obtained by creating a “dead” luciferase reporter plasmid [26].


Generation of knockout rabbits using transcription activator-like effector nucleases.

Wang Y, Fan N, Song J, Zhong J, Guo X, Tian W, Zhang Q, Cui F, Li L, Newsome PN, Frampton J, Esteban MA, Lai L - Cell Regen (Lond) (2014)

In vitro and in vivo testing of TALENs. A. Schematic depiction of the SSA test as explained in step 4 of the procedure. B. Flow cytometry analysis of HEK 293 T cells transfected with a GFP “dead” reporter shows increased GFP activity with co-transfection of a specific pair of TALEN plasmids compared to the control. C. T7 endonuclease I test result. Lanes 1 and 2 are negative controls, 3 and 5 are samples A and B before treatment, respectively, 4 and 6 are samples A and B after treatment, respectively. White arrows indicate the expected fragments after cleavage by T7 endonuclease I; bp stands for base pairs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230510&req=5

Fig3: In vitro and in vivo testing of TALENs. A. Schematic depiction of the SSA test as explained in step 4 of the procedure. B. Flow cytometry analysis of HEK 293 T cells transfected with a GFP “dead” reporter shows increased GFP activity with co-transfection of a specific pair of TALEN plasmids compared to the control. C. T7 endonuclease I test result. Lanes 1 and 2 are negative controls, 3 and 5 are samples A and B before treatment, respectively, 4 and 6 are samples A and B after treatment, respectively. White arrows indicate the expected fragments after cleavage by T7 endonuclease I; bp stands for base pairs.
Mentions: Construct a “dead” reporter plasmid by inserting the DNA sequence targeted by the TALENs’ EBEs and the spacer sequence (~48 base pairs) into the GFP (green fluorescent protein) coding sequence (Figure 3A). A similar readout can be obtained by creating a “dead” luciferase reporter plasmid [26].

Bottom Line: Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research.They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci.This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Regenerative Biology of the Chinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, 510530 China.

ABSTRACT
Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

No MeSH data available.


Related in: MedlinePlus