Genome wide functional genetics in haploid cells

Some organisms such as yeast or males of social insects are haploid, i.e. they carry a single set of chromosomes, while haploidy in mammals is exclusively restricted to mature germ cells. A single copy of the genome provides the basis for genetic analyses where any recessive mutation of essential genes will show a clear phenotype due to the absence of a second gene copy. Most prominently, haploidy in yeast has been utilized for recessive genetic screens that have markedly contributed to our understanding of development, basic physiology, and disease. Somatic mammalian cells carry two copies of chromosomes (diploidy) that obscure genetic analysis. Near haploid human leukemic cells however have been developed as a high throughput screening tool. Although deemed impossible, we and others have generated mammalian haploid embryonic stem cells from parthenogenetic mouse embryos. Haploid stem cells open the possibility of combining the power of a haploid genome with pluripotency of embryonic stem cells to uncover fundamental biological processes in defined cell types at a genomic scale. Haploid genetics has thus become a powerful alternative to RNAi or CRISPR based screens.     

Gene Transfer and Genome-Wide Insertional Mutagenesis by Retroviral Transduction in Fish Stem Cells

Retrovirus (RV) is efficient for gene transfer and integration in dividing cells of diverse organisms. RV provides a powerful tool for insertional mutagenesis (IM) to identify and functionally analyze genes essential for normal and pathological processes. Here we report RV-mediated gene transfer and genome-wide IM in fish stem cells from medaka and zebrafish. Three RVs were produced for fish cell transduction: rvLegfp and rvLcherry produce green fluorescent protein (GFP) and mCherry fluorescent protein respectively under control of human cytomegalovirus immediate early promoter upon any chromosomal integration, whereas rvGTgfp contains a splicing acceptor and expresses GFP only upon gene trapping (GT) via intronic in-frame integration and spliced to endogenous active genes. We show that rvLegfp and rvLcherry produce a transduction efficiency of 11~23% in medaka and zebrafish stem cell lines, which is as 30~67% efficient as the positive control in NIH/3T3. Upon co-infection with rvGTgfp and rvLcherry, GFP-positive cells were much fewer than Cherry-positive cells, consistent with rareness of productive gene trapping events versus random integration. Importantly, rvGTgfp infection in the medaka haploid embryonic stem (ES) cell line HX1 generated GTgfp insertion on all 24 chromosomes of the haploid genome. Similar to the mammalian haploid cells, these insertion events were presented predominantly in intergenic regions and introns but rarely in exons. RV-transduced HX1 retained the ES cell properties such as stable growth, embryoid body formation and pluripotency gene expression. Therefore, RV is proficient for gene transfer and IM in fish stem cells. Our results open new avenue for genome-wide IM in medaka haploid ES cells in culture.     

A review of mitosis in the fission yeast Schizosaccharomyces pombe

Mitosis and cell division are the final events of the cell cycle, resulting in the precise segregation of chromosomes into two daughter cells. A highly controlled and accurate segregation of the chromosomes is required to ensure that each daughter cell receives a complete genome and remains viable. The fission yeast, Schizosaccharomyces pombe, is a unicellular eukaryotic organism which is particularly convenient for investigating these problems. It is very amenable to genetic analysis and its predominantly haploid life cycle has allowed the isolation of recessive temperature-sensitive mutants unable to complete the cell cycle. Classical genetic analysis of these mutants has been used to identify over 40 gene functions that are required for cell cycle progress in S. pombe. Many of these genes have now been cloned and sequenced and in some cases the encoded gene product has been identified. This approach, coupling classical and molecular genetics, allows identification of the molecules important in the mitotic processes and provides a means for establishing what functional roles they may play.     

The evolutionary dynamics of haplodiploidy: Genome architecture and haploid viability

Haplodiploid reproduction, in which males are haploid and females are diploid, is widespread among animals, yet we understand little about the forces responsible for its evolution. The current theory is that haplodiploidy has evolved through genetic conflicts, as it provides a transmission advantage to mothers. Male viability is thought to be a major limiting factor; diploid individuals tend to harbor many recessive lethal mutations. This theory predicts that the evolution of haplodiploidy is more likely in male heterogametic lineages with few chromosomes, as genes on the X chromosome are often expressed in a haploid environment, and the fewer the chromosome number, the greater the proportion of the total genome that is X‐linked. We test this prediction with comparative phylogenetic analyses of mites, among which haplodiploidy has evolved repeatedly. We recover a negative correlation between chromosome number and haplodiploidy, find evidence that low chromosome number evolved prior to haplodiploidy, and that it is unlikely that diplodiploidy has reevolved from haplodiploid lineages of mites. These results are consistent with the predicted importance of haploid male viability.     

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.     

Karyotype Characterization of In Vivo- and In Vitro-Derived Porcine Parthenogenetic Cell Lines

Mammalian haploid cell lines provide useful tools for both genetic studies and transgenic animal production. To derive porcine haploid cells, three sets of experiments were conducted. First, genomes of blastomeres from 8-cell to 16-cell porcine parthenogenetically activated (PA) embryos were examined by chromosome spread analysis. An intact haploid genome was maintained by 48.15% of blastomeres. Based on this result, two major approaches for amplifying the haploid cell population were tested. First, embryonic stem-like (ES-like) cells were cultured from PA blastocyst stage embryos, and second, fetal fibroblasts from implanted day 30 PA fetuses were cultured. A total of six ES-like cell lines were derived from PA blastocysts. No chromosome spread with exactly 19 chromosomes (the normal haploid complement) was found. Four cell lines showed a tendency to develop to polyploidy (more than 38 chromosomes). The karyotypes of the fetal fibroblasts showed different abnormalities. Cells with 19–38 chromosomes were the predominant karyotype (59.48–60.91%). The diploid cells were the second most observed karyotype (16.17%–22.73%). Although a low percentage (3.45–8.33%) of cells with 19 chromosomes were detected in 18.52% of the fetus-derived cell lines, these cells were not authentic haploid cells since they exhibited random losses or gains of some chromosomes. The haploid fibroblasts were not efficiently enriched via flow cytometry sorting. On the contrary, the diploid cells were efficiently enriched. The enriched parthenogenetic diploid cells showed normal karyotypes and expressed paternally imprinted genes at extremely low levels. We concluded that only a limited number of authentic haploid cells could be obtained from porcine cleavage-stage parthenogenetic embryos. Unlike mouse, the karyotype of porcine PA embryo-derived haploid cells is not stable, long-term culture of parthenogenetic embryos, either in vivo or in vitro, resulted in abnormal karyotypes. The porcine PA embryo-derived diploid fibroblasts enriched from sorting might be candidate cells for paternally imprinted gene research.     

Evolutionary implications of permanent odd polyploidy in the stable sexual, pentaploid of Rosa canina L

In Rosa canina (2n = 5x = 35), the pollen and ovular parents contribute, respectively, seven and 28 chromosomes to the zygote. At meiosis I, 14 chromosomes form seven bivalents and 21 chromosomes remain as univalents. Fluorescent in situ hybridization to mitotic and pollen mother cells (PMC) of R. canina showed that 10 chromosomes (two per genome) carry ribosomal DNA (rDNA) loci. Five chromosomes carry terminal 18S-5.8S-26S rDNA loci; three of these also carry paracentric 5S rDNA loci and were designated as marker chromosomes 1. Five chromosomes carry only 5S rDNA loci and three of these were designated as marker chromosomes 2. The remaining four of the 10 chromosomes with rDNA loci were individually identifiable by the type and relative sizes of their rDNA loci and were numbered separately. At PMC meiosis, two marker chromosomes 1 and two marker chromosomes 2 formed bivalents, whereas the others were unpaired. In a gynogenetic haploid of R. canina (n = 4x = 28), obtained after pollination with gamma-irradiated pollen, chromosomes at meiosis I in PMC remained predominantly unpaired. The data indicate only one pair of truly homologous genomes in R. canina. The 21 unpaired chromosomes probably remain as univalents through multiple generations and do not recombine. The long-term evolutionary consequence for the univalents is likely to be genetic degradation through accumulated mutational change as in the mammalian Y chromosome and chromosomes of asexual species. But there is no indication that univalents carry degenerate 5S rDNA families. This may point to a recent evolution of the R. canina meiotic system.     

Xist敲降可以改善孤雄单倍体囊胚质量

单倍体胚胎干细胞研究一直以来吸引着研究者们的注意,它可以用作基因修饰的工具或是用于药物筛选。随着孤雄胚胎干细胞系的成功建立,更扩展了单倍体胚胎干细胞的应用前景。但单倍体孤雄胚胎发育率低,胚胎质量差,制约着孤雄单倍体胚胎干细胞的建系。为改善孤雄单倍体胚胎发育潜能及胚胎干细胞建系效率低的问题,我们检测了小鼠(Mus musculus domesticus)孤雄单倍体胚胎的体外发育过程和该过程中Xist基因表达情况。结果发现,孤雄单倍体胚胎囊胚发育率只有10%~14%,发育至囊胚所需时间差异较大,从3.5~5.5 d不等。通过核糖核酸荧光原位杂交实验(RNA-FISH)跟踪Xist基因表达情况,发现其在发育至囊胚阶段的胚胎中呈抑制状态,而在早期胚胎中多呈表达状态。通过si RNA扰低Xist表达,虽然没有改变孤雄单倍体胚胎发育到囊胚的比例,但显著提高了囊胚质量,并提高了接种胚胎内细胞团(ICM)后建立细胞系的效率。结果说明,Xist基因的表达可能是导致小鼠孤雄单倍体胚胎质量差、干细胞建系率低的原因之一。     

Engineering of polyploid Saccharomyces cerevisiae for secretion of large amounts of fungal glucoamylase

We engineered Saccharomyces cerevisiae cells that produce large amounts of fungal glucoamylase (GAI) from Aspergillus awamori var. kawachi. To do this, we used the delta-sequence-mediated integration vector system and the heat-induced endomitotic diploidization method. delta-Sequence-mediated integration is known to occur mainly in a particular chromosome, and the copy number of the integration is variable. In order to construct transformants carrying the GAI gene on several chromosomes, haploid cells carrying the GAI gene on different chromosomes were crossed with each other. The cells were then allowed to form spores, which was followed by dissection. Haploid cells containing GAI genes on multiple chromosomes were obtained in this way. One such haploid cell contained the GAI gene on five chromosomes and exhibited the highest GAI activity (5.93 U/ml), which was about sixfold higher than the activity of a cell containing one gene on a single chromosome. Furthermore, we performed heat-induced endomitotic diploidization for haploid transformants to obtain polyploid mater cells carrying multiple GAI genes. The copy number of the GAI gene increased in proportion to the ploidy level, and larger amounts of GAI were secreted.     

Isopropyl-N(3-chlorophenyl) carbamate (CIPC) induced chromosomal loss in soybean: a new tool for plant somatic cell genetics

Summary The properties and uses of tissue culture partial haploid soybean cell lines are explored. Partial haploid lines were prepared by CIPC treatment of a genetic heterozygote, and compared to this heterozygote and to homozygous cell lines corresponding to the parental genotypes from which the heterozygote was derived. Cell lines which lack chromosomes were characterized physiologically and with respect to a variety of isozyme markers. Often the loss of chromosomes revealed a phenotype corresponding to a recessive parental genotype. In some cases, however, new phenotypes were observed indicating a complex genotype and suggests the interaction of several genes. The implications of this for plant breeding are discussed.CIPC also was used as a tool to dissect a complex phenotype which arose as the result of mutagenesis. A mutant cell line which required asparagine for growth but also had acquired the ability to grow on allantoin as a sole source of nitrogen was treated with CIPC to remove chromosomes. The requirement for asparagine could be separated from the ability to use allantoin, demonstrating that these phenotypes were the result of separate mutations.     

What the papers say: Differential roles of paternal and maternal genomes during embryogenesis in the mouse

Although female and male gametes are presumably equivalent in their genetic contribution to embryos, they carry specific information, perhaps reversibly imprinted into the genomes during oogenesis and spermatogenesis, as to their maternal or paternal origin. This information is crucial for embryogenesis and, in the absence of at least one haploid set of chromosomes from each parent, embryos do not develop to term. The paternal genome is probably required for proliferation of extraembryonic tissues and the maternal genome for some stages of embryogeneis. Furthermore, the effects of certain mutations are determined by the paternal or maternal origin of the inheritance.     

A model for random genetic damage directing selection of diploid or aneuploid tumours

Objectives: To test whether genetic instability may determine whether tumours become aneuploid or diploid. Materials and methods: We have identified genes needed for cell survival or replication by combining Affymetrix gene expression array data from 12 experimental cell lines with in silico GEO+GNF and expO databases. Specific loss of heterozygosis (LOHs), chromosomal abnormalities (called derivative chromosomes) and numbers of normal homologues were identified by SNP and SKY analyses. Random gene losses were calculated under the assumption that bi‐allelic MMR gene inactivation causes a 20‐fold increase in rate of gene loss. Results: There were ～1.23 × 10⁴ genes widely dispersed throughout the genome and possibly expressed by all cells for survival or proliferation, many of these genes performed housekeeping functions. Conservation of the genes may explain the complete haploid genomes found for 15 different cell types and derivative chromosomes selectively retained in aneuploid cancer cell lines after LOH formations, and normal homologue losses. Loss of cell survival/replication genes was calculated to be higher in colon stem cells of carriers of MMR gene mutations than carriers of APC gene mutations. Conclusion: Random loss of cell survival/replication genes was calculated to be low enough for colon stem cells with APC gene mutations to ‘select’ LOH and derivative chromosome combinations favouring tumour cell proliferation. However, cell survival/replication gene loss was calculated to be too high for colonic stem cells lacking MMR genes to survive chromosomal instability, explaining why MMR mutations only produce tumours with diploid chromosome cells.     

Repair of DNA double-strand breaks requires two homologous DNA duplexes

DNA repair and cell survival in haploid and its diploid derivative strains ofSaccharomyces cerevisiae were studied after 100 krad X-ray irradiation. The cells were in theG ₁ stage of the cell cycle, where haploid cells had only one copy of genetic material per genome and diploid had two copies. It was found that diploid could repair double-strand breaks in its DNA after 48 hr of liquid holding which was accompanied by a four-fold rise in survival. In contrast a haploid strain failed to repair its DNA and showed no increase in survival after liquid holding. It is concluded that (1) repair of DNA double-strand breaks requires the availability of two homologous DNA duplexes, (2) restoration of cell viability during liquid holding is connected with repair of DNA double-strand breaks and (3) this repair is a slow process possibly associated with slow finding and conjugation of homologous chromosomes.     

Modification of an existing chromosomal inversion to engineer a balancer for mouse chromosome 15

Chromosomal inversions are valuable genetic tools for mutagenesis screens, where appropriately marked inversions can be used as balancer chromosomes to recover and maintain mutations in the corresponding chromosomal region. For any inversion to be effective as a balancer, it should exhibit both dominant and recessive visible traits; ideally the recessive trait should be a fully penetrant lethality in which inversion homozygotes die before birth. Unfortunately, most inversions recovered by classical radiation or chemical mutagenesis techniques do not have an overt phenotype in either the heterozygous or the homozygous state. However, they can be modified by relatively simple procedures to make them suitable as an appropriately marked balancer. We have used homologous recombination to modify, in embryonic stem cells, the recessive-lethal In(15)21Rk inversion to endow it with a dominant-visible phenotype. Several ES cell lines were derived from inversion heterozygotes, and a keratin-14 (K14) promoter-driven agouti minigene was introduced onto the inverted chromosome 15 in the ES cells by gene targeting. Mice derived from the targeted ES cells carry the inverted chromosome 15 and, at the same time, exhibit lighter coat color on their ears and tails, making this modified In(15)21Rk useful as a balancer for proximal mouse chromosome 15.     

Euploid segregation through multipolar mitosis in mammalian cell cultures

Cultures were made of kidney cells of male Rhesus monkey and a karyological analysis of these cells was carried out at various times after plating using the chromosome banding method of Seabringht (1972), in order to study the segregation phenomena which take place in cell cultures in vitro through multipolar mitoses and to identify segregating cells. — Several segregating cells were found: haploid cells, diploid cells with two X chromosomes and triploid cells which were strictly euploid. Polyploidization-segregation cycles in the cell cultures and their mechanism and chronological sequence were analyzed. — On the basis of these results it was suggested that the genome is organized in haploid sets capable of segregating as units and of producing strictly euploid segregating daughter cells.     

The genome of Oscheius tipulae: determination of size, complexity, and structure by DNA reassociation using fluorescent dye.

This work describes the physicochemical characterization of the genome and telomere structure from the nematode Oscheius tipulae CEW1. Oscheius tipulae is a free-living nematode belonging to the family Rhabditidae and has been used as a model system for comparative genetic studies. A new protocol that combines fluorescent detection of double-stranded DNA and S1 nuclease was used to determine the genome size of O. tipulae as 100.8 Mb (approximately 0.1 pg DNA/haploid nucleus). The genome of this nematode is made up of 83.4% unique copy sequences, 9.4% intermediate repetitive sequences, and 7.2% highly repetitive sequences, suggesting that its structure is similar to those of other nematodes of the genus Caenorhabditis. We also showed that O. tipulae has the same telomere repeats already found in Caenorhabditis elegans at the ends and in internal regions of the chromosomes. Using a cassette-ligation-mediated PCR protocol we were able to obtain 5 different putative subtelomeric sequences of O. tipulae, which show no similarity to C. elegans or C. briggsae subtelomeric regions. DAPI staining of hermaphrodite gonad cells show that, as detected in C. elegans and other rhabditids, O. tipulae have a haploid complement of 6 chromosomes.     

Integration of a B chromosome into the A genome of a wasp,revisited

A previous study showed that in the haplodiploid solitary wasp Trypoxylon albitarse, most individuals carry one B chromosome per haploid genome, the same dosage as the standard (A) chromosomes, indicating a possible regularization of B-chromosome meiotic behaviour and its integration into the A genome. In a new sampling, we have analysed 15 populations (including 9 out of the 10 previously analysed) to test the evolution of this integration process. The new results provide a direct report of the invasion process in the Porto Firme population, where B frequency has dramatically increased in only four generations. In the populations from the Viçosa region, however, B frequency has remained stable, although the principal B type, the metacentric one, has increased in frequency at the expense of the acrocentric one in several populations. The implications of these new results on the hypothesis of the integration of these B chromosomes, as regular members of the A genome, are discussed.     

A PiggyBac-based recessive screening method to identify pluripotency regulators

Phenotype driven genetic screens allow unbiased exploration of the genome to discover new biological regulators. Bloom syndrome gene (Blm) deficient embryonic stem (ES) cells provide an opportunity for recessive screening due to frequent loss of heterozygosity. We describe a strategy for isolating regulators of mammalian pluripotency based on conversion to homozygosity of PiggyBac gene trap insertions combined with stringent selection for differentiation resistance. From a screen of 2000 mutants we obtained a disruptive integration in the Tcf3 gene. Homozygous Tcf3 mutants showed impaired differentiation and enhanced self-renewal. This phenotype was reverted in a dosage sensitive manner by excision of one or both copies of the gene trap. These results provide new evidence confirming that Tcf3 is a potent negative regulator of pluripotency and validate a forward screening methodology to identify modulators of pluripotent stem cell biology.