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

Yue Huang 0 1 Stephen J. Pettitt 1 Ge Guo 1 Guang Liu 0 Meng Amy Li 1 Fengtang Yang 1 Allan Bradley 1 0 State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Department of Medical Genetics, Peking Union Medical College & Chinese Academy of Medical Sciences , Dong Dan San Tiao 5, Beijing 100005, China 1 The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus , Hinxton, Cambridge CB10 1SA, UK 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 lossof-function mutants for forward genetic applications. - Mammalian cell lines provide a convenient model for mammalian cell biology, particularly in high-throughput applications where using mice is not feasible. Forward genetic screens using various cell lines have identified genes required for many cellular processes (1). Gain-of-function screens, where genes are overexpressed or ectopically expressed, have been successful for many phenotypes (2,3). However, loss-of-function screens using mutant cells that lack expression of a particular gene are harder to conduct in diploid mammalian cells, as in most cases both alleles of a gene must be knocked out to see a phenotype. This difficulty has meant that loss-of-function screens have not been applied as widely as in yeast, Drosophila or Caenorhabditis elegans, where homozygous mutants can easily be obtained on a genome-wide scale (4,5). This problem of obtaining functional null mutants can be solved using a number of technologies, each with limitations. First, a cell line that is functionally hemizygous can be used. Chinese hamster ovary cells are one such cell type in which many loci are functionally hemizygous and these cells have been used to isolate, for example, X-ray sensitive mutants, with a single random hit (6). However, the extent of hemizygosity in the genome of these cells is unknown and it is likely that not all genes are accessible. A near-haploid human leukemia cell line has also been described, which can be used to isolate loss-of-function mutants in the haploid portion of the genome with a single hit (7,8). Further derivatives of this cell line (9), and the recent report of a haploid ES cell line (10), show promise for screens in the future. RNA interference (RNAi) screens have also been used in mammalian cells (11). These act at the mRNA level and thus the zygosity of the gene is irrelevant. Many RNAi screens have been performed in mammalian cell lines, and the reagents are applicable to a variety of cell types enabling many phenotypes to be studied. Common problems encountered are incomplete knockdown at the protein level, and off-target effects where transcripts other than the one predicted are affected, which can lead to variable results (12,13). The most robust way to make loss-of-function mutations in a relatively normal cell line would be serially target both alleles in mouse embryonic stem (ES) cells. Although resources of targeting vectors and targeted heterozygous mutant ES cells are increasing in size [www.knockoutmouse.org and ref. (14)], this is still a very time-consuming task on the scale required for genome-wide genetic screens of homozygous mutations. We have previously used ES cells deficient for the Bloom syndrome gene [Blm, ref. (15)] to obtain homozygous mutants. Blm-deficient cells (referred to here as Blm cells for simplicity) have an increased frequency of crossing over following mitotic recombination relative to wild-type cells (Figure 1A). In practice, this means that cells carrying a heterozygous mutation segregate homozygous mutations at division at a low rate of the order of 10 4 events/locus/cell/division. Screens of mutant libraries made in Blm cells have been successful for phenotypes where null mutants are selectable, for example resistance to 6-thioguanine (mismatch repair mutants), aerolysin (glycosylphosphatidylinositol anchor synthesis mutants) or retroviral infection (1618). Reporter systems can also be used to make the phenotype artificially selectable (19,20). The requirement for a selectable phenotype is due to the fact that each potentially interesting homozygous cell in the library is outnumbered by the order of one thousand cells heterozygous for the insertion, which are unlikely to display a loss-of-function phenotype. We were therefore interested in extending this method to other non-selectable phenotypes by increasing the proportion of homozygous cells in the library. We present here a method to isolate homozygous cells from these libraries independent of their phenotype. We use the piggyBac (PB) transposon (21) to cause loss-of-function insertion mutations and also to deliver a selection construct that carries two drug resistance genes but can express only one at a time. The expressed gene can be switched using Cre recombinase, and only cells with two copies of this construct can acquire resistance to both corresponding drugs simultaneously. This allows selection for the increase in copy number of the mutation that occurs after loss of heterozygosity (LOH), and thus for homozygous mutants. We have isolated homozygous mutants in 48 genes using this method, which should be applicable on a larger scale to generate clonal libraries of ES cells with null mutations for genetic screens. MATERIALS AND METHODS Construction of the gene trap vectors The PB transposon vector, which contains 313 bp of the 50 inverted terminal DNA repeat (TR) and 235 bp of the 30 inverted TR, has been described previously (19). For the deletion homozygosity selection vector (DHSV), the PB 50 and 30 TRs were polymerase chain reaction (PCR) amplified and cloned upstream and downstream of the selection cassettes. The SA- geo-bpA gene trap cassette was derived from RGTV1 (18). The Bsd-bpA fragment was derived from pY (...truncated)