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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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Characterization of the peripheral B-cell compartment of mutant offspring from ZFN-treated rabbits by FACS analysis.A. The top panels show representative dot plot profiles of peripheral blood mononuclear cells (PBMCs) of a wild type rabbit stained with antibodies specific for surface IgM (left) and surface IgG (right). Lower panels show Δ1/Δ7 PBMCs stained with the same antibodies. B. The upper dot plot shows wild type PBMCs immunostained with RACT30A, an antibody that recognizes a rabbit B-cell specific surface protein [25]. The lower panel shows Δ1/Δ7 PBMCs stained the same way. A and B: Only live PBMCs were included in these analyses. C. Dot plot profiles showing wild type (left) and Δ7/Δ7 (right) PBMCs, stained intracellularly for the B cell receptor component CD79a. The percentage of gated B cells (IgM+, IgG+, rabbit B cell marker+, or CD79a+) of all PBMCs is indicated in the upper right of each dot plot.
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pone-0021045-g002: Characterization of the peripheral B-cell compartment of mutant offspring from ZFN-treated rabbits by FACS analysis.A. The top panels show representative dot plot profiles of peripheral blood mononuclear cells (PBMCs) of a wild type rabbit stained with antibodies specific for surface IgM (left) and surface IgG (right). Lower panels show Δ1/Δ7 PBMCs stained with the same antibodies. B. The upper dot plot shows wild type PBMCs immunostained with RACT30A, an antibody that recognizes a rabbit B-cell specific surface protein [25]. The lower panel shows Δ1/Δ7 PBMCs stained the same way. A and B: Only live PBMCs were included in these analyses. C. Dot plot profiles showing wild type (left) and Δ7/Δ7 (right) PBMCs, stained intracellularly for the B cell receptor component CD79a. The percentage of gated B cells (IgM+, IgG+, rabbit B cell marker+, or CD79a+) of all PBMCs is indicated in the upper right of each dot plot.

Mentions: The Δ1 (male founder 22826) and Δ7 (female founder 22830) mutations in exon 1 both result in frameshifts in the IgM coding region and were predicted to disrupt expression of full-length protein. The founder male 22826 was intercrossed with the founder female 22830 to generate Δ1/Δ7 compound heterozygous animals. Three mutated F1 animals were obtained, one carrying the Δ1 mutation, one carrying the Δ7 mutation, and one Δ1/Δ7 compound heterozygote. The Δ7 mutation was also bred to homozygosity in an F2 generation. Serum IgM and IgG levels in these animals were determined by ELISA at 10 weeks of age. IgM and IgG levels were similar to wild type in Δ1 and Δ7 heterozygotes, but were undetectable in Δ1/Δ7 and Δ7/Δ7 rabbits (Table 2). The peripheral B-cell population in Δ1/Δ7 and Δ7/Δ7 rabbits was characterized by FACS analysis; only ∼0.1% of PBMCs stained positive for surface IgM (sIgM), and sIgG positive PBMCs were completely absent. In contrast, control wild type animals showed 21.4% of PBMC positive for sIgM and 0.7% positive for sIgG, typical for rabbit blood. A rabbit pan B-cell specific antibody [25] also revealed significantly fewer stained cells in the Δ1/Δ7 animal (1.5%) than in wild type (22.9%), see Figure 2 A and B. A second and well-defined B cell-specific marker is CD79a, constituting a part of the B cell receptor. Using an antibody that binds to the highly conserved intracellular domain of CD79a [26], no B cell receptor positive cells could be detected in Δ1/Δ7 and Δ7/Δ7 rabbits, whereas 41.4% of all PBMCs were marked as B cells in control animals (Figure 2 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)

Characterization of the peripheral B-cell compartment of mutant offspring from ZFN-treated rabbits by FACS analysis.A. The top panels show representative dot plot profiles of peripheral blood mononuclear cells (PBMCs) of a wild type rabbit stained with antibodies specific for surface IgM (left) and surface IgG (right). Lower panels show Δ1/Δ7 PBMCs stained with the same antibodies. B. The upper dot plot shows wild type PBMCs immunostained with RACT30A, an antibody that recognizes a rabbit B-cell specific surface protein [25]. The lower panel shows Δ1/Δ7 PBMCs stained the same way. A and B: Only live PBMCs were included in these analyses. C. Dot plot profiles showing wild type (left) and Δ7/Δ7 (right) PBMCs, stained intracellularly for the B cell receptor component CD79a. The percentage of gated B cells (IgM+, IgG+, rabbit B cell marker+, or CD79a+) of all PBMCs is indicated in the upper right of each dot plot.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0021045-g002: Characterization of the peripheral B-cell compartment of mutant offspring from ZFN-treated rabbits by FACS analysis.A. The top panels show representative dot plot profiles of peripheral blood mononuclear cells (PBMCs) of a wild type rabbit stained with antibodies specific for surface IgM (left) and surface IgG (right). Lower panels show Δ1/Δ7 PBMCs stained with the same antibodies. B. The upper dot plot shows wild type PBMCs immunostained with RACT30A, an antibody that recognizes a rabbit B-cell specific surface protein [25]. The lower panel shows Δ1/Δ7 PBMCs stained the same way. A and B: Only live PBMCs were included in these analyses. C. Dot plot profiles showing wild type (left) and Δ7/Δ7 (right) PBMCs, stained intracellularly for the B cell receptor component CD79a. The percentage of gated B cells (IgM+, IgG+, rabbit B cell marker+, or CD79a+) of all PBMCs is indicated in the upper right of each dot plot.
Mentions: The Δ1 (male founder 22826) and Δ7 (female founder 22830) mutations in exon 1 both result in frameshifts in the IgM coding region and were predicted to disrupt expression of full-length protein. The founder male 22826 was intercrossed with the founder female 22830 to generate Δ1/Δ7 compound heterozygous animals. Three mutated F1 animals were obtained, one carrying the Δ1 mutation, one carrying the Δ7 mutation, and one Δ1/Δ7 compound heterozygote. The Δ7 mutation was also bred to homozygosity in an F2 generation. Serum IgM and IgG levels in these animals were determined by ELISA at 10 weeks of age. IgM and IgG levels were similar to wild type in Δ1 and Δ7 heterozygotes, but were undetectable in Δ1/Δ7 and Δ7/Δ7 rabbits (Table 2). The peripheral B-cell population in Δ1/Δ7 and Δ7/Δ7 rabbits was characterized by FACS analysis; only ∼0.1% of PBMCs stained positive for surface IgM (sIgM), and sIgG positive PBMCs were completely absent. In contrast, control wild type animals showed 21.4% of PBMC positive for sIgM and 0.7% positive for sIgG, typical for rabbit blood. A rabbit pan B-cell specific antibody [25] also revealed significantly fewer stained cells in the Δ1/Δ7 animal (1.5%) than in wild type (22.9%), see Figure 2 A and B. A second and well-defined B cell-specific marker is CD79a, constituting a part of the B cell receptor. Using an antibody that binds to the highly conserved intracellular domain of CD79a [26], no B cell receptor positive cells could be detected in Δ1/Δ7 and Δ7/Δ7 rabbits, whereas 41.4% of all PBMCs were marked as B cells in control animals (Figure 2 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