Generation of a mouse mutant by oligonucleotide-mediated gene modification in ES cells

Abstract

Oligonucleotide-mediated gene targeting is emerging as a powerful tool for the introduction of subtle gene modifications in mouse embryonic stem (ES) cells and the generation of mutant mice. However, its efficacy is strongly suppressed by DNA mismatch repair (MMR). Here we report a simple and rapid procedure for the generation of mouse mutants using transient down regulation of the central MMR protein MSH2 by RNA interference. We demonstrate that under this condition, unmodified single-stranded DNA oligonucleotides can be used to substitute single or several nucleotides. In particular, simultaneous substitution of four adjacent nucleotides was highly efficient, providing the opportunity to substitute virtually any given codon. We have used this method to create a codon substitution (N750F) in the Rb gene of mouse ES cells and show that the oligonucleotide-modified Rb allele can be transmitted through the germ line of mice.

Figure 1

Oligonucleotide-mediated base substitution. (a) A single copy of a defective neomycin gene (neo) carrying an T to A point mutation in the start codon (Target 2) was inserted into the Rosa26 locus of Msh2^−/−, Msh3^−/−, Msh6^−/−, Msh2^+/− and wild-type ES cells. Upper case indicates codons. (b) Activity of the neo gene can be restored by single-stranded DNA oligonucleotides that substitute 1–4 nt to create a new ATG start codon (mismatching bases are indicated in red). Upper case indicates codons. (c) The efficiency of oligonucleotide-mediated base substitution in Msh3^−/− (red bars), Msh6^−/− (blue bars) and pS-MSH2 transfected wild-type (green bars) or Msh2^+/− (orange bars) ES cells is the number of G418-resistant colonies per 10⁵ cells that were plated after exposure to the oligonucleotides. Targeting data of Msh2^−/− (black bars) and wild-type (white bars) ES cells are taken from Ref. (26) and shown as controls. For each oligonucleotide, the mean value is given of at least three independent experiments. Error bars show standard deviation. * P < 0.001, **P < 0.05 as compared with untreated wild-type cells (Student's t-test).

Figure 2

Oligonucleotide-mediated base insertion. (a) A single copy of a defective neomycin gene (neo) carrying a GT insertion behind the start codon (target 1) was inserted into the Rosa26 locus of Msh2^−/−, Msh6^−/−, Msh2^+/− and wild-type ES cells. Upper case indicates codons. (b) Activity of the neo gene can be restored by single-stranded DNA oligonucleotides inserting either 1 or 4 nt to regenerate the open reading frame (inserted bases are shown in red). Upper case indicates codons. (c) The efficiency of oligonucleotide-mediated base insertion in Msh6^−/− (blue bars) and pS-MSH2 transfected wild-type (green bars) or Msh2^+/− (orange bars) ES cells is the number of G418-resistant colonies per 10⁵ cells that were plated after exposure to the oligonucleotides. Targeting data of Msh2^−/− (black bars) and wild-type (white bars) ES cells are taken from Ref. (26) and shown as controls. For each oligonucleotide, the mean value is given of at least three independent experiments. Error bars show standard deviation. * denotes P < 0.001 as compared with untreated wild-type cells (Student's t-test).

Figure 3

MMR protein levels after MSH2 suppression. Western blot analysis of wild-type ES cells transfected with pS-MSH2 followed by puromycin selection for 2 days. Whole-cell extracts were analyzed for MSH2, MSH3 and MSH6 for six consecutive days. Whole-cell extracts of Msh2^−/− cells were used as negative control. WT, wild-type cells not treated with pS-MSH2; Asterisk indicates non-specific band; l.c., non-specific band as loading control.

Figure 4

Oligonucleotide-mediated codon substitution in Rb. (a) Single-stranded DNA oligonucleotide Rb-CTTT was designed to replace an asparagine by a phenylalanine at position 750 in exon 22 of the Rb gene. Mismatching bases are indicated in red. Arrows indicate the location of PCR primers. (b) Primer pair 1/2 was used to amplify a 738 bp fragment from pools of cells. This fragment was used in a second PCR round using the nested primer pairs 3/4 or 5/6 of which primers 3 and 6 are specific for the CTTT mutation. (c) Sequence analysis of Rb mRNA in a purified mutant ES cell clone revealed the presence of the CTTT mutation, replacing the asparagine at position 750 by a phenylalanine. (d) PCR-based detection of Rb^N750F mutation in genomic DNA. PCRs were conducted with primer pairs 1/2 and 3/4, yielding a 213-bp product specific for the CTTT mutation. Lane M, molecular mass standards; lane 1, Rb^+/N750F ES cell clone; lane 2, Rb^+/N750F mouse; lane 3, wild-type littermate; lane 4, water control; * indicates non-specific band.