Figure 1.
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.
Table 1.
Injection of ZFN-encoding mRNA into fertilized rabbit oocytes.
Figure 2.
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.
Table 2.
IgM and IgG serum levels at 10 weeks of age.
Figure 3.
Targeted sequence replacement at the IgM locus.
The structure of the IgME1 gene targeting vector, regions of homology with the IgM locus, and the predicted structure of the targeted locus are shown. Exons are marked and numbered as open boxes. The ZFN 18255/18257 cleavage site within exon 1 is indicated by a vertical arrow. The 5′ and 3′ junction PCR products are indicated; with positions of primers marked by arrows, and restriction sites for MscI and ApaI marked by (M) and (A), respectively. The hybridization probe and the diagnostic fragments used for Southern analysis are indicated in the lower part of the diagram. HinDIII (H) and BspHI (B) restriction sites are marked.
Figure 4.
PCR analysis of gene targeted blastocysts and fetuses.
Upper panel, representative 5′ junction PCR products amplified from blastocysts. Upper panel left, 5′ junction PCR from fetuses. Predicted 5′ PCR fragment size: 1.758 kb. Upper panel right, restriction digests of 5′ PCR fragment amplified from fetus 7. Predicted restriction fragment sizes: ApaI - 1242, 516 bp; MscI - 853, 687, 219 bp. Middle and bottom panels, 5′ and 3′ junction PCR products amplified from fetuses 1–12. Predicted 3′ PCR fragment size: 1.683 kb. M - size markers.
Table 3.
Screening of rabbit blastocysts for targeted gene replacement.