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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

A Pair of TALENs used for targeting therabbit Rag 1 gene. Red circles show a 5′-T preceding the TALEN EBE binding sequences (in red). The spacer has a length of 16 base pairs. We used the FOKI variants ELD and KKR [22].
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Fig2: A Pair of TALENs used for targeting therabbit Rag 1 gene. Red circles show a 5′-T preceding the TALEN EBE binding sequences (in red). The spacer has a length of 16 base pairs. We used the FOKI variants ELD and KKR [22].

Mentions: Design the TALEN effector binding elements (EBEs) using standard principles applied in your laboratory. We routinely design 2 pairs of EBEs (15-17 base pairs each) for each target gene. We use the web-based public program TAL Effector Nucleotide Targeter 2.0 (https://tale-nt.cac.cornell.edu) for designing the EBEs. After obtaining candidates with this program, we adhere to 3 main principles derived from the reports by Cermak et al. and Doyle et al.[20, 23] (Figure 2). First, the EBEs (on the sense and antisense genomic DNA strand) should be preceded by a 5′-T. Second, the average G nucleotide composition of the left EBE should be less than 25%, and the same applies to the average C composition of the right EBE. Third, the optimum spacer length between the 2 EBEs should be 15-17 base pairs.


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)

A Pair of TALENs used for targeting therabbit Rag 1 gene. Red circles show a 5′-T preceding the TALEN EBE binding sequences (in red). The spacer has a length of 16 base pairs. We used the FOKI variants ELD and KKR [22].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: A Pair of TALENs used for targeting therabbit Rag 1 gene. Red circles show a 5′-T preceding the TALEN EBE binding sequences (in red). The spacer has a length of 16 base pairs. We used the FOKI variants ELD and KKR [22].
Mentions: Design the TALEN effector binding elements (EBEs) using standard principles applied in your laboratory. We routinely design 2 pairs of EBEs (15-17 base pairs each) for each target gene. We use the web-based public program TAL Effector Nucleotide Targeter 2.0 (https://tale-nt.cac.cornell.edu) for designing the EBEs. After obtaining candidates with this program, we adhere to 3 main principles derived from the reports by Cermak et al. and Doyle et al.[20, 23] (Figure 2). First, the EBEs (on the sense and antisense genomic DNA strand) should be preceded by a 5′-T. Second, the average G nucleotide composition of the left EBE should be less than 25%, and the same applies to the average C composition of the right EBE. Third, the optimum spacer length between the 2 EBEs should be 15-17 base pairs.

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