Modeling disease mutations by gene targeting in one-cell mouse embryos - PubMed (original) (raw)

Modeling disease mutations by gene targeting in one-cell mouse embryos

Melanie Meyer et al. Proc Natl Acad Sci U S A. 2012.

Abstract

Gene targeting by zinc-finger nucleases in one-cell embryos provides an expedite mutagenesis approach in mice, rats, and rabbits. This technology has been recently used to create knockout and knockin mutants through the deletion or insertion of nucleotides. Here we apply zinc-finger nucleases in one-cell mouse embryos to generate disease-related mutants harboring single nucleotide or codon replacements. Using a gene-targeting vector or a synthetic oligodesoxynucleotide as template for homologous recombination, we introduced missense and silent mutations into the Rab38 gene, encoding a small GTPase that regulates intracellular vesicle trafficking. These results demonstrate the feasibility of seamless gene editing in one-cell embryos to create genetic disease models and establish synthetic oligodesoxynucleotides as a simplified mutagenesis tool.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Generation of Rab38 G19V mutants by ZFNRab38 and gene-targeting vector. (A) The targeting vector is designed for the insertion of a G19V mutation into the first exon of Rab38. The structure of the Rab38+ locus, the targeted Rab38IDG-Cht allele, and the location of the ZFNRab38 binding sites of the Rab38 5′ probe and of PCR primers P-for and P-rev are shown. The positions of SexAI (S), BsaJI (B), and ApaLI (A) restriction sites and of SexAI restriction fragments are indicated. (B) Comparison of Rab38 codons 17–20 of the Rab38 wild type allele, (Rab38+) the natural Rab38 chocolate (Rab38cht) allele, and the targeting vector (IDG-Cht). Nucleotides and amino acids differing from the wild type sequence are shown in red. The presence of a thymidine at the second position of codon 19 creates a valine codon, erases a BsaJI, and generates a SexAI site. The IDG-Cht G19V replacement was marked with an additional silent replacement at the third position of codon 19. (C) PCR amplification of a 213 bp region covering the first exon of Rab38 from C57BL/6 (B6), homozygous Rab38cht (Cht/Cht), heterozygous Rab38cht (Cht/+) control mice and pup R1.1 derived from embryos injected with ZFNRab38 and targeting vector (upper image). PCR products were digested with BsaJI (lower image) to determine the Rab38 genotype of pup R1.1 as Rab38+/Rab38IDG-Cht. (D) Comparison of sequences within the first exon of the Rab38 wild type gene, the targeting vector (IDG-Cht) and of BsaJI resistant PCR products amplified with primers P-for and P-rev from genomic DNA of pups R1.1, R3.5 and R3.9. The position of codon 19 and the ZFNRab38 binding regions are indicated. Nucleotides differing from the wild type sequence (blue background) are shown on a yellow background. (E) Southern blot analysis of SexAI digested genomic DNA using the Rab38 5′ probe shows a 10.5 kb and a 6.0 kb fragment from the Rab38+ loci of two C57BL/6 (B6) control mice and an additional 2.1 kb band derived from the targeted Rab38IDG-Cht allele of pups R1.1, R3.5, and R3.9. Homozygous Rab38IDG-Cht mutants (#47, #86) show the 10.5 kb and 2.1 kb bands, but not the 6.0 kb band. (F) Comparison of the coat color of a wild type C57BL/6N mouse and a homozygous Rab38IDG-Cht (#47) mouse.

Fig. 2.

Fig. 2.

Targeting of the Rab38 gene by ZFNRab38 and a synthetic oligodesoxynucleotide. (A) The targeting oligonucleotide ODNIDG-WT is designed for the introduction of a silent nucleotide replacement in codon 18 of a Rab38+ or Rab38cht allele. The structure of the Rab38+ and the Rab38cht (cht) locus, of the targeted Rab38IDG-WT allele, the location of the ZFNRab38 binding sites of the Rab38 5′-probe and of PCR primers P-for and P-rev are shown. The positions of SexAI (S), BsaJI (B), and ApaLI (A) restriction sites and of SexAI and ApaLI restriction fragments are indicated. The Rab38cht allele (cht) differs from Rab38+ (+) by the presence of a SexAI site instead of a BsaJI site in the first exon. (B) Comparison of Rab38 codons 17–19 and 30/31 of the Rab38 wild type allele (Rab38+), the Rab38cht allele and the targeting ODNIDG-WT. Nucleotides and amino acids differing from the wild type sequence are shown in red. For ODNIDG-WT, the presence of a thymidine at the third position of codon 31 erases an ApaLI site. (C) PCR amplification with primer pair P-for/P-rev of a 213 bp region covering the first exon of Rab38 from DNA from a C57BL/6 (B6) and a homozygous Rab38IDG-Cht (IDG-Cht/IDG-Cht) control mouse and pups C7.3 and C7.4 derived from Rab38+/Rab38cht embryos injected with ZFNRab38 and targeting ODNIDG-WT. The Rab38 genotype was determined by digestion of the PCR products with BsaJI, or ApaLI. PCR products derived from a Rab38+ allele (B6) or a targeted Rab38IDG-WT allele (recombined within codon 18) are digested by BsaJI into fragments of 153 bp and 60 bp; product from Rab38IDG-Cht alleles (IDG-Cht/IDG-Cht) is resistant to BsaJI digestion. Digestion with ApaLI of PCR products from Rab38+ and Rab38IDG-Cht alleles results into fragments of 115 bp and 98 bp that appear within a single band; product from a Rab38IDG-Cht allele (recombined within codon 30/31) is resistant to ApaLI digestion. The analysis of PCR products showed in both pups C7.3 and C7.4 the presence of an ApaLI resistant Rab38 allele. The more diffuse appearance of the ApaLI resistant PCR product of sample 7.4 is likely explained by the presence of a 27 bp deletion (see D) resulting in a reduced size of 186 bp and either an incomplete digestion or the presence of hybrid molecules formed with full-length wild type DNA strands. Pup C7.3 but not C7.4 harbors a BsaJI resistant Rab38cht allele. M: 100 bp size ladder. (D) Comparison of sequences within the first exon of the Rab38+ and Rab38cht alleles, the targeting ODNIDG-WT, and of ApaLI-resistant PCR products amplified with primers P-for and P-rev from genomic DNA of pups C7.3 and C7.4. The position of codon 18 and the ZFNRab38 binding regions are indicated. Nucleotides differing from the wild type sequence (blue background) are shown on a yellow background; nucleotide deletions are indicated by a dash. (E) Southern blot analysis of SexAI digested genomic DNA using the Rab38 5′-probe shows the presence of Rab38cht alleles (2.1 kb band) in a homozygous Rab38IDG-Cht control mouse (IDG-Cht) and pup C7.3 but its absence in pup C7.4. (F) Southern blot analysis of ApaLI-digested genomic DNA using the Rab38 5′-probe shows a 6.6 kb fragment from the Rab38+ alleles of a C57BL/6 control mouse (B6) and a 10.5 kb band for the Rab38IDG-Cht alleles from a homozygous Rab38IDG-Cht control (IDG-Cht/IDG-Cht). Both pups C7.3 and C7.4 exhibit a 6.6 kb band from an unmodified Rab38+ (C7.4) or Rab38cht C7.3) allele and a 10.5 kb band derived from an ApaLI resistant Rab38IDG-WT allele in pup C7.3 and a Rab38Δ allele in pup C7.4.

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