Derivation of haploid embryonic stem cells from mouse embryos

Abstract

Most animals are diploid, but haploid-only and male-haploid (such as honeybee and ant) species have been described. The diploid genomes of complex organisms limit genetic approaches in biomedical model species such as mice. To overcome this problem, experimental induction of haploidy has been used in fish. Haploid development in zebrafish has been applied for genetic screening. Recently, haploid pluripotent cell lines from medaka fish (Oryzias latipes) have also been established. In contrast, haploidy seems less compatible with development in mammals. Although haploid cells have been observed in egg cylinder stage parthenogenetic mouse embryos, most cells in surviving embryos become diploid. Here we describe haploid mouse embryonic stem cells and show their application in forward genetic screening.

Figure 1. Derivation of haploid ESCs

Flow analysis of DNA after PI staining of (a) diploid control ESCs, (b) haploid ESC line HAP-1 at passage 7 (p7) and (c) HAP-1 (p11) after sorting at p7. (d) Colony morphology of haploid ESCs (HAP-1). (ef) Chromosome spreads of HAP-3 (e) and HAP-1 (f), (Scale bar = 10 μm). (g) CGH analysis of HAP-1 and HAP-2 ESCs and control male CBA kidney DNA. Relative copynumber is plotted at 200 kb resolution using a log2 scale. Genomic positions indicated by blue bars (top) are enlarged at 40 kb resolution in (h); CBA control (black), HAP-1 (red) and HAP-2 (green).

Figure 2. Expression analysis of haploid ESCs

(a) Immunofluorescence shows Nanog protein (red) in haploid (HAP-1) and diploid ESCs, and Gata4 (green) in differentiated cells (Scale bar = 10 μm). (b) Expression of pluripotency markers in haploid and diploid (set to 1) ESCs by real time PCR. Errorbars represent standard deviation (n=3). (c) 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 probesets (r is the Paerson correlation coefficient; red lines indicate 2-fold up- and down-regulation). (d) Diagram of more than 2-fold up- and down-regulated genes in haploid ESCs.

Figure 3. Developmental potential of haploid ESCs

(a) 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). (b) 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. (c) Live born chimeric mice were obtained from GFP marked HAP-2 ESCs. (d) Chimeric mice obtained from injection of HAP-1 ESCs into C57BL/6 blastocysts (black) show coat colour contribution from the ESCs (agouti).