Diploid Embryonic Stem Cells Research Articles

Generation of live mice from haploid ESCs with germline-DMR deletions or switch

Genomic imprinting is required for sexual reproduction and embryonic development of mammals, in which, differentially methylated regions (DMRs) regulate the parent-specific monoallelic expression of imprinted genes. Numerous studies on imprinted genes have highlighted their critical roles in development. However, what imprinting network is essential for development is still unclear. Here, we establish a stepwise system to reconstruct a development-related imprinting network, in which diploid embryonic stem cells (ESCs) are derived by fusing between parthenogenetic (PG)- and androgenetic (AG)-haploid embryonic stem cells (haESCs) with different DMR deletions (termed Ha-Ha-fusion system), followed by tetraploid complementation to produce all-haESC fetuses. Diploid ESCs fused between PG-haESCs carrying 8 maternally-derived DMR deletions and AG-haESCs with 2 paternally-derived DMR deletions give rise to live pups efficiently, among which, one lives to weaning. Strikingly, diploid ESCs derived from the fusion of PG-haESCs with 7 maternal DMR deletions and AG-haESCs with 2 paternal DMR deletions and maternal Snrpn-DMR deletion also support full-term embryonic development. Moreover, embryos reconstructed by injection of AG-haESCs with hypomethylated H19-DMR into oocytes with H19-DMR deletion develop into live mice sustaining inverted allelic gene expression. Together, our findings indicate that restoration of monoallelic expression of 10 imprinted regions is adequate for the full-term development of all-haESC pups, and it works irrespective of their parental origins. Meanwhile, Ha-Ha-fusion system provides a useful tool for deciphering imprinting regulation networks during embryonic development.

P-806 Heritable genome editing in haploid mouse embryonic stem cells

Abstract Study question Can we edit a specific gene by CRISPR-Cas9 in haploid mouse embryonic stem cells? Summary answer We successfully carried out heritable genome editing of a coat pigmentation gene in haploid mouse embryonic stem cells via electroporation. What is known already Embryonic stem cells have been widely used as a substrate to carry out genetic studies due to their availability, low cost and ease of handling. CRISPR-Cas9 has been successful in editing genes in the human model. Gene editing occurred by homology directed repair or non-homologous end joining. The utilization of a haploid genome would confirm the feasibility of non-homologous end joining repair mechanism. Study design, size, duration In the past 3 months, diploid as positive control and experimental haploid embryonic stem cells were independently cultured for genome editing experiments. Electroporation was used as a means to introduce CRISPR-Cas9 into cells. The negative control cohort consisted of embryonic stem cells electroporated without delivering any CRISPR solution. CRISPR-Cas9 was designed to knock out the Tyr gene to create an albino phenotype. Participants/materials, setting, methods Diploid embryonic stem cells were derived from C57BL/6 mice. The haploid counterpart instead was obtained from haploid androgenetic embryos using spermatozoa from Oct4-EGFP mice. CRISPR solution was prepared by combining Tyr gRNA and Cas9 protein. For each experiment, one million cells were suspended in CRISPR solution and an electroporation buffer. Then, electric pulses were applied for electroporation with 4 different parameters. Gene modification at the target site was confirmed using T7E1 cleavage assay. Main results and the role of chance A 584 base pairs region around the CRISPR target site was amplified in all the diploid and haploid mouse embryonic stem cells. After performing T7E1 cleavage assay, editing efficiency was calculated following the manufacturer protocol. Precisely, after testing out different electroporation conditions on diploid mESCs, 1350V/20ms/2 pulses together with the combination of three sgRNAs (exon 1 and exon 2) and Cas9 protein, provided the best genome editing efficiency at 36.8% with a viability of 80%. Interestingly, when delivering only one type of gRNA (exon 2) together with Cas9 protein, we saw an efficiency rising to 77.6%. We then used the same condition to edit the Tyr gene in the genome of haploid androgenetic stem cells and confirmed that also in these cells, we were able to obtain a comparable genome editing efficiency at 68.4%. Limitations, reasons for caution While these experimental observations are limited, it is encouraging that it is possible to target and edit a haploid genome with a single allele. Wider implications of the findings The utilization of a haploid cell culture model paves the way to utilize CRISPR-Cas9 for gamete genome editing. This will provide a consistent and reliable method to correct inheritable genetic conditions on gametes allowing uniform gene editing of all cell progeny. Trial registration number Not applicable

Delayed DNA replication in haploid human embryonic stem cells.

Haploid human embryonic stem cells (ESCs) provide a powerful genetic system but diploidize at high rates. We hypothesized that diploidization results from aberrant DNA replication. To test this, we profiled DNA replication timing in isogenic haploid and diploid ESCs. The greatest difference was the earlier replication of the X Chromosome in haploids, consistent with the lack of X-Chromosome inactivation. We also identified 21 autosomal regions that had delayed replication in haploids, extending beyond the normal S phase and into G2/M. Haploid-delays comprised a unique set of quiescent genomic regions that are also underreplicated in polyploid placental cells. The same delays were observed in female ESCs with two active X Chromosomes, suggesting that increased X-Chromosome dosage may cause delayed autosomal replication. We propose that incomplete replication at the onset of mitosis could prevent cell division and result in re-entry into the cell cycle and whole genome duplication.

Generation and applications of mammalian haploid embryonic stem cells

Mammalian embryonic stem cells (ESCs) are usually diploid, which greatly limit their applications in genetic studies due to existence of two copies of each gene. Recently, the haploid embryonic stem cells (haESCs) have been established from different species, enabling large-scale forward or reverse genetic screening in mammals. The haESCs, same as diploid ESCs, sustain the abilities of self-renewal, proliferation and differentiation, greatly enhancing genetic analyses at the cellular level. Meanwhile, the allodiploid ESCs derived by fusion of haESCs from mice and rats provide a unique system to study the evolution of genes. Besides, the ability of mouse haESCs that can be used as sperm replacement for generation of animals opens new avenues for functional genetics in vivo , such as investigating the roles of imprinted genes during prenatal embryonic development, generation of mouse models carrying complex gene modifications to mimic human diseases and screening for critical genes or amino acids of a specific protein involved in a developmental process of interest. Genome Tagging Project (GTP) proposed based on haESCs was initiated recently with the goal to tag every protein in haESCs, followed by generation of tagged mice at genome-wide scale. GTP will provide a standard platform for the protein study, which may enable precise description of protein atlas and regulatory networks during embryonic development.

Genetic and epigenetic features direct differential efficiency of Xist-mediated silencing at X-chromosomal and autosomal locations

Xist is indispensable for X chromosome inactivation. However, how Xist RNA directs chromosome-wide silencing and why some regions are more efficiently silenced than others remains unknown. Here, we explore the function of Xist by inducing ectopic Xist expression from multiple different X-linked and autosomal loci in mouse aneuploid and female diploid embryonic stem cells in which Xist-mediated silencing does not lead to lethal functional monosomy. We show that ectopic Xist expression faithfully recapitulates endogenous X chromosome inactivation from any location on the X chromosome, whereas long-range silencing of autosomal genes is less efficient. Long interspersed elements facilitate inactivation of genes located far away from the Xist transcription locus, and genes escaping X chromosome inactivation show enrichment of CTCF on X chromosomal but not autosomal loci. Our findings highlight important genomic and epigenetic features acquired during sex chromosome evolution to facilitate an efficient X chromosome inactivation process.

ID: 1065 Establishment of parthenogenetic diploid embryonic stem cells in mice

Parthenogenesis is a process in which zygotes are produced without sperm presence. Due to lack of paternal genes, parthenogenetic embryos cannot develop to full-term; however, these embryos show a great potential to generate histocompatible stem cells (parthenogenetic embryonic stem – pES cells) for transplantation. In this research, parthenogenetic activation in the mouse was carried out using strontium chloride (SrCl2) combined with cytochalasin B (CB). The rate of embryo development, blastocyst quality and expression of acetylation of histone H4 lysine 12 (H4K12Ac) were investigated, while parthenogenetic blastocysts were used to establish pES cells. The results showed that rate of in vitro blastulation of parthenogenetic embryos was lower than that of fertilized ones (45.1% vs 98.0%, respectively). In addition, blastocysts developed from parthenogenetic embryos also expressed lower quality, which was demonstrated by lower total cell number. Moreover, H4K12Ac expression significantly decreased in the inner cell mass (ICM) of parthenogenetic blastocysts compared to fertilized ones, indicating a possible reason for lower blastocyst quality. Following embryo collection and activation, two ES cell lines – fertilized (fES) and pES cell lines have been successfully established and maintained long term in vitro. To sum up, differences in blastocyst quality and H4K12Ac expression in ICM cells of blastocyst may contribute to aberrant developmental and embryonic stem cell formation in parthenogenetic embryos.

Tetraploid embryonic stem cells can contribute to the development of chimeric fetuses and chimeric extraembryonic tissues

Our study examined the in vivo chimeric and survival capacities of chimeras created by injecting tetraploid embryonic stem cells (ESCs) expressing green fluorescent protein (GFP) into diploid embryos. At 3.5 days post-coitum (dpc) and 4.5 dpc, the tetraploid ESCs were able to contribute to the inner cell mass (ICM) just as diploid ESCs tagged with GFP. At 6.5 dpc, 8.0 dpc and 10.5 dpc, the tetraploid ESCs manifested in the same location as the diploid ESCs. The GFP cells in the extraembryonic tissues and fetuses of tetraploid ESC chimeras were tetraploid as determined by fluorescence activated cell sorting (FACS). Furthermore, tetraploid ESCs contributed to the development of the placenta, embryolemma and umbilical cord at 13.5 dpc and 16.5 dpc; however, very less GFP cells were found in the fetuses of tetraploid ESC chimeras. We further found that the proliferation of tetraploid ESCs was slower than that of diploid ESCs. In addition, the relative mRNA expression in the three germ layers and the trophoblast was abnormal in the EBs of tetraploid ESCs compared with diploid ESCs. In short, slower proliferation and abnormal differentiation potential of tetraploid ESCs might be two of the reasons for their poor survival and chimeric capacities.

Single-Cell Dynamic Analysis of Mitosis in Haploid Embryonic Stem Cells Shows the Prolonged Metaphase and Its Association with Self-diploidization.

SummaryThe recent establishment of mammalian haploid embryonic stem cells (ESCs) provides new possibilities for genetic screening and for understanding genome evolution and function. However, the dynamics of mitosis in haploid ESCs is still unclear. Here, we report that the duration of mitosis in haploid ESCs, especially the metaphase, is significantly longer than that in diploid ESCs. Delaying mitosis by chemicals increased self-diploidization of haploid ESCs, while shortening mitosis stabilized haploid ESCs. Taken together, our study suggests that the delayed mitosis of haploid ESCs is associated with self-diploidization.

Tetraploid Embryonic Stem Cells Maintain Pluripotency and Differentiation Potency into Three Germ Layers.

Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals.

Cytogenetic analysis and Dlk1-Dio3 locus epigenetic status of mouse embryonic stem cells during early passages.

Mouse embryonic stem (ES) cells are widely used in early development studies and for transgenic animal production; however, a stable karyotype is a prerequisite for their use. We derived 32 ES cell lines of outbred mice (129×BALB (1B), C57BL×1B, and DD×1B F1 hybrids). Pluripotency was assessed by utilizing stem-cell-marker gene expression, teratoma formation assays and the formation of chimeras. It was shown that only 21 of the 32 ES cell lines had a diploid modal number of chromosomes of 40. In these lines, the percentage of diploid cells varied from 30.3 to 78.9%, and trisomy of chromosomes 1, 8 and 11 was observed in some cells in 16.7, 36.7 and 20.0% of the diploid ES cell lines, respectively. Some cells had trisomy of chromosomes 6, 9, 12, 14, 18 and 19. In situ hybridization with an X chromosome paint probe revealed that 7 of the 11 XX-cell lines had X chromosome rearrangements in some cells. Analysis of the methylation status of the Dlk1-Dio3 locus showed that imprinting was altered in 4 of the 18 ES cell lines. Thus, mouse ES cell lines are prone to chromosome abnormalities even at early passages. Therefore, routine cytogenetic and imprinting analyses are necessary for ES cell characterization.

A highly efficient method for generation of therapeutic quality human pluripotent stem cells by using naive induced pluripotent stem cells nucleus for nuclear transfer.

Even after several years since the discovery of human embryonic stem cells and induced pluripotent stem cells (iPSC), we are still unable to make any significant therapeutic benefits out of them such as cell therapy or generation of organs for transplantation. Recent success in somatic cell nuclear transfer (SCNT) made it possible to generate diploid embryonic stem cells, which opens up the way to make high-quality pluripotent stem cells. However, the process is highly inefficient and hence expensive compared to the generation of iPSC. Even with the latest SCNT technology, we are not sure whether one can make therapeutic quality pluripotent stem cell from any patient’s somatic cells or by using oocytes from any donor. Combining iPSC technology with SCNT, that is, by using the nucleus of the candidate somatic cell which got reprogrammed to pluripotent state instead that of the unmodified nucleus of the candidate somatic cell, would boost the efficiency of the technique, and we would be able to generate therapeutic quality pluripotent stem cells. Induced pluripotent stem cell nuclear transfer (iPSCNT) combines the efficiency of iPSC generation with the speed and natural reprogramming environment of SCNT. The new technique may be called iPSCNT. This technique could prove to have very revolutionary benefits for humankind. This could be useful in generating organs for transplantation for patients and for reproductive cloning, especially for childless men and women who cannot have children by any other techniques. When combined with advanced gene editing techniques (such as CRISPR-Cas system) this technique might also prove useful to those who want to have healthy children but suffer from inherited diseases. The current code of ethics may be against reproductive cloning. However, this will change with time as it happened with most of the revolutionary scientific breakthroughs. After all, it is the right of every human to have healthy offspring and it is the question of reproductive freedom and existence.

Abstract A36: A transposon-based genetic screen in haploid mouse embryonic stem cells identifies Parp1 as a major mediator of olaparib toxicity

Abstract Haploid embryonic stem cells have recently been isolated from activated mouse oocytes. Due to the ease of random gene disruption in haploid cells, these have great promise for forward genetic screens. We have used the piggyBac transposon, a highly active insertional mutagen, to generate stable libraries of mutants in these cells. These libraries comprise at least 200,000 mutants. We have exposed these libraries to several drugs to generate resistant clones. Mapping the transposon integration sites in these clones can identify genes required for toxicity of the drugs to normal cells. In a proof of principle screen we isolated clones resistant to 6-thioguanine that had insertions in components of the DNA mismatch repair pathway, which is known to be required for toxicity of this purine analogue. In a screen for mutants resistant to the poly-(ADP-ribose) polymerase (PARP) 1/2 inhibitor olaparib, we identified two mutants with different insertions in Parp1 that were more than 100-fold resistant to olaparib than wild type cells. These mutants lacked Parp1 protein expression and radiation-induced poly-(ADP-ribose) formation and were also resistant to other PARP inhibitors. Removal of the transposon by re-expressing transposase in the cells resulted in reversion of the phenotype. Knockdown of PARP1 by siRNA in human cell lines also led to olaparib resistance. The finding that PARP1 itself is required for toxicity of olaparib supports the hypothesis that PARP1 is a component of the toxic lesion. There were several other resistant mutants isolated in the screen, but no other genes had multiple resistant clones with insertions. Removal of the transposon did not revert the phenotype in these clones, suggesting that they arose from background mutations in cell culture. Interestingly many of these clones also lacked Parp1 protein expression, suggesting that inactivation of Parp1 may also be the mechanism of resistance. Therefore Parp1 may be the major determinant of olaparib toxicity to wild type cells. Conditional gene targeting technology is well-established in diploid embryonic stem cells. By combining targeted mutation in a cancer gene with random transposon mutagenesis, it may also be possible to use this screening system to identify synthetic lethal interactions. We have successfully used gene targeting to modify these cells (at the Hprt locus) and are developing strategies to monitor the loss of mutants from a pooled culture by high throughput sequencing of transposon integration sites. Citation Format: Stephen J. Pettitt, Farah L. Rehman, Ilirjana Bajrami, Helen Pemberton, Rachel Brough, Iwanka Kozarewa, Christopher J. Lord, Alan Ashworth. A transposon-based genetic screen in haploid mouse embryonic stem cells identifies Parp1 as a major mediator of olaparib toxicity. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A36.

Diploid, but not haploid, human embryonic stem cells can be derived from microsurgically repaired tripronuclear human zygotes

Human embryonic stem cells have shown tremendous potential in regenerative medicine, and the recent progress in haploid embryonic stem cells provides new insights for future applications of embryonic stem cells. Disruption of normal fertilized embryos remains controversial; thus, the development of a new source for human embryonic stem cells is important for their usefulness. Here, we investigated the feasibility of haploid and diploid embryo reconstruction and embryonic stem cell derivation using microsurgically repaired tripronuclear human zygotes. Diploid and haploid zygotes were successfully reconstructed, but a large proportion of them still had a tripolar spindle assembly. The reconstructed embryos developed to the blastocyst stage, although the loss of chromosomes was observed in these zygotes. Finally, triploid and diploid human embryonic stem cells were derived from tripronuclear and reconstructed zygotes (from which only one pronucleus was removed), but haploid human embryonic stem cells were not successfully derived from the reconstructed zygotes when two pronuclei were removed. Both triploid and diploid human embryonic stem cells showed the general characteristics of human embryonic stem cells. These results indicate that the lower embryo quality resulting from abnormal spindle assembly contributed to the failure of the haploid embryonic stem cell derivation. However, the successful derivation of diploid embryonic stem cells demonstrated that microsurgical tripronuclear zygotes are an alternative source of human embryonic stem cells. In the future, improving spindle assembly will facilitate the application of triploid zygotes to the field of haploid embryonic stem cells.

The embryos’ geneology-sorting fact from fancy

The embryos’ geneology-sorting fact from fancy

Triploid and diploid embryonic stem cell lines derived from tripronuclear human zygotes

Human embryonic stem cells (hESCs) are self-renewing, pluripotent cells that are valuable research tools and hold promise for use in regenerative medicine. The need for new hESC lines motivated our attempts to find a new resource for the derivation of hESC lines. The aim of this work was to establish more hESC lines from abnormal fertilized zygotes and to meet the emerging requirements for their use in cell replacement therapies, disease modeling, and basic research. A total of 130 tripronuclear human zygotes was collected 18-20 h post-insemination and cultured in a modified culture medium. The inner cell mass of 12 blastocysts were isolated by a mechanical method in order to establish embryonic stem cell lines. We established four hESC lines derived from 130 trinuclear zygotes, one of which was triploid and the others were diploid. The efficiency of deriving hESC lines is 3.08 %. The ratio of deriving triploid and diploid hESC lines is 1:3. All of these hESC lines exhibited similar markers of undifferentiated hESCs and had the typical morphology of hESCs, a capacity for long-term proliferation, and pluripotent differentiation potential both in vivo and in vitro. These abnormal zygotes, which otherwise would have been discarded, can serve as an alternative source for normal euploid hESC lines.

Generation of Medaka Fish Haploid Embryonic Stem Cells

Haploid embryonic stem (ES) cells combine haploidy and pluripotency, enabling direct genetic analyses of recessive phenotypes in vertebrate cells. Haploid cells have been elusive for culture, due to their inferior growth and genomic instability. Here, we generated gynogenetic medaka embryos and obtained three haploid ES cell lines that retained pluripotency and competitive growth. Upon nuclear transfer into unfertilized oocytes, the haploid ES cells, even after genetic engineering, generated viable offspring capable of germline transmission. Hence, haploid medaka ES cells stably maintain normal growth, pluripotency, and genomic integrity. Mosaic oocytes created by combining a mitotic nucleus and a meiotic nucleus can generate fertile fish offspring. Haploid ES cells may offer a yeast-like system for analyzing recessive phenotypes in numerous cell lineages of vertebrates in vitro.

FISH analysis of regional replication of homologous chromosomes in hybrid cells obtained by fusion of embryonic stem cells with somatic cells

The paper concerns FISH-analysis of regional replication of parental chromosomes 1, 3 and 6 in hybrid cells obtained by fusion between Mus musculus embryonic stem cells (ESC) and M. caroli splenocytes. The data demonstrated that parental chromosomes in the hybrid cells with near-diploid karyotype showed synchronous replication in 70-75% of tested cells that was comparable with diploid ESC and diploid fibroblasts. Synchronous replication of parental chromosomes in hybrid cells with near-triploid karyotype was observed in 46-57% of tested cells. However, it was correct in the case of hybrid cells with three copies of the tested chromosomes whereas the ratio of synchronous replication in triploid cells with two copies was comparable or similar to that in diploid cells. Hybrid cells with near-tetraploid karyotype showed high ratio of asynchronous replication (over 50%) comparable to those tetraploid ESC and tetraploid fibroblasts but significantly distinguished from diploid cells. Thus, most hybrid cells with two copies of tested parental chromosomes showed their synchronous replication. Unfortunately, the FISH-analysis is poor informative when it is used for studying cells with more than two copies of tested chromosomes.

Hybrid cells generated by fusion of embryonic stem cells with di-and tetraploid fibroblasts have alternative parental phenotypes

Hybrid cells generated by fusion of embryonic stem cells (ESCs) with diploid somatic cells have the phenotype and level of pluripotency that are comparable to those of ESCs, including the capacity for forming chimeras [1, 2]. The basis of the dominance of the ESC genome in the hybrid cells is reprogramming of genome of the diploid somatic partner (fibroblast, thymocyte, etc.), which converts the profile of the activities of its genes into the profile similar to that of ESCs. The character of gene expression of the pluripotent partner in the hybrid cells of this type slightly differs from its character in the parental ESCs [3‐5]. This unidirectional dominance of the ESC genome over the somatic genome is an unusual phenomenon, because both genomes are in the same nucleus, where they may exchange trans -regulating signals. As a first step in the study of the phenomenon of dominance of the ESC phenotype in the hybrid cells, we decided to evaluate the effect of the ploidy of cells of the somatic partner on the phenotype of hybrid cells by comparison of the characteristics of hybrids generated by fusion of di- and tetraploid fibroblasts with diploid ESCs. The experiments performed allowed us to discover alternative dominance of parental genomes in the hybrid cells, which depends on the ploidy of the somatic partner: the cell hybrids formed by a diploid ESC and a diploid fibroblast had the characteristics of an ESC and, conversely, hybrid cells formed by a diploid ESC and a tetraploid fibroblast had the characteristics of a fibroblast. Deciphering of the mechanisms of the all-or-nothing “switching” between the dominances of the two genomes may help to control this process. Cultures of diploid fetal fibroblasts were obtained by the standard method [6] from 12-day-old embryos of DD/c mice (strain dMEF) and C57Bl/6-I(I)1RK mice with a heterozygous double insertion in chromosome 1, which was previously described in [7] (the iMEF strain). A primary culture of embryonic fibroblasts proliferates for a limited time period. However, in rare cases, the cells of primary cultures obtain the capacity for unlimited proliferation, which is frequently accompanied by tetraploidy. These cells were used to obtain tetraploid dtMEF fibroblasts from the dMEF strain and itMEF cells from the iMEF strain, the proportion of cells with a tetraploid set of chromosomes reaching 95%. In the cell hybridization experiments, we used ESC line E14Tg2aSc4TP6.3 with a deficient activity of hypoxanthine phosphoribosyltransferase (HPRT) and transformed with a construct that contained the genes of green fluorescent protein (GFP) and puromycin resistance [8]. To induce cell fusion, growing diploid or tetraploid fibroblasts (about 70% of the monolayer) were covered with ESCs at a ratio of 1 : 1; within 1 h, the growth medium was removed, and the cells were treated for 1.5

Embryonic Stem Cells Contribute to Mouse Chimeras in the Absence of Detectable Cell Fusion

Embryonic stem (ES) cells are capable of differentiating into all embryonic and adult cell types following mouse chimera production. Although injection of diploid ES cells into tetraploid blastocysts suggests that tetraploid cells have a selective disadvantage in the developing embryo, tetraploid hybrid cells, formed by cell fusion between ES cells and somatic cells, have been reported to contribute to mouse chimeras. In addition, other examples of apparent stem cell plasticity have recently been shown to be the result of cell fusion. Here we investigate whether ES cells contribute to mouse chimeras through a cell fusion mechanism. Fluorescence in situ hybridization (FISH) analysis for X and Y chromosomes was performed on dissociated tissues from embryonic, neonatal, and adult wild-type, and chimeric mice to follow the ploidy distributions of cells from various tissues. FISH analysis showed that the ploidy distributions in dissociated tissues, notably the tetraploid cell number, did not differ between chimeric and wild-type tissues. To address the possibility that early cell fusion events are hidden by subsequent reductive divisions or other changes in cell ploidy, we injected Z/EG (lacZ/EGFP) ES cells into ACTB-cre blastocysts. Recombination can only occur as the result of cell fusion, and the recombined allele should persist through any subsequent changes in cell ploidy. We did not detect evidence of fusion in embryonic chimeras either by direct fluorescence microscopy for GFP or by PCR amplification of the recombined Z/EG locus on genomic DNA from ACTB-cre::Z/EG chimeric embryos. Our results argue strongly against cell fusion as a mechanism by which ES cells contribute to chimeras.

Production of chimeras by aggregation of embryonic stem cells with diploid or tetraploid mouse embryos

The production of mouse chimeras is a common step in the establishment of genetically modified animal strains. Chimeras also provide a powerful experimental tool for following cell behavior during both prenatal and postnatal development. This protocol outlines a simple and economical technique for the production of large numbers of mouse chimeras using traditional diploid morula<-->diploid embryonic stem (ES) cell aggregations. Additional steps are included to describe the procedures necessary to produce specialized tetraploid chimeras using tetraploid morula<-->diploid ES cell aggregations. This increasingly popular form of chimera produces embryos of nearly complete ES cell derivation that can be used to speed transgenic production or ask developmental questions. Using this protocol, mouse chimeras can be generated and transferred to pseudopregnant surrogate mothers in a 5-d period.