Zinc-finger nuclease mediated disruption of Rag1 in the LEW/Ztm rat

Abstract

Background: Engineered zinc-finger nucleases (ZFN) represented an innovative method for the genome manipulation in vertebrates. ZFN introduced targeted DNA double strand breaks (DSB) and initiated non-homologous end joining (NHEJ) after pronuclear or cytoplasmatic microinjection into zygotes. Resulting frame shift mutations led to functional gene ablations in zebra fish, mice, pigs and also in laboratory rats. Therefore, we targeted the rat Rag1 gene essential for the V(D)J recombination within the immunoglobulin production process and for the differentiation of mature B and T lymphocytes to generate an immunodeficient rat model in the LEW/Ztm strain.

Results: After microinjection of Rag1 specific ZFN mRNAs in 623 zygotes of inbred LEW/Ztm rats 59 offspring were born from which one carried a 4 bp deletion. This frame shift mutation led to a premature stop codon and a subsequently truncated Rag1 protein confirmed by the loss of the full-length protein in Western Blot analysis. Truncation of the Rag1 protein was characterized by the complete depletion of mature B cells. The remaining T cell population contained mature CD4+/CD3+/TCRαβ+ as well as CD8+/CD3+/TCRαβ+ positive lymphocytes accompanied by a compensatory increase of natural killer cells in the peripheral blood. Reduction of T cell development in Rag1 mutant rats was associated with a hypoplastic thymus that lacked follicular structures. Histological evaluation also revealed the near-complete absence of lymphocytes in spleen and lymph nodes in the immunodeficient Rag1 mutant rat.

Conclusion: The Rag1 mutant rat will serve as an important model for transplantation studies. Furthermore, it may be used as a model for reconstitution experiments related to the immune system, particularly with respect to different populations of human lymphocytes, natural killer cells and autoimmune phenomena.

Figure 1

Introduction of a frame shift mutation. A The 11097 base pairs of genomic Rag1 are separated in two exons. Of these only Exon 2 contains the complete coding sequence (red bar). Rag1 specific zinc-finger nucleases recognizing 18 bp upstream and downstream of the 6 bp cutting site between position 5233 and 5267 introduced DSBs through the FokI endonuclease domain and initiated NHEJ. BCel-I endonuclease digest of the 310 bp heteroduplex DNA derived from the hybridization of the DNA from LEW wild type rats and founder 58 into 174 bp and 136 bp fragments proofed the mutagenesis of one Rag1 allele in founder 58. G/C: heteroduplex control DNA; LEW: LEW/Ztm wild type DNA.

Figure 2

Analysis of the mutant Rag1 allele. A Heterozygosity at the genomic Rag1 locus of founder 58 was confirmed by sequencing. The start of the mixed signal indicated the Rag1 mutation identified as A to C exchange (green upper case) plus 4 bp deletion at the ZFN cut site (red lower case) and the ZFN binding regions (black upper case). The introduced frame shift caused a premature stop codon leading to a truncation of the Rag1 protein to a 198 aa N-terminal residue. B Full length Rag1 protein consists the functional important zinc-binding dimerization domain (ZDD) containing the RING finger (RING) and the zinc finger A (ZFA) as well as the c-terminal core domain with the nonamer-binding region (NBR) and the zinc finger B (ZFB). Mutated Rag1 lost all domains essential for Rag1 function in V(D)J recombination.

Figure 3

Off-target mutation analysis. A Cel-I digest of heteroduplex DNA revealed no additional off-target mutations at the 10 loci with highest homology to Rag1; 1: Vom2r-ps62; 2: Atrn; 3: Vwa5b1; 4: KIAA2012; 5: Zfn9; 6: Dctn1; 7: Mylip; 8: Manl1; 9: LOC689553; 10: Kir3d1l; G/C: control heteroduplex DNA. B Genes, gene IDs, sequence homologies and localization of Rag1 related sequences to exclude off-target mutations; upper case: ZFN binding sites; lower case: ZFN cut site; homolog base pairs in red; (-): Gene located on the minus strand.

Figure 4

Genotyping and macroscopical examination. A Genotyping of the offspring derived from founder 58 using PCR amplification and StyI-HF digest. Half of the 1052 bp PCR products from heterozygous animal are digested into 603 bp and 449 bp fragments, while the Rag1 PCR product from wild type offspring was digested completely; N: negative control. 58: PCR product from founder 58 as positive control. B Normal thymus of wild type animal of the strain LEW-Rag1em1/Ztm-Ragl^emlZtm. C Hypoplastic thymus in a Rag1 mutant rat. D Comparison of the thymus sizes of wild type and Rag1 mutant rats. E Comparison of thymus weigths, *** : P < 0.0001.

Figure 5

Histological evaluation of lymphatic organs of wild type and Rag1 mutant rats. A and B thymus; C and D spleen; E and F lymph nodes. H/E staining.

Figure 6

Analysis of Rag1 expression in lymphatic organs. RT-PCR of wild type, heterozygous and Rag1 mutant rats: The Rag1 PCR product (upper panel) was digested with StyI-HF to distinguish between translation of wild type and mutated Rag1 (mid panel). GAPDH used as housekeeping gene (lower panel). B Western Blot analysis: Translation of Rag1 in the thymus from wild type, heterozygous and Rag1 mutant rats compared to GAPDH as housekeeping protein. C Comparison of NK cells, B and T cell ratio in the peripheral blood of wild type, heterozygous and Rag1 mutated rats determined by FACS analysis. *** P < 0.0001; ** P < 0.005.

Figure 7

FACS analysis of NKC, B and T lymphocytes in the blood: A wild type, B heterozygous and C Rag1 mutant rat; blue: CD161 positive NKC; green: CD45RA positive B cells; red: T cell receptor positive lymphocytes.

Figure 8

Mature CD4 or CD8 positive T lymphocytes in the peripheral blood of Rag1 mutant rats. A Absence of CD4⁺/CD8⁺ lymphocytes in wild type and Rag1 mutant rats. B and C Reduction of CD4⁺/CD3⁺ and CD8⁺/CD3⁺ T cells. D and E While there are no differences in the population of CD25⁺/CD4⁺ T cells the number of CD25⁺/CD8⁺ lymphocytes was reduced. orange: autofluorescent cells; green: FITC positive lymphocytes; blue: PE positive lymphocytes.