We have developed a method for enriching libraries of Blm-deficient ES cells carrying single-copy transposon mutations for homozygous mutants. The method is based on selection for two copies of the transposon construct in the rare homozygous mutants, which is achieved by a switchable drug selection cassette. Using our method of single-copy mutagenesis, clonal expansion, cassette switching by Cre recombinase and selection for two copies, we obtained double-drug resistant populations enriched in homozygous mutants. The proportion of homozygous mutants increased from a predicted 0.1% in the expanded unselected population to a measured average of 26%, depending on the vector used, in the double-resistant population. This increased proportion means that homozygous mutants can be obtained by picking relatively small numbers of subclones. It is possible that this number is an underestimate where clones were genotyped by absence of wild-type locus by PCR, as this may be very sensitive to contamination either between clones or from wild-type feeder cells.
Genotyping of subclones from the double-drug resistant populations revealed two classes of mutants. We found that while some subclones had lost the wild-type locus, consistent with a homozygous mutant, others retained a wild-type locus that could be amplified by PCR. Further analysis of some of these clones revealed numerical chromosome abnormalities, either genome wide (tetraploidy) or affecting the chromosome bearing the transposon insertion (trisomy). We conclude that our method faithfully selects for two copies of the construct, and there are two routes by which Blm
cells can increase mutation copy number—the predicted LOH pathway and by numerical chromosome instability (). Often we isolated similar numbers of these two classes of double-resistant subclone, suggesting that each of these events occurs at a similar low rate. In some cases, this may be exacerbated by a growth advantage linked to trisomy, particularly for chromosomes 8, 11 and 16 (40–42
). However, we did successfully isolate homozygous mutants from insertions on these chromosomes.
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 (more ...)
The system of selection for copy number increase in Blm
-deficient ES cells could be applied more broadly to obtain large collections of mutants for study, for example in genetic screens. Using the procedure described above, we isolated homozygous mutants for 48 out of 108 cell lines tested ( and Supplementary Tables S1
). We screened relatively small numbers of subclones (usually six or fewer); therefore it is possible that homozygous mutants could be isolated for some of the remaining 60 cell lines by screening further subclones.
We were unable to isolate homozygous mutants for some clones, either because no double-resistant cells were obtained or because all double-resistant subclones analyzed retained the wild-type locus. This could be simply because the events leading to double resistance are rare and may not occur in every expanded clone, or because only a chromosome instability event and not an LOH event occurs. Another explanation could be that the gene mutated is essential for ES cell survival or growth, and therefore homozygous cells do not survive or are at a growth disadvantage compared with wild-type retaining cells. This is likely to be the case for some clones that we tested, for example Faf1
, a mutant of which has been reported to cause early embryonic lethality and for which no homozygotes were isolated using our system (43
). It should also be noted that when the DHSV vector is used, double-resistant cells may not be recovered if Cre is very efficient and causes deletion in both copies of the resistant cassette. In the case of TNN, the theoretical chance of isolating a cell with two copies is 50% if the Cre reaction reaches an effective equilibrium—this represents the probability that both copies are in different orientations. However, we did not notice an overall difference in the number of double-resistant clones isolated with each vector in practice.
mutant alleles used here are constitutive, rather than inducible, mutations. Since Blm is a key regulator of homologous recombination (44
), there is cause for concern that a constitutive mutation may result in ongoing genome instability. We have not observed high levels of gross chromosomal instability, for example genuine homozygotes isolated from cell lines that also gave aneuploid subclones usually have a normal diploid karyotype (at the microscopic level). An inducible Blm
) is an obvious potential improvement to the system, and a similar system has been developed using such an allele (45
). Even in this case, Blm function must be still disrupted to allow LOH, hence an inducible allele will not improve genome stability during the homozygote isolation process, although it would allow for formal separation of the effects of the transposon-induced mutations from the Blm
-deficient genetic background.
Even better validation of mutations could come from resources of targeted mouse ES cell knockouts and targeting vectors, which are increasing in size to the point where obtaining a heterozygous mutant in a gene of interest will become trivial. These ES cell lines can be used to generate homozygous mutants after disruption of the remaining wild-type allele using the vectors provided (14
). Given the ease of obtaining mutants from the public resources in a low-passage cell line with a ‘clean’ genetic background, this represents the best option for further functional study. Mammalian screening methods such as the one we describe here are an excellent complement to these resources, as they provide rapid functional information to prioritize study of the available mutants.
In conclusion, we describe a system using PB
transposon constructs that cause null mutations with wide genome coverage in mouse ES cells that are a versatile model mammalian cell type. The mutations generated are stable, but reversion can be induced and selected for by a second round of transposition to allow phenotype rescue experiments (26
). This gives a robust link between genotype and phenotype, representing a key advantage compared to RNAi-based screens where causally linking the phenotype to the targeted gene can be problematic. Moreover, other mutations or reporter constructs can easily be introduced into the Blm
-deficient cells to create customized libraries for more sophisticated screens. By providing easy access to elusive homozygous mammalian mutant cells, our method significantly expands the scope of genetic screens and future functional studies in mammalian cells.