Mammalian haploid embryonic stem cells

Abstract

The invention relates to mammalian haploid embryonic stem cells and methods for the production of such stem cells. The inventions also relates to a cell culture and a cell line of mammalian haploid embryonic stem cells.

Claims

1. An isolated mammalian haploid embryonic stem cell line, wherein the stem cell line is pluripotent, proliferates, and is capable of maintaining a haploid karyotype for at least 15 passages during proliferation in culture, and wherein the stem cell line is derived from a mouse, a rat, or a human.

2. The isolated mammalian haploid embryonic stem cell line of claim 1 wherein the cell line is capable of proliferation when cultured in conditions selected from: a) N2B27 chemically defined medium with LIF and CHIR99021 and PD0325901 (2i inhibitors); and/or b) High glucose DMEM medium with 15% fetal calf serum supplemented with Glutamin, beta mercapto ethanol, penicillin-streptomycine, non-essential amino acids and 500 units per milliliter recombinant mouse LIF; and/or c) ES cell medium including LIF and fetal calf serum; and/or d) Serum free medium with BMP and LIF; and/or e) high glucose media supplemented with serum or a serum replacement, and cytokines; and/or f) DMEM with 15% serum and LIF.

3. The isolated mammalian haploid embryonic stem cell line of claim 1, wherein the proliferation conditions further include a fibroblast feeder layer.

4. The isolated mammalian haploid embryonic stem cell line of claim 1, wherein the isolated mammalian haploid embryonic stem cell line is capable of maintaining a haploid karyotype for at least 20 passages during proliferation in culture.

5. A mutagenized haploid embryonic stem cell derived from a mammalian haploid embryonic stem cell of the isolated mammalian haploid embryonic stem cell line of claim 1.

6. A haploid differentiated cell obtained from differentiation of a mammalian haploid embryonic stem cell of the isolated mammalian haploid embryonic stem cell line of claim 1.

7. The isolated mammalian haploid embryonic stem cell line of claim 1, wherein the isolated mammalian haploid embryonic stem cell line is derived from a rat.

8. The isolated mammalian haploid embryonic stem cell line of claim 1, wherein the isolated mammalian haploid embryonic stem cell line is derived from a mouse.

9. The isolated mammalian haploid embryonic stem cell line of claim 1, wherein the isolated mammalian haploid embryonic stem cell line is derived from a human.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention will now be described, by way of example only, with reference to the accompanying drawings, in which

(2) FIG. 1 shows a strategy for the derivation of the mammalian haploid embryonic stem cells.

(3) FIGS. 2A and 2B show the characterisation of a haploid mouse embryonic stem (ES) cell line. FIG. 2A shows three representative pictures of metaphase spreads of a haploid mouse ES cell line showing a haploid set of 20 chromosomes. DNA was stained with DAPI. FIG. 2B shows a FACS analysis of fixed ES cells after DNA staining with propidium iodide. Position of haploid (1 n), diploid (2 n), and tetraploid (4 n) DNA content is indicated. The haploid line (upper panel) shows a predominantly 1 n DNA content with some G2 cells after DNA replication showing as 2 n. The contamination with diploid cells is low as judged from the 4 n peak corresponding to G2 cells. A diploid control ES cell line (lower panel) shows a 2 n G1 peak and a 4 n G2 peak.

(4) FIG. 3 shows immunofluorescence staining of haploid and control diploid ES cells for detecting Nanog, a marker of pluripotent ES cells, and Gata4, an early ES differentiation marker. Colonies of both haploid and control diploid ES cells express the pluripotency marker Nanog but do not express markers that are indicative of differentiation.

(5) FIG. 4 shows analytic flow profiles after DNA staining with PI for 129Sv derived diploid control embryonic stem cells, the haploid H129-1 ES cell line at passage 6 and at passage 12 after two rounds of sorting the 1 n peak.

(6) FIGS. 5A-5H show derivation of haploid ESCs. Flow analysis of DNA after PI staining of (FIG. 5A) diploid control ESCs, (FIG. 5B) haploid ESC line HAP-1 at passage 7 (p7) and (FIG. 5C) HAP-1 (p11) after sorting at p7. (FIG. 5D) Colony morphology of haploid ESCs (HAP-1). (FIG. 5E AND 5F) Chromosome spreads of HAP-3 (FIG. 5E) and HAP-1 (FIG. 5F), (Scale bar=10 μm). (FIG. 5G) CGH analysis of HAP-1 and HAP-2 ESCs and control male CBA kidney DNA. Relative copy number is plotted at 200 kb resolution using a log 2 scale. Genomic positions indicated by blue bars (top) are enlarged at 40 kb resolution in (FIG. 5H); CBA control (black), HAP-1 (red) and HAP-2 (green).

(7) FIGS. 6A-6D show expression analysis of haploid ESCs. (FIG. 6A) Immunofluorescence shows Nanog protein (red) in haploid (HAP-1) and diploid ESCs, and Gata4 (green) in differentiated cells (Scale bar=10 μm). (FIG. 6B) Expression of pluripotency markers in haploid and diploid (set to 1) ESCs by real time PCR. Error bars represent standard deviation (n=3). (FIG. 6C) Scatter plot showing log2 transformed average expression values from gene expression profiles of 3 haploid (HAP-1, HAP-2 and HTG-1) and three diploid J1 ESC lines for 45,001 probe sets (r is the Pearson correlation coefficient; red lines indicate 2-fold up- and down-regulation). (FIG. 6D) Diagram of more than 2-fold up- and down-regulated genes in haploid ESCs.

(8) FIGS. 7A-7D show developmental potential of haploid ESCs. (FIG. 7A) GFP marked haploid HAP-2 ESCs (p18) contribute to chimeric embryos at E12.5. 6 out of 9 embryos showed GFP contribution. A GFP negative embryo is shown as a control (below). (FIG. 7B) Representative flow analyses of DNA content of all cells (above) and GFP positive cells (below) extracted from a chimeric E12.5 embryo are shown. All 6 embryos gave similar results. (FIG. 7C) Live born chimeric mice were obtained from GFP marked HAP-2 ESCs. (FIG. 7D) Chimeric mice obtained from injection of HAP-1 ESCs into C57BU6 blastocysts (black) show coat colour contribution from the ESCs (agouti).

(9) FIGS. 8A-8B show derivation of CBA/B6 hybrid haploid ES cells in KSR. FIG. 8A shows a flow profile of DNA content after PI staining recorded from diploid control shows a 2 n and 4 n peak. FIG. 8B shows a flow profile of DNA content of an ES cell line (HAP-9, p8) derived from haploid blastocysts using Knockout Serum Replacement shows an additional 1 n peak.

(10) FIGS. 9A-9B show derivation of haploid ES cells from a mixed genetic background in 2i medium. FIG. 9A shows a flow profile shows the DNA content of a diploid control ES cell line after PI staining. FIG. 9B shows a flow profile of a haploid ES cell line (HTG-1, p8) derived from blastocysts with a mixed genetic background shows a nearly pure haploid cell population with 1 n and 2 n peaks but no 4 n peak.

(11) FIGS. 10A-10B show CGH analysis of HTG haploid ES cells. FIG. 10A shows genomic overview of copy number variations (CNVs) in 200 kb resolution in HTG-1 and HTG-2 haploid ES cells and control male kidney DNA from the mixed strain background of which the ES cells were derived. Average values of log2 ratios are plotted using somatic C57BL6 DNA as a reference. Blue bars on top indicate the positions of regions enlarged in panel b. FIG. 10B shows zoom in views of regions with CNVs on chromosomes 4, 7, 14 and X are shown. Signals from somatic kidney DNA from HTG genetic background mouse strain (black), HTG-1 (green) and HTG-2 (red) ES cells are overlaid and shown at 40 kb resolution. The positions of CNVs overlap indicating they are likely resulting from genomic variation between the strain of origin and C57BL6 mouse strains.

(12) FIG. 11 shows CGH analysis of haploid ES cells. CGH profiles of HAP-1, HAP-2, HTG-1 and HTG-2 haploid mouse ES cells are shown along genomic coordinates. Kidney DNA from a CBA male mouse and from a male of the transgenic mixed background strain from which HTG ES cells were derived were analysed as controls. All hybridizations were performed using a C57BL6 male DNA reference. Average values of log 2 ratios are plotted at a 40 kb resolution.

(13) FIG. 12 shows analysis of similarity between haploid and diploid ES cells. An overview of the gene expression profiles of haploid and diploid ES cells is shown. Gene expression profiles were clustered using all genes and the Paerson correlation coefficient was calculated (indicated by red color). Three different haploid ES cell lines (HAP-1, HAP-2, and HTG-1) cluster together showing highly similar expression profiles. Gene expression of haplioid ES cells is highly similar to control diploid J1 ES cells but different from mouse embryonic fibroblasts (MEF) or neural and mesodermal progenitors. The dendrogram (top and right) was generated by hierarchical clustering by Euclidean distance and complete linkage analysis (rep1, rep2, rep3 indicate biological replicates).

(14) FIGS. 13A-13B show stable integration of a GFP transgene into haploid ES cells. Flow profiles show the (FIG. 13A) DNA content and (FIG. 13B) DNA content combined with GFP intensity of HAP-2 ES cells transfected with a piggyBac vector for expressing GFP. The 1 n/GFP positive population was purified for analysis of the developmental potential of haploid ES cells.

(15) FIGS. 14A-14B show the developmental potential of haploid ES cells. FIG. 14A shows a GFP image and merged GFP brightfield image of a chimeric E9.5 embryo from injection of GFP labeled HTG-2 ES cells (p23) into C57BL6 host blastocysts. FIG. 14B shows immunofluorescence analysis of Nestin (green) and Oct4 (white) expression in HAP-2 (p31) ES cells and HAP-2 derived neural stem (NS) cells. A merged image with DNA stained with DAPI (blue) is shown below.

(16) FIGS. 15A-15B show the genetrap insertions recovered in missmatch repair screen. FIG. 15A shows a schematic representation of the piggyBac genetrap vector is shown (SA, splice acceptor). FIG. 15B shows a Genome browser view shows BLAST hits for sequences recovered by Splinkerette PCR from 6-TG resistant clones obtained from a missmatch repair screen using haploid mouse ES cells (see text). Seven clones were analysed and three insertions into genes previously identified to mediate 6-TG sensitivity were identified. Two independent insertions in the Msh2 and one insertion in the Hprt gene are shown. The integration sites were mapped to intron three and fifteen of Msh2 on chromosome 17 at base position 88,081,425 and 88,121,546, respectively. A further insertion was identified in intron 1 of the Hprt gene at base position 50,349,004 on the X chromosome. All gene trap insertions were in forward orientation trapping the gene transcripts as expected. Genomic positions and gene structure are based on the NCBI37/mm9 assembly of the mouse genome.

(17) FIG. 16 shows a segmentation table of the CGH analysis of haploid ES cells. Segmentation analysis of the CGH profiles of HAP-1, HAP-2, HTG-1 and HTG-2 haploid mouse ES cells and CBA and HTG control male somatic samples was performed using the NimbleScan software (Roche). Copynumber deviations with a cut-off log 2 value of 0.8 were determined and listed with chromosome and genomic position.

(18) FIG. 17 shows differentially regulated genes in haploid ES cells (>2 fold; p<0.05).

DETAILED DESCRIPTION OF THE INVENTION

Example 1

(19) Oocyte Activation

(20) Oocytes were isolated in the morning from mouse oviducts after hormonal induction of ovulation. Ovulation was induced by intraperitoneal injection of PMS and hCG 48 hours later (this superovulation procedure is described in Manipulating the mouse embryo, ISBN 0-87969-384-3). A potential other method for obtaining unfertilized oocytes could be through natural matings with vasectomised males.

(21) Activation of oocytes was carried out by incubation in M16 medium to which 5 mM (millimolar) SrCl.sub.2 and 2 mM EGTA were added. Incubation times of 1 to 3 hour were found to be efficient. Shorter incubation led to lower activation rates whereas 5 hour incubation appeared to affect subsequent development. After activation oocytes were cultured in in groups of 50 using microdrop culture. For this 80 microliter drops of M16 medium were overlayed with mineral oil. All culture media were preequilibrated and incubation was at 37° C., 5% CO.sub.2 in a tissue culture incubator (SANYO). Blastocyst stage embryos were obtained at day 3 to 4 and used for ES cell derivation.

(22) Activation of freshly ovulated oocytes in vitro is mediated by induction of Calcium (Ca) waves. This can be achieved by prolonged culture in medium without Ca. The addition of Strontium (Sr) has been used to increase activation rates and enhance the development of diploid parthenogenetic embryos (Bianchi et al., 2010). Complexing the Ca with EGTA has made it possible to use preformulated media in combination with Sr for efficient activation of mouse oocytes (Kishigami and Wakayama, 2007). Similar protocols are established based on permeabilization of the oocyte membrane for Ca by ionomycine treatment. These protocols are more relevant for other mammalian species including human where Sr does not lead to efficient oocyte activation.

(23) ES Cell Derivation in Chemically Defined Medium

(24) For derivation of ES cells in chemically defined medium 8-cell embryos were cultured for two days in M16 supplemented with the following inhibitors: 1 μM PD0325901 and 3 μM CHIR99021 (M16-2i). 2i is a composition that enhances the efficiency of ES cell line derivation and thereby reduces the number of embryos required (see, for example, Nature. 2008 May 22; 453(7194):519-23. The ground state of embryonic stem cell self-renewal. Ying Q L, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A).

(25) This step expanded the inner cell mass of the blastocyst. The zona pellucida was then removed with acidic Tyrode's solution (Hogan et al., 1994). Optionally, the trophectoderm was removed by immunosurgery. Isolated inner cell masses or embryos were then cultured on mitotically inactivated embryonic fibroblast feeders in chemically defined N2B27 medium containing above 2i inhibitors and LIF (N2B27-2i plus LIF). LIF is a cytokine that inhibits embryonic stem cell differentiation and stimulates stem cell growth. Outgrowths were passaged repeatedly every 3 to 4 days until ES cell lines emerged. In 2i ES cell lines emerged after the first passage.

(26) ES cell culture in defined medium with signal inhibition (Ying et al., 2008) can be used to derive ES cells from recalcitrant mouse strains (Nichols et al., 2009) and rats (Buehr et al., 2008). Recently, similar approaches have led to a culture of an embryonic stem cell state from humans that have similar properties with mouse ES cells (Hanna et al., 2010).

(27) ES Cell Derivation in Standard Conditions

(28) For ES cell derivation the zona pellucida was removed from morula and blastocyst stage embryos with acidic Tyrode's solution (Hogan et al., 1994). After removal of the trophectoderm by immunosurgery (Hogan et al., 1994) the inner cell mass was explanted and cultured in high glucose DMEM supplemented with fetal bovine serum or serum replacement (KSR) and LIF. Outgrowths were passaged every 3-4 days until ES cell lines were obtained.

(29) Enrichment for Cells with in Content Using Cell Sorting

(30) HOECHST 33342 stained living cells were sorted using a DAKO MoFlo high speed sorter. Cells with a haploid (1 n) DNA content were selected. Diploid cell lines (2 n) were used as controls. For analysis the FlowJo Flow Cytometry Analysis Software was used (Tree Star Inc.).

(31) Additionally, ES cell lines can be subcloned.

(32) Characterization of Haploid ES Cell Lines

(33) DNA content was investigated by analytical FACS (Fluorescence Activated Cell Sorting). Karyotypes were identified by metaphase chromosome spreads. Haploid ES cell lines display a characteristic colony morphology and growth rate comparable to diploid mouse ES cells. In addition these cells express markers of mouse ES cells (FIG. 3).

(34) Forward Genetic Screen to Identify Genes Involved in Mismatch Repair.

(35) This proof of principle screen was designed according to a screen performed previously (Genome Res. 2009 April; 19(4):667-73. Epub 2009 Feb. 20. A piggyBac transposon-based genome-wide library of insertionally mutated Blm-deficient murine ES cells. Wang W, Bradley A, Huang Y).

(36) 5×10.sup.6 haploid ES cells were co-transfected with a gene trap cassette embedded in a PiggyBac vector and a vector carrying the PiggyBac transposase gene. Integration of the gene trap cassette into a transcribed locus brings a puromycin resistance cassette under control of the endogenous promoter. This allows selection for successful integration of gene trap cassettes into the genome using puromycin (Sigma; P8833) at a concentration of 2 microgram per millilitre. The pool of puromycin resistant haploid ES cells was used for further screening. Cells were seeded at a density of 1.5×10.sup.6 per 15 cm plate and selection for mutations in MMR genes was performed using 2 μM, 2-amino-6-mercaptopurine (Sigma, 6-TG). Selection was initiated 24 h after plating and continued for 8 d. Colonies were picked and expanded before analysis. PiggyBac integrations were mapped by Splinkerette PCR.

(37) (Mikkers, H., et al (2002) High-throughput retroviral tagging to identify components of specific signaling pathways in cancer. Nat. Genet. 32:153-159.) Results are shown in table 2.

(38) Conclusion

(39) The above results describe the successful production of a mouse haploid embryonic stem cell. As discussed previously, to date there has been no prior description of a mammalian haploid cell and cell line. Indeed, it was previously believed that the derivation of such cells was impossible. Moreover, the above-described methods allow haploid embryonic stem cells to be derived from unfertilised oocytes. As a result, such cells do not contain tumour derived mutations, genomic rearrangements or oncogenes. We therefore describe a means for deriving embryonic stem cells with a normal haploid karyotype. Haploid ES cells provide a platform for highly efficient forward genetic screening in mammalian cells. Further, we believe that haploid ES cells are maintained most purely if they are sorted every few passages. Haploid ES cells also have a tendency to diploidize, which could enable the generation of developmental models.

(40) TABLE-US-00001 TABLE 1 Derivation of haploid mouse ES cell lines ES cell max. ES cell lines with haploid derivation genetic Oocytes Nr of lines haploid contribution protocol background activated blastocysts obtained contribution (%) 2i/immuno- CBA/B6 132 30 27 6 40 surgery 2i CBA/B6 22 10 5 1 15 2i mixed 50 32 3 3 60 KSR/ CBA/B6 273 48 22 6 10 immuno- surgery

(41) TABLE-US-00002 TABLE 2 Forward genetic screen in haploid ES cells to identify genes involved in mismatch repair cell line number genes identified known MMR genes 1 Msh2 2 Fanci 3 Msh2 novel MMR candidates or false positives 4 Zfp462, Lypd1, nlgn1, Slc44a3 5 Acbd6, Lypd1, nlgn1 6 Usp14, Atl1, 7SK 7 Wdr40a, Sorbs2

Example 2

(42) Derivation of Haploid Embryonic Stem Cells from the 129Sv Inbred Mouse Strain

(43) 140 oocytes were collected from superovulated 129Sv female mice and activated using strontium chloride and EGTA in M16 medium for 2 hours. Embryos were cultured in M16 medium until the blastocyst stage. 13 blastocysts were obtained, the zona was removed and the inner cell masses were cultured in 2i medium in the presence of LIF and BSA. Form a total of 10 embryonic stem cell lines obtained, 3 showed a substantial content of cells with a haploid genome equivalent. The haploid cell content was estimated between 40% and 60%. Sorting of the haploid 1 n peak allowed the establishment of pure haploid 129Sv mouse embryonic stem cells cultures. FIG. 4 shows analytic flow profiles after DNA staining with PI for 129Sv derived diploid control embryonic stem cells, the haploid H129-1 ES cell line at passage 6 and at passage 12 after two rounds of sorting the 1 n peak.

Example 3

(44) Most animals are diploid but haploid-only and male-haplo species have been described.sup.1. Diploid genomes of complex organisms limit genetic approaches in biomedical model species such as in mice. To overcome this problem experimental induction of haploidy has been used in fish.sup.2,3. In contrast, haploidy appears less compatible with development in mammals.sup.4,5. Here we describe haploid mouse embryonic stem cells and show their application in forward genetic screening.

(45) Experimentally induced haploid development in zebrafish has been utilized for genetic screening.sup.2. Recently, haploid pluripotent cell lines from medaka fish have also been established.sup.3. In contrast to fish, haploidy is not compatible with development in mammals. Although haploid cells have been observed in egg cylinder stage parthenogenetic mouse embryos.sup.6 the majority of cells in surviving embryos become diploid. Previous attempts to establish pluripotent stem cell lines from haploid embryos have resulted in the isolation of parthenogenetic embryonic stem cells (ESCs) with a diploid karyotype.sup.4. These studies reported the development of apparently normal haploid mouse blastocysts with a defined inner cell mass (ICM).sup.4,5. In order to investigate the haploid ICM, we cultured haploid mouse blastocysts in chemically defined medium with inhibitors of mitogen activated protein kinase kinase (MEK) and glycogen synthase kinase 3 (GSK3). This 2i medium.sup.7 has previously been used for isolating ESCs from recalcitrant mouse strains.sup.8 and rats.sup.9 and may help to maintain certain characteristics of early mouse epiblast cells.sup.10,11.

(46) We generated haploid mouse embryos by activation of unfertilized oocytes isolated from superovulated B6CBAF1 hybrid female mice using strontium chloride. After culture in M16 medium 30 blastocysts (22%) were obtained from 132 activated oocytes and used for ESC derivation. After removal of the zona and trophectoderm, ICMs were cultured in gelatinized 96 well dishes in 2i medium in the presence of LIF. 27 ESC lines were obtained (93%). Individual ESC lines were expanded and their DNA content was analysed by flow analysis using diploid ESCs as controls (FIGS. 5A and 5B). In 6 ESC lines, at least 10% of the cells had a haploid DNA content and the proportion of haploid cells could reach a conservatively estimated 60% (FIG. 5B). Further enrichment was achieved by flow sorting of cells with a haploid DNA content after staining with HOECHST 33342 (FIG. 5C). This allowed expansion of haploid ESC lines for over 35 passages.

(47) We further tested the requirements for deriving haploid mouse ESCs (Table 3). These experiments showed that removal of the trophectoderm by immunosurgery was not essential. Haploid ESCs could also be established using DMEM medium supplemented with Knockout Serum Replacement (KSR) and LIF showing that derivation without kinase inhibitors is possible (Table 3 and FIGS. 8A and 8B). We further succeeded in isolating haploid ESCs from the 129Sv inbred mouse strain and two genetically modified mouse lines. In the latter several alleles had been bred to homozygosity and maintained on a mixed genetic background for several generations (Table 3, and FIGS. 9A and 9B). In summary, we derived 25 haploid ESC lines in 7 independent experiments. Haploid ESC cultures could also be maintained on feeders in serum containing DMEM supplemented with LIF.

(48) Haploid ESCs exhibited a typical mouse ESC colony morphology (FIG. 5D). Chromosome spreads showed 20 chromosomes corresponding to the haploid mouse chromosome set (FIGS. 5E and 5F). For further characterizing the genetic integrity we performed comparative genomic hybridization (CGH) of 4 haploid ESC lines and control DNA from the CBA strain and the mixed transgenic mouse line from which HTG-1 and HTG-2 ESCs were derived (FIGS. 5G and 5H and FIGS. 10A, 10B, and 11). Copy number variations (CNVs) that were detected in the genome of haploid ESC lines were also present in the strains of origin (FIG. 16). Albeit some CNVs appeared haploid ESC specific, inspection of the actual signals (FIG. 11) suggested that these CNVs were also present in the CBA or HTG control DNAs but not detected with the threshold applied. CNVs between the C57BL6 and CBA strain of mice were consistent with a previously reported analysis.sup.12. Taken together these data show that haploid ESCs maintained an intact haploid genome without amplifications or losses.

(49) On the molecular level, haploid ESCs expressed pluripotency markers including Oct4, Rex1, KIf4, Sox2 and Nanog (FIGS. 6A and 6B). Genome-wide expression analysis showed a high correlation (Pearson correlation coefficient r=0.97 over all genes) between haploid ESCs and control diploid male ESCs (FIG. 6C and FIG. 12). In haploid ESCs 279 and 194 genes were more than 2-fold up- or down-regulated (p<0.05), respectively (FIG. 17). Among these, 99 X-linked genes were overexpressed and 4 Y-linked genes were lost in haploid ESCs consistent with different sex chromosome constitutions (FIG. 6D). Thus, haploid ESCs largely maintain a mouse ESC transcription profile.

(50) This prompted us to investigate the developmental potential of haploid ESCs. For this we introduced a piggyBac transposon vector for expressing green fluorescent protein (GFP) into HAP-2 ESCs. Flow sorting of cells for GFP fluorescence and DNA staining with Hoechst 33342 yielded a haploid ESC population that expressed GFP at high level showing that a haploid genome content was maintained during the transfection procedure (FIGS. 13A and 13B). GFP marked haploid ESCs contributed substantially to chimeric embryos when injected into C57BL/6 blastocysts (FIG. 7A). The great majority of GFP positive cells extracted from chimeric embryos had a diploid DNA content (FIG. 7B) indicating that haploid ESCs contributed extensively to development after diploidization. We also obtained 2 male and 2 female live born chimeras with a substantial contribution from haploid ESCs (FIG. 7C). These mice developed normally with apparent coat colour chimerism. Similar results were obtained with the HAP-1 and HTG-2 ESCs (FIG. 7D and FIG. 14A). Furthermore, the diploid fraction of HAP-2 ESCs at passage 31 could be differentiated into Nestin positive cells following a neural in vitro differentiation protocol.sup.13 (FIG. 14B). Taken together these findings demonstrate that haploid ESCs maintain a wide differentiation potential. These chimeric mice have transmitted the GFP transgene from the GFP transgenic HAP-1 haploid embryonic stem cell line to two of their offspring. This is evidence for the ability of introducing mutations or genetic modifications into mice via haploid ES cells.

(51) To investigate the utility of haploid ESCs for genetic screening we performed a pilot screen for mismatch repair genes following a previously published strategy.sup.14. For this, 5×10.sup.6 haploid ESCs were co-transfected with a gene trap piggyBac transposon vector (FIG. 15A) and a plasmid for expressing an optimized piggyBac transposase.sup.15. Gene trap insertions were selected with puromycin. A pool of 1×10.sup.7 cells was then cultured in the presence of 2-amino-6-mercaptopurine (6-TG) which is toxic to mismatch repair proficient cells. After 8 days 20 6-TG resistant colonies were isolated and the integration sites were mapped using Splinkerette PCR.sup.16. Of 7 clones analysed we identified two independent insertions in Msh2 and one in Hprt (FIG. 15B). Msh2 is a known mismatch repair gene and Hprt is required for converting 6-TG into a toxic metabolite.sup.14. Thus, identification of mutations in autosomal genes was possible suggesting a potential for haploid ESCs in forward genetic screening in mammals.

(52) The difficulty in obtaining haploid ESC lines in previous attempts might be explained by aberrant gene regulation such as aberrant dosage compensation and genomic imprinting. However, diploid ESCs from mouse and human parthenogenetic embryos have been established.sup.17,18. Misregulation of X inactivation has been observed to some extent in haploid mouse embryos.sup.5 and has also been shown to reduce the efficiency of producing cloned mice.sup.19. Thus, it is conceivable that X inactivation is initiated aberrantly in haploid embryos during some ESC derivation procedures. Direct capture of naive pluripotent cells from ICM outgrowths as accentuated by the use of 2i conditions.sup.11 could have contributed to the success of our study.

(53) Previously, near-haploid cells have been observed in human tumours (for literature review see.sup.20) and a near-haploid human tumour derived cell line has been described.sup.21,22. These tumour cells carry genomic rearrangements and mutations that might stabilize the haploid genome. An interesting aspect of haploid ESCs is their developmental potential. We have observed rapid diploidization when haploid ESCs differentiate. The resulting diploid parthenogenetic cells can contribute to development.sup.23. It is interesting to speculate whether differentiated haploid lineages can be generated perhaps through suppression of X inactivation and whether it is possible to derive haploid human ESCs.

(54) Methods Summary

(55) For the derivation of haploid ESCs mouse oocytes were activated in M16 medium as described.sup.24. ESC culture in chemically defined 2i medium has been described previously.sup.7,8. Cell sorting for DNA content was performed after staining with 15 μg/ml Hoechst 33342 (Invitrogen) on a MoFlo flow sorter (Beckman Coulter) selecting the haploid 1 n peak. For analytic flow profiles cells were fixed in ethanol, RNase treated, and stained with propidium iodide (PI). For karyotype analysis cells were arrested in metaphase with demecolcine (Sigma). After incubation in hypotonic KCI buffer cells were fixed in methanol-acetic acid (3:1) and chromosome spreads were prepared and stained with DAPI. RNA was extracted using the RNeasy Kit (Quiagen). Transcription profiles were generated using Affymetrix GeneChip 430.2 arrays. Sample preparation, hybridization, and basic data analysis were performed by Imagenes (Berlin, Germany). Further analysis was performed using the Genespring GX software (Agilent). For CGH analysis genomic DNA was isolated from haploid ESC lines and hybridized to NimbleGen 3×720K whole-genome tiling arrays by Imagenes (Berlin, Germany) using C57BL/6 kidney DNA as a reference. For chimera experiments GFP labelled HAP-1 (p29), HAP-2 (p18) and HTG-2 (p23) ESCs were injected into C57BL/6 host blastocysts. Live born chimaeras were analysed for expression of GFP at postnatal day 2. Genetic screening was performed following a previously published strategy.sup.25. In brief, HAP-1 ESCs were co-transfected with 2 μg piggyBac transposase expression vector.sup.15 and 1 μg piggyBac gene trap vector (FIGS. 15A and 15B) using Lipofectamine 2000 (Invitrogen). Selection for transposon insertions was performed using 2 μg/ml puromycin for 8 days. 1×10.sup.7 puromycin resistant ESCs were plated in two 15 cm dishes and mutations in mismatch repair genes were selected using 0.3 μg/ml 6-TG (Sigma). piggyBac integration sites in seven 6-TG resistant clones were mapped by Splinkerette PCR.sup.16.

REFERENCES FOR EXAMPLE 3

(56) 1. Otto, S. P. & Jarne, P. Evolution. Haploids-hapless or happening? Science 292, 2441-3 (2001). 2. Wiellette, E. et al. Combined haploid and insertional mutation screen in the zebrafish. Genesis 40, 231-40 (2004). 3. Yi, M., Hong, N. & Hong, Y. Generation of medaka fish haploid embryonic stem cells. Science 326, 430-3 (2009). 4. Kaufman, M. H., Robertson, E. J., Handyside, A. H. & Evans, M. J. Establishment of pluripotential cell lines from haploid mouse embryos. J Embryol Exp Morphol 73, 249-61 (1983). 5. Latham, K. E., Akutsu, H., Patel, B. & Yanagimachi, R. Comparison of gene expression during preimplantation development between diploid and haploid mouse embryos. Biol Reprod 67, 386-92 (2002). 6. Kaufman, M. H. Chromosome analysis of early postimplantation presumptive haploid parthenogenetic mouse embryos. J Embryol Exp Morphol 45, 85-91 (1978). 7. Ying, Q. L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519-23 (2008). 8. Nichols, J. et al. Validated germline-competent embryonic stem cell lines from nonobese diabetic mice. Nat Med 15, 814-8 (2009). 9. Buehr, M. et al. Capture of authentic embryonic stem cells from rat blastocysts. Cell 135, 1287-98 (2008). 10. Nichols, J., Silva, J., Roode, M. & Smith, A. Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo. Development 136, 3215-22 (2009). 11. Nichols, J. & Smith, A. The origin and identity of embryonic stem cells. Development 138, 3-8 (2011). 12. Cutler, G., Marshall, L. A., Chin, N., Baribault, H. & Kassner, P. D. Significant gene content variation characterizes the genomes of inbred mouse strains. Genome Res 17, 1743-54 (2007). 13. Pollard, S. M., Benchoua, A. & Lowell, S. Neural stem cells, neurons, and glia. Methods Enzymol 418, 151-69 (2006). 14. Li, M. A., Pettitt, S. J., Yusa, K. & Bradley, A. Genome-wide forward genetic screens in mouse ES cells. Methods Enzymol 477, 217-42 (2010). 15. Cadinanos, J. & Bradley, A. Generation of an inducible and optimized piggyBac transposon system. Nucleic Acids Res 35, e87 (2007). 16. Mikkers, H. et al. High-throughput retroviral tagging to identify components of specific signaling pathways in cancer. Nat Genet 32, 153-9 (2002). 17. Mai, Q. et al. Derivation of human embryonic stem cell lines from parthenogenetic blastocysts. Cell Res 17, 1008-19 (2007). 18. Revazova, E. S. et al. Patient-specific stem cell lines derived from human parthenogenetic blastocysts. Cloning Stem Cells 9, 432-49 (2007). 19. Inoue, K. et al. Impeding Xist expression from the active X chromosome improves mouse somatic cell nuclear transfer. Science 330, 496-9 (2010). 20. Sukov, W. R. et al. Nearly identical near-haploid karyotype in a peritoneal mesothelioma and a retroperitoneal malignant peripheral nerve sheath tumor. Cancer Genet Cytogenet 202, 123-8 (2010). 21. Kotecki, M., Reddy, P. S. & Cochran, B. H. Isolation and characterization of a near-haploid human cell line. Exp Cell Res 252, 273-80 (1999). 22. Carette, J. E. et al. Haploid genetic screens in human cells identify host factors used by pathogens. Science 326, 1231-5 (2009). 23. Jiang, H. et al. Activation of paternally expressed imprinted genes in newly derived germline-competent mouse parthenogenetic embryonic stem cell lines. Cell Res 17, 792-803 (2007). 24. Kishigami, S. & Wakayama, T. Efficient strontium-induced activation of mouse oocytes in standard culture media by chelating calcium. J Reprod Dev 53, 1207-15 (2007). 25. Guo, G., Wang, W. & Bradley, A. Mismatch repair genes identified using genetic screens in Blm-deficient embryonic stem cells. Nature 429, 891-5 (2004).

(57) TABLE-US-00003 TABLE 3 Derivation of haploid mouse ESC lines Names of ESC ESC lines with haploid Exp. lines used genetic Oocytes number of ESC lines contribution No derivation protocol in study background activated blastocysts obtained (max. % haploid before sorting) 1 2i/immunosurgery HAP-1 to 6.sup.† B6CBAF1 132 30 27 6 (>60%) 2 2i HAP-7 B6CBAF1 22 10 5 1 (>15%) 3 2i HTG-1 to 3.sup.‡ mixed TG* 50 32 3 3 (>90%) 4 KSR/immunosurgery HAP-8 to 13 B6CBAF1 273 48 22 6 (>10%) 5 2i H129B6-1 to 5 129B6F1 250 37 8 5 (>10%) 6 2i HTX-1 mixed TG** 70 11 1 1 (>40%) 7 2i H129-1 to 3 129Sv 140 13 10 3 (>60%) .sup.†Contribution to chimeric mice was confirmed for the HAP-1 and HAP-2 haploid ESC lines. .sup.‡Contribution to chimeric mice was confirmed for the HTG-1 haploid ESC line. *Derived from ROSA26.sup.nlsrtTA LC1 Xist.sup.2LOX homozygous female mice. **Derived from ROSA26.sup.nlsrtTA tetOPXist homozygous female mice.sup.30.

Methods for Example 3

(58) Derivation of Haploid ESCs

(59) Oocytes were isolated from superovulated females and activated in M16 medium using 5 mM strontium chloride and 2 mM EGTA as described.sup.24. Embryos were subsequently cultured in M16 or KSOM medium microdrops covered by mineral oil. Under these conditions around 80% of oocytes reached the 2-cell stage on the next morning. Thereafter development of preimplantation embryos was variable with a large number of embryos showing unequal sized blastomeres or unusual embryo morphology. Removal of the zona, immunosurgery for removal of the trophectoderm and ESC derivation was performed as described previously.sup.7,8. ESCs were cultured in chemically defined 2i medium plus LIF as described.sup.7,8 with minor modifications. 2i medium was supplemented with non essential amino acids and 0.35% BSA fraction V. Culture of ESCs on feeders was performed as previously described.sup.26. Knockout serum replacement (KSR) was obtained from Invitrogen. Cell sorting for DNA content was performed after staining with 15 μg/ml Hoechst 33342 (Invitrogen) on a MoFlo flow sorter (Beckman Coulter). The haploid 1 n peak was purified. Diploid cells did arise in cultures to various extents in all ESC lines. Periodic purification by flow sorting every four to five passages allowed us to maintain cultures containing a great majority of haploid ESCs in all cases. Analytic flow profiles of DNA content were recorded after fixation of the cells in ethanol, RNase digestion, and staining with propidium iodide (PI) on a Cyan analyser (Beckman Coulter). For karyotype analysis cells were arrested in metaphase using demecolcine (Sigma). After incubation in hypotonic KCI buffer cells were fixed in methanol-acetic acid (3:1) and chromosome spreads were prepared and stained with DAPI. Immunostaining was performed as described.sup.27 using Nanog (Abcam; 1:100), Oct4 (Santa Cruz; 1:100), Nestin (Developmental Studies Hybridoma bank, Iowa City; 1:30) and Gata4 (Santa Cruz; 1:200) antibodies.

(60) Microarray Analysis

(61) RNA from biological triplicates of diploid ESCs and three independently derived haploid

(62) ESCs (HAP-1 p21, HAP-2 p24, HTG-1 p11) was extracted using the RNeasy kit (Quiagen). Gene expression analysis on Affymetrix GeneChip 430 2.0 arrays was performed by Imagenes Ges.m.b.H. (Berlin, Germany). Additional gene expression profiles of neural progenitors, mesodermal progenitors and mouse embryonic fibroblasts (MEF) were obtained from a previously published dataset (GEO accession number GSE12982.sup.28). The data was analysed using Genespring GX software (Agilent Technologies). Data were normalized using the RMA algorithm. Lists showing differentially regulated genes (>2 fold change; p<0.05) are provided in FIG. 17. p-values were established by an unpaired t test followed by FDR adjustment by the Benjamini Hochberg method. Hierarchical clustering was performed based on the Euclidean distances and complete linkage analysis. The relatedness of transcription profiles was determined by calculating the Pearson correlation coefficient (r). DNA samples for comparative genomic hybridization (CGH) experiments were extracted and sent to Imagenes Ges.m.b.H. (Berlin, Germany) for CGH analysis using NimbleGen 3×720K mouse whole-genome tiling arrays with an average probe spacing of 3.5 kb.

(63) Adult male C57BL/6 kidney DNA was used as a reference. A genomic overview of these analyses is presented in FIG. 5G and FIGS. 10A and 10B at 200 kb resolution and selected zoomed in regions at 40 kb resolution. The complete data set at 40 kb resolution is included in FIG. 11.

(64) Accession of Datasets

(65) Gene expression and CGH data sets can be accessed as the GEO reference series GSE30879 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE30879). This series includes the GSE30744 (Expression analysis of haploid and diploid ES cells in 2i medium) and the GSE30749 (CGH analysis of haploid ES cells) data sets.

(66) Quantitative Gene Expression Analysis

(67) RNA was extracted using the RNeasy kit (Quiagen) and converted into cDNA using the Quantitect reverse transcription kit (Quiagen). Real time PCR was performed on a StepOnePlus machine (Applied Biosystems) using the Fast Sybr green master mix (Applied Biosystems) and previously published primers.sup.27. The ddCt method was used for quantification of gene expression. Expression levels were normalized to L32 ribosomal protein mRNA and values in diploid control ESCs were set to 1.

(68) Embryo Analysis

(69) Haploid ESCs were co-transfected with a piggyBac vector carrying a CAG-GFP-IRES-hygro transgene and a piggyback transposase expression plasmid. Stable integrants were selected using 150 μg/ml Hygromycin for 7 days. The haploid fraction of HAP-1 (p29), HAP-2 (p18) and HTG-2 (p23) GFP positive cells were purified by flow sorting (FIGS. 13A and 13B). GFP labelled ESCs were expanded and injected into C57BL/6 host blastocysts which were transferred to recipient females. Embryos were analysed at E9.5 and E12.5. Dissociation to single cells was performed by incubation in 0.25% Trypsin/EDTA for 15 min. Prior to PI staining cells were fixed in 4% PFA and permeabilized in PBS/0.25% Triton X-100. Live born chimeras were analysed at postnatal day 2 (P2) for expression of GFP using UV illumination. Images were obtained using a Canon Powershot S5 IS camera with a FHS/EF-3GY2 filter (BLS). All mouse experiments were conducted in accordance with institutional guidelines of the University of Cambridge. All necessary UK home office licenses were in place.

(70) Gene Trap Screen

(71) The screen was performed based on a previously published protocol.sup.25. 5×10.sup.6 HAP-1 ESCs were co-transfected with 2 μg piggyBac transposase plasmid.sup.15 and 1 μg piggyBac gene trap vector (FIG. 15A) using Lipofectamine 2000. piggyBac insertions into expressed genes were selected with 2 μg/ml puromycin for 8 days. 1×10.sup.7 ESCs corresponding to approximately 5,000 puromycin resistant colonies were then plated onto two 15 cm dishes. Selection for mismatch deficient integrants was performed using 0.3 μg/ml 6-TG (Sigma). 20 colonies were picked and piggyBac integration sites of seven clones were identified by Splinkerette PCR and mapped using iMapper.sup.29.

REFERENCES USED ONLY IN THE METHODS SECTION

(72) 26. Wutz, A. & Jaenisch, R. A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation. Mol Cell 5, 695-705 (2000). 27. Leeb, M. et al. Polycomb complexes act redundantly to repress genomic repeats and genes. Genes Dev 24, 265-76 (2010). 28. Shen, X. et al. EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol Cell 32, 491-502 (2008). 29. Kong, J., Zhu, F., Stalker, J. & Adams, D. J. iMapper: a web application for the automated analysis and mapping of insertional mutagenesis sequence data against Ensembl genomes. Bioinformatics 24, 2923-5 (2008). 30. Savarese, F., Flahndorfer, K., Jaenisch, R., Busslinger, M. & Wutz, A. Hematopoietic precursor cells transiently reestablish permissiveness for X inactivation. Mol Cell Biol 26, 7167-77 (2006).