Isolation of homozygous mutant mouse embryonic stem cells using a dual selection system

Abstract

Obtaining random homozygous mutants in mammalian cells for forward genetic studies has always been problematic due to the diploid genome. With one mutation per cell, only one allele of an autosomal gene can be disrupted, and the resulting heterozygous mutant is unlikely to display a phenotype. In cells with a genetic background deficient for the Bloom's syndrome helicase, such heterozygous mutants segregate homozygous daughter cells at a low frequency due to an elevated rate of crossover following mitotic recombination between homologous chromosomes. We constructed DNA vectors that are selectable based on their copy number and used these to isolate these rare homozygous mutant cells independent of their phenotype. We use the piggyBac transposon to limit the initial mutagenesis to one copy per cell, and select for cells that have increased the transposon copy number to two or more. This yields homozygous mutants with two allelic mutations, but also cells that have duplicated the mutant chromosome and become aneuploid during culture. On average, 26% of the copy number gain events occur by the mitotic recombination pathway. We obtained homozygous cells from 40% of the heterozygous mutants tested. This method can provide homozygous mammalian loss-of-function mutants for forward genetic applications.

Figure 1.

Copy number selection for recovery of homozygous mutants. (A) Mechanism of LOH in Blm-deficient cells. Blue and red chromosomes represent homologs. Crossing over between homologous chromosomes is normally suppressed in mitosis (top). In the absence of Blm, crossovers can occur in G2 phase, forming recombinant chromatids. If these segregate to different daughter cells, LOH occurs distal to the point of crossover. If the cells carry a heterozygous mutation (represented by the star), this becomes homozygous after LOH, and the copy number of the mutation increases from one in the starting cell to two in the homozygous daughter. (B and C) Design of vectors for copy number-based selection. The vectors DHSV (B) and TNN (C) are shown integrated into an intron, with grey boxes representing exons of the disrupted gene; note that TNN can trap genes transcribed in either direction. SA, splice acceptor; PB5 and PB3, PB repeats.

Figure 2.

Isolation of homozygous mutants by selection for copy number increase. (A) Transposition using limiting amounts of transposon donor plasmid results in most G418 resistant subclones having one transposon per cell. (B) Selection scheme, showing predicted number and type of transposon present at each stage. (C) Typical result of double-selection stage. Well 5 has two non-allelic transposon integrations to begin with and thus all cells can become double resistant. (D) Genotyping scheme—primer positions (1–3) are shown relative to the transposon integration site. (E) All double-resistant subclones (derived from an integration in Dhx35) fail to amplify the wild-type PCR product. (F and G) Examples of double-resistant populations in which some (F) and all (G) subclones have a wild-type allele.

Figure 3.

Abnormal karyotype of clones that retain a wild-type allele. (A) Structure of Myo10 locus analyzed in (B–D). (B) PCR genotyping as in Figure 2D. All double-resistant subclones retain the wild-type allele. (C) All these subclones have a single-transposon integration site (top) but contain both pre- (In: insertion) and post-Cre (Del: deleted) forms of the transposon (bottom). (D) Spectral karyotype showing tetraploidy in a double-resistant subclone. (E) An example of a less severe aberration—only chromosome 6, which bears the insertion at the Grid2 locus, is present in excess. Left, FISH showing three copies of chromosome 6 with (white arrows) and without (yellow arrow) transposon insertions (red dots). Right, spectral karyotype.

Figure 4.

Measurement of LOH rate at different loci. (A) Position of loci investigated on chromosome 11. (B) Number of FIAU resistant colonies obtained (one-tenth of each culture was plated). Data are shown in the original random order, and the rate calculation shown in Table 2. (C) Cumulative frequency plot of all Vega-curated gene start sites (37). The positions of the loci investigated here are marked.

Figure 5.

Insertion of DHSV at Atm causes DNA damage sensitivity. (A) Structure of mutant Atm locus. (B) Triple primer PCR genotyping using primers a, b and c shown in (A). Clones 2, 3 and 5 lack the wild-type band, consistent with being homozygous mutants. (C) RT–PCR showing loss of Atm transcript. Internal primers to LacZ were included as a positive control. (D) Atm mutants are hypersensitive to adriamycin. Mean surviving fraction (colonies) is plotted. NN5, n =2; Atm^−/−, n = 3. Error bars show SEM.

Figure 6.

Robust disruption of transcription in homozygous mutants. (A) PCR genotyping of DHSV insertion in Ddef2 using primers for mutant (Junction, left) and wild-type alleles. All double-resistant subclones are homozygous mutants. (B) RT–PCR showing amplification of fusion transcript (left) and loss of wild-type transcript (right) in double-resistant subclones. (C and D) Insertions of the TNN vector also disrupt transcription. PCR and RT–PCR genotyping is shown for insertions in Dym (C) and Arrb2 (D). Asterisks indicate homozygous mutant subclones, all of which have lost wild-type transcript expression. Minus indicates no template control, Actb is an internal control.

Figure 7.

Pathways for copy number gain in Blm-deficient cells. Most cell divisions do not result in copy number increase (left). The two pathways of mitotic recombination (middle) and aneuploidy acquisition (right) result in copy number gain and contribute to the double-resistant population.