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Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.

Flisikowska T, Thorey IS, Offner S, Ros F, Lifke V, Zeitler B, Rottmann O, Vincent A, Zhang L, Jenkins S, Niersbach H, Kind AJ, Gregory PD, Schnieke AE, Platzer J - PLoS ONE (2011)

Bottom Line: Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed.Two major goals have been achieved.Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species.

View Article: PubMed Central - PubMed

Affiliation: Chair of Livestock Biotechnology, Technische Universität München, Freising, Germany.

ABSTRACT
Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+) and IgG(+) B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.

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ZFN-mediated disruption of rabbit IgM.A. Recognition sequences in the rabbit IgM locus for ZFNs used in this study. B. Screening data using a budding yeast proxy system (see Materials and Methods) for ZFNs shown in panel A. The first sample represents positive control ZFNs that target the human CCR5 locus [51]. The grey and black bars represent reporter gene correction levels at low and high levels of ZFN synthesis, respectively. As detailed in Doyon et al. [18], the ZFN-encoding transgene is inducible, and this allows measurement of ZFN activity at different levels of the nuclease. C. DNA target sequences and recognition helices for the ZFNs used in the present work. The binding regions of left (ZFN-L) and right (ZFN-R) zinc finger protein array are underlined. Bases in capital letters are those that contribute to binding of the ZFP arrays. D. Summary of rabbit IgM mutant alleles obtained and characterized in offspring from ZFN microinjections. Stillborn animals are marked with an asterisk. The genotype of each allele is indicated to the right of the DNA sequence; inserted sequences are highlighted in red.
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pone-0021045-g001: ZFN-mediated disruption of rabbit IgM.A. Recognition sequences in the rabbit IgM locus for ZFNs used in this study. B. Screening data using a budding yeast proxy system (see Materials and Methods) for ZFNs shown in panel A. The first sample represents positive control ZFNs that target the human CCR5 locus [51]. The grey and black bars represent reporter gene correction levels at low and high levels of ZFN synthesis, respectively. As detailed in Doyon et al. [18], the ZFN-encoding transgene is inducible, and this allows measurement of ZFN activity at different levels of the nuclease. C. DNA target sequences and recognition helices for the ZFNs used in the present work. The binding regions of left (ZFN-L) and right (ZFN-R) zinc finger protein array are underlined. Bases in capital letters are those that contribute to binding of the ZFP arrays. D. Summary of rabbit IgM mutant alleles obtained and characterized in offspring from ZFN microinjections. Stillborn animals are marked with an asterisk. The genotype of each allele is indicated to the right of the DNA sequence; inserted sequences are highlighted in red.

Mentions: ZFNs directed against exons 1–4 of rabbit IgM (Figure S1) were designed using an archive of pre-validated zinc finger modules as described [14], [15], [17]–[20]. The ZFNs were ranked for activity using a budding yeast based system previously shown to identify nucleases active in editing endogenous loci in zebrafish and rat [18], [20], and the highest-ranking ZFN for each exon was selected for in-vivo use. Target sequence, structure and recognition helices of the selected ZFNs are shown in Figure 1 A–C.


Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.

Flisikowska T, Thorey IS, Offner S, Ros F, Lifke V, Zeitler B, Rottmann O, Vincent A, Zhang L, Jenkins S, Niersbach H, Kind AJ, Gregory PD, Schnieke AE, Platzer J - PLoS ONE (2011)

ZFN-mediated disruption of rabbit IgM.A. Recognition sequences in the rabbit IgM locus for ZFNs used in this study. B. Screening data using a budding yeast proxy system (see Materials and Methods) for ZFNs shown in panel A. The first sample represents positive control ZFNs that target the human CCR5 locus [51]. The grey and black bars represent reporter gene correction levels at low and high levels of ZFN synthesis, respectively. As detailed in Doyon et al. [18], the ZFN-encoding transgene is inducible, and this allows measurement of ZFN activity at different levels of the nuclease. C. DNA target sequences and recognition helices for the ZFNs used in the present work. The binding regions of left (ZFN-L) and right (ZFN-R) zinc finger protein array are underlined. Bases in capital letters are those that contribute to binding of the ZFP arrays. D. Summary of rabbit IgM mutant alleles obtained and characterized in offspring from ZFN microinjections. Stillborn animals are marked with an asterisk. The genotype of each allele is indicated to the right of the DNA sequence; inserted sequences are highlighted in red.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113902&req=5

pone-0021045-g001: ZFN-mediated disruption of rabbit IgM.A. Recognition sequences in the rabbit IgM locus for ZFNs used in this study. B. Screening data using a budding yeast proxy system (see Materials and Methods) for ZFNs shown in panel A. The first sample represents positive control ZFNs that target the human CCR5 locus [51]. The grey and black bars represent reporter gene correction levels at low and high levels of ZFN synthesis, respectively. As detailed in Doyon et al. [18], the ZFN-encoding transgene is inducible, and this allows measurement of ZFN activity at different levels of the nuclease. C. DNA target sequences and recognition helices for the ZFNs used in the present work. The binding regions of left (ZFN-L) and right (ZFN-R) zinc finger protein array are underlined. Bases in capital letters are those that contribute to binding of the ZFP arrays. D. Summary of rabbit IgM mutant alleles obtained and characterized in offspring from ZFN microinjections. Stillborn animals are marked with an asterisk. The genotype of each allele is indicated to the right of the DNA sequence; inserted sequences are highlighted in red.
Mentions: ZFNs directed against exons 1–4 of rabbit IgM (Figure S1) were designed using an archive of pre-validated zinc finger modules as described [14], [15], [17]–[20]. The ZFNs were ranked for activity using a budding yeast based system previously shown to identify nucleases active in editing endogenous loci in zebrafish and rat [18], [20], and the highest-ranking ZFN for each exon was selected for in-vivo use. Target sequence, structure and recognition helices of the selected ZFNs are shown in Figure 1 A–C.

Bottom Line: Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed.Two major goals have been achieved.Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species.

View Article: PubMed Central - PubMed

Affiliation: Chair of Livestock Biotechnology, Technische Universität München, Freising, Germany.

ABSTRACT
Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+) and IgG(+) B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.

Show MeSH
Related in: MedlinePlus