Effects of 5-azacytidine on the development of parthenogenetic mouse embryos

This study describes the effects of 5-azacytidine (5-azaC) on the development of diploid parthenogenetic embryos (PE) of CBA, C57BL/6 and (CBA × C57BL/6)F₁ mice in vitro at the 1-cell or the blastocyst stage or in vivo after implantation. Our findings indicate that genomic imprinting is modulated by genetic background. Non-fertilized C57BL/6 eggs form diploid parthenogenetic blastocysts at a much higher frequency than CBA eggs. Eggs from F₁ hybrid females form parthenogenetic blastocysts at an approximately intermediate level between these inbred strains of mice. C57BL/6 PE do not develop to the somite stages. In contrast, CBA PE and F₁ PE develop to various somite stages. Following administration of 5–azaC at 1.0 μmol/L in vitro at the 1- -cell stage, the number of implantations of C57BL/6 PE transferred to pseudopregnant females increased. In contrast, the number of implantations and somite F₁ PE did not significantly change following exposure to 5–azaC. However, administration of 5-azaC at the 1-cell stage stimulates development of somite F₁ PE. Administration of 5-azaC at 0.2 and 1.0 μmol/L in vitro at the blastocyst stage did not change the number of implantations of C57BL/6 PE. However, the number of implantations and somite CBA PE decreased. After injection of 5azaC at 0.24mg/kg in vivo at day 8 of gestation, some F₁ PE developed to 26–35 somites compared with a maximum of 25 somites in controls. The different effects of 5-azaC on the development of PE depend upon the mouse strain used and the stage of development.     

印迹基因修饰使孤雌胚胎干细胞获得四倍体补偿能力

孤雌胚胎干细胞(Parthenogenetic embryonic stem cells,pESCs)的遗传物质全部来源于母源基因组,因缺失父源基因而不具备四倍体补偿的能力。为了使pESCs也具备发育到个体的能力,呈现与受精卵来源ESCs类似的多能性,文中借助CRISPR/Cas9系统对孤雌来源的pESCs中的2个重要母源印迹基因的差异甲基化区域(Differentially methylated region,DMR)进行单等位基因敲除(H19-DMR,IG-DMR),获得双基因敲除的(DKO)pESCs。结果表明,pESCs虽然来源于母源基因组,但是其形态特征、多能干性标记分子的表达水平、体外神经分化能力与受精卵来源的ESCs基本一致。最后,通过基因修饰的DKOpESCs可以通过四倍体补偿获得发育到期的胎儿,表明经过印迹基因修饰的pESCs也具有发育到一个完整个体的多能性。从而为再生医学研究提供了一类具有主要组织相容性复合基因匹配且多能性良好的资源细胞。     

Leukemia Inhibitory Factor (LIF) Improves and Prolongs the Development of Parthenogenetic Mouse Embryos

We studied the effect of the growth factor LIF on the development of parthenogenetic mouse embryos (CBA × C57BL/6)F1. LIF was added to the culture medium at 10, 50, 100, and 250 ng/ml at the morula stage and parthenogenetic embryos were cultured in vitro until the late blastocyst stage and then transplanted in the uterus of pseudopregnant females, which were then sacrificed on day 12 of pregnancy. All the LIF doses used improved the development of parthenogenetic mouse embryos at the preimplantation stages and increased the amount of blastocysts by 15%, on average, as compared to the control. LIF at 50 and 100 ng/ml increased approximately twice the number of embryos that reached the somite stages. Some of them reached the stage of 32–45 somites and had fore and hind limb buds. No such embryos were found in the control. Well formed placenta was observed in 6% of the embryos treated with LIF and the most pronounced effect was recorded at 100 ng/ml. The data we obtained suggest that exogenous LIF can improve pre- and postimplantation development of parthenogenetic mouse embryos due, possibly, to increased survival rate of embryonic stem cells derived from the inner cell mass of blastocysts. LIF improves not only the development of the parthenogenetic embryoper se, but also the formation of its extraembryonic envelopes, which leads to the development of a larger placenta in LIF-treated parthenogenetic embryos, as compared to the control.     

Haploid embryonic stem cells: an ideal tool for mammalian genetic analyses

Identification of the function of all genes in the mammalian genome is critical in understanding basic mechanisms of biology. However, the diploidy of mammalian somatic cells has greatly hindered efforts to elucidate the gene function in numerous biological processes by mutagenesis-based genetic approaches. Recently, mouse haploid embryonic stem (haES) cells have been successfully isolated from parthenogenetic and androgenetic embryos, providing an ideal tool for genetic analyses. In these studies, mouse haES cells have already shown that they could be used in cell-based forward or reverse genetic screenings and in generating gene-targeting via homologous recombination. In particular, haES cells from androgenetic embryos can be employed as novel, renewable form of fertilization agent for yielding live-born mice via injection into oocytes, thus showing the possibility that genetic analysis can be extended from cellular level to organism level.     

Simple and efficient production of mice derived from embryonic stem cells aggregated with tetraploid embryos

Six newly derived hybrid mouse embryonic stem (ES) cell lines and two inbred ES cell lines were tested for their ability to produce completely ES cell-derived mice by aggregation of ES cells with tetraploid embryos. Forty-five ES cell-tetraploid pups were generated from six hybrid ES cell lines and no pups from two inbred ES cell lines. These pups were found to have increased embryonic and placental weights than control mice. Twenty-two pups survived to adulthood and produced normal offsprings, and the other 23 pups died of several reasons including respiratory distress, abdomen ulcer-like symptoms, and foster failure. The 22 adult ES cell-tetraploid mice were completely ES cell-derived as judged by coat color and germline transmission, only two of them was found to have tetraploid component in liver, blood, and lung as analyzed by microsatellite loci. Our data suggested that genetic heterozygosity is a crucial factor for postnatal survival of ES cell-tetraploid mice, and tetraploid embryo aggregation using hybrid ES cells is a simple and efficient procedure for immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps.     

Aggregation of pre-implantation embryos improves establishment of parthenogenetic stem cells and expression of imprinted genes

Parthenogenetic embryonic stem cells (PgES) might advance cell replacement therapies and provide a valuable in vitro model system to study the genomic imprinting. However, the differential potential of PgES cells was limited. It could result from relative low heterology of PgES cells compared with ES cells from fertilization (fES), which produce different expression of most imprinted genes. Here, we described the establishment of PgES cells by aggregating parthenogenetic embryos at the 8-cell stage (aPgES cells), which may increase heterozygy. We found that derivation of aPgES cells in association with an increased number of inner cell mass cells by aggregating was more efficient than that of PgES cells from a single parthenogenetic blastocyst. The aPgES cells have normal karyotype, stain positive for alkaline phosphatase, express high levels of ES cell markers and can differentiate into teratomas composed of the three germ layers. Moreover, compared with PgES cells, the more highly upregulated paternally expressed imprinted genes were observed in aPgES cells, the same change was not shown in aPg blastocysts. This suggested that the aggregation induced effect could modify the expression of paternally expressed imprinted genes. Our studies showed that aPgES cells, the expression of imprinted genes in which more closely resemble fES cells than PgES cells, would contribute to all organs and avoiding immuno-rejection, which may provide invaluable material for regeneration medicine.     

Identification of genes showing altered expression in preimplantation and early postimplantation parthenogenetic embryos

Uniparental embryos have been instrumental in studying imprinting because contributions from the parental genomes can be determined unambiguously. In this study, we set out to identify imprinted genes showing differential expression between parthenogenetic and fertilized embryos during preimplantation and early postimplantation stages of development. We identified three genes-apolipoprotein E, pyruvate kinase-3, and protein phosphatase 1 gamma-that represent excellent candidates for imprinted genes, based on the results of the differential screen, their function in differentiation and the cell cycle, and their location within imprinted chromosomal regions. In addition, two novel genes expressed in trophoblast were identified, 1661 and RA81. These genes, together with four known imprinted genes, H19, Igf2r, Igf2, and Snrpn, showed evidence of expression from both parental alleles in early stage embryos, indicating a role for postfertilization processes in regulating imprinted gene function. © 1995 Wiley-Liss, Inc.     

A fast and efficient method for transiently transfecting ES cells: application to the development of systems for conditional gene expression

Classically, mouse embryonic stem (ES) cells are transfected by electroporation, a method that requires a large number of cells. Here we describe a protocol using a liposome based transfection agent that is a very simple, rapid and cost effective way of transiently transfecting very low numbers of ES cells. We found this method very useful in screening a large number of ES clones when working with inducible expression systems in which at least two elements are required for regulated expression of the gene of interest. After stable transfection of the first component, clones can be easily and rapidly screened for expression of the gene of interest by transiently transfecting the second component of the system using this protocol.     

Erasure of methylation imprinting of Igf2r during mouse primordial germ-cell development

During germ cell differentiation in mice, the genome undergoes specific epigenetic modifications. These include demethylation of imprinted genes and subsequent establishment of parental allele-specific methylation. The mouse Igf2r gene is an imprinted gene that shows maternal-specific expression. Maternal-specific methylation of differentially methylated region 2 (DMR2) of this gene may be necessary for its maternal-specific expression. Before the allele-specific methylation is established, DMR2 is demethylated in both male and female primordial germ cells (PGCs) by 13.5 days post coitum (dpc), indicating that the demethylation of this region occurs earlier in PGC development. The timing of the demethylation has been, however, unknown. In this study, we attempted to determine the timing of methylation erasure of Igf2r DMR2 in developing PGCs, using transgenic mice expressing green fluorescent protein specifically in the germ line. We purified migrating PGCs from the transgenic mice and examined the methylation status of DMR2. The results show that some CpG sites within DMR2 start demethylation at 9.5 dpc in some migrating PGCs, before the cells colonize genital ridges, and the progression of demethylation is rapid after colonization of the genital ridges. To examine whether the gonadal environment is involved in demethylation, we analyzed the methylation of DMR2 after culturing migrating PGCs in the absence of a gonadal environment. These culture experiments support the idea that a gonadal environment is not required for demethylation of the region in at least a fraction of PGCs.     

Combined activin A/LiCl/Noggin treatment improves production of mouse embryonic stem cell-derived definitive endoderm cells

Induction of definitive endoderm (DE) cells is a prerequisite for the whole process of embryonic stem (ES) cells differentiating into hepatic or pancreatic progenitor cells. We have established an efficient method to induce mouse ES cell-derived DE cells in suspension embryonic body (EB) culture. Similar to previous studies, mouse ES cell-derived DE cells, which were defined as Cxcr4(+) c-Kit(+) , Cxcr4(+) E-cadherin(+) cells or Cxcr4(+) PDGFRa(-) cells, could be induced in the serum-free EBs at Day 4 of induction. The activations of Wnt, Nodal, and FGF signaling pathways in differentiating EBs promoted DE cell differentiation, while activation of BMP4 signaling inhibited the process. In the present study, we found that chemical activation of canonical Wnt signaling pathway by LiCl could synergize with Activin A-mediated Nodal signaling pathway to promote induction of DE cells, and inhibition of Bmp4 signaling by Noggin along with Activin A/LiCl further improved the efficiency of DE cell differentiation. The derived DE cells were proved for their capacities to become hepatic progenitor cells or pancreatic progenitor cells. In conclusion, we significantly improved the efficiency of generating mouse ES cell-derived DE cells by combined Activin A/LiCl/Noggin treatment. Our work will be greatly helpful to generate ES cell-derived hepatic cells and ES cell-derived pancreatic cells for future regenerative medicine.     

Database for exchangeable gene trap clones: Pathway and gene ontology analysis of exchangeable gene trap clone mouse lines

Gene trapping in embryonic stem (ES) cells is a proven method for large‐scale random insertional mutagenesis in the mouse genome. We have established an exchangeable gene trap system, in which a reporter gene can be exchanged for any other DNA of interest through Cre/mutant lox‐mediated recombination. We isolated trap clones, analyzed trapped genes, and constructed the database for Exchangeable Gene Trap Clones (EGTC) [ http://egtc.jp ]. The number of registered ES cell lines was 1162 on 31 August 2013. We also established 454 mouse lines from trap ES clones and deposited them in the mouse embryo bank at the Center for Animal Resources and Development, Kumamoto University, Japan. The EGTC database is the most extensive academic resource for gene‐trap mouse lines. Because we used a promoter‐trap strategy, all trapped genes were expressed in ES cells. To understand the general characteristics of the trapped genes in the EGTC library, we used Kyoto Encyclopedia of Genes and Genomes (KEGG) for pathway analysis and found that the EGTC ES clones covered a broad range of pathways. We also used Gene Ontology (GO) classification data provided by Mouse Genome Informatics (MGI) to compare the functional distribution of genes in each GO term between trapped genes in the EGTC mouse lines and total genes annotated in MGI. We found the functional distributions for the trapped genes in the EGTC mouse lines and for the RefSeq genes for the whole mouse genome were similar, indicating that the EGTC mouse lines had trapped a wide range of mouse genes.     

Ectopic expression of Fgf-4 in chimeric mouse embryos induces the expression of early markers of limb development in the lateral ridge

The biological consequences of constitutive fibroblast growth factor-4 (fgf-4) expression were investigated in chimeric embryos prepared between wild-type host embryos and murine ES cells transfected with a construct in which expression of the murine fgf-4 gene was directed by the phosphoglycerate kinase (PGK-1) promoter. The embryos exhibit abnormalities of the limbs and the anterior central nervous system (CNS). The limb phenotype comprised the induction of growth along the lateral ridge between the definitive fore and hind limbs resembling the early phases of limb development. The CNS defects comprised a complete absence, or marked reduction in forebrain and midbrain structures and rudimentary or absent eye development. Constitutive expression of fgf-4 was also accompanied by ectopic expression of the sonic hedgehog (shh) and msx-1 genes in the lateral ridge. These findings indicate that FGF exhibits multiple activities in early development which include the ability to induce the expression of early markers of limb development in the lateral ridge. © 1996 Wiley-Liss, Inc.     

Changes in allele‐specific association of histone modifications at the imprinting control regions during mouse preimplantation development

Allele‐specific association of histone modification is observed at the regulatory region of imprinted genes and has been suggested to work as an epigenetic marker for monoallelic gene expression, along with the allelic CpG methylation of DNA. Although the parent‐origin‐specific epigenetic status in imprinted genes is thought to be established during preimplantation development, little is known about the allelic specificity of histone modifications during this period because of the limited volume of material available for analysis. In this study, we first revealed the allelic enrichment of histone modifications and variant histones at the imprinting control regions (ICRs) of four‐cell to blastocyst stage preimplantation embryos by using carrier chromatin immunoprecipitation and sequence polymorphism analysis of immunoprecipitated DNA. We found relative enrichment of histone H3 lysine 9 dimethylation at the imprinted alleles of ICRs and obtained the results suggesting that histone modifications at ICRs are established during a late preimplantation stage. genesis, 47:611–616, 2009. © 2009 Wiley‐Liss, Inc.     

Sequential gene activation and gene imprinting during early embryo development in maize

Gene imprinting is a widely observed epigenetic phenomenon in maize endosperm; however, whether it also occurs in the maize embryo remains controversial. Here, we used high‐throughput RNA sequencing on laser capture microdissected and manually dissected maize embryos from reciprocal crosses between inbred lines B73 and Mo17 at six time points (3–13 days after pollination, DAP) to analyze allelic gene expression patterns. Co‐expression analysis revealed sequential gene activation during maize embryo development. Gene imprinting was observed in maize embryos, and a greater number of imprinted genes were identified at early embryo stages. Sixty‐four strongly imprinted genes were identified (at the threshold of 9:1) on manually dissected embryos 5–13 DAP (more imprinted genes at 5 DAP). Forty‐one strongly imprinted genes were identified from laser capture microdissected embryos at 3 and 5 DAP (more imprinted genes at 3 DAP). Furthermore, of the 56 genes that were completely imprinted (at the threshold of 99:1), 36 were not previously identified as imprinted genes in endosperm or embryos. In situ hybridization demonstrated that most of the imprinted genes were expressed abundantly in maize embryonic tissue. Our results shed lights on early maize embryo development and provide evidence to support that gene imprinting occurs in maize embryos.     

Genomic Imprinting and Problem of Parthenogenesis in Mammals

Genomic imprinting belongs by its nature to problems of epigenetics, which studies hereditary changes in gene expression not related to defective sequences of DNA nucleotides. Epigenetic mechanisms of control, including genomic imprinting, are involved in many processes of normal and pathological development of humans and animals. Disturbances of genomic imprinting may lead to various consequences, such as formation of developmental anomalies and syndromes in humans, appearance of the large offspring syndrome and increased mortality upon cloning of mammals, and death of parthenogenetic embryos soon after implantation and beginning of organogenesis. The death of diploid parthenogenetic or androgenetic mammalian embryos is determined by the absence of expression of the genes of imprinted loci of the maternal or paternal genome, which leads to significant defects in development of tissues and organs. A review is provided of the studies aimed at search of possible normalization of misbalanced gene activity and modulation of genomic imprinting effects during parthenogenetic development in mammals.__________Translated from Ontogenez, Vol. 36, No. 4, 2005, pp. 300–309.Original Russian Text Copyright © 2005 by Platonov.     

A novel triple fusion reporter system for use in gene trap mutagenesis

Gene trapping is an insertional mutagenesis strategy that allows for simultaneous gene identification and mutation in embryonic stem (ES) cells. Gene trap vectors both disrupt coding sequence and report on the genes' endogenous expression. The most popular gene trap reporter to date combines beta-galactosidase expression with neomycin resistance in a fusion protein known as beta-geo. Here we describe a refinement to this reporter that also incorporates real time fluorescent readouts. We have constructed a series of gene trap vectors incorporating a novel tripartite fusion protein consisting of EGFP, beta-galactosidase, and the neomycin or hygromycin resistance activities. Our results indicate that these triple fusions can function efficiently as reporters of endogenous trapped gene expression and subcellular localization. We show that these fusion proteins constitute versatile gene trap reporters whose activity can be detected in real time by fluorescence and in fixed tissue with a sensitive enzymatic activity.     

Self-renewal and differentiation of mouse embryonic stem cells as measured by Oct4 gene expression: Effects of lif,serum-free medium,retinoic acid,and dbcAMP

Summary In this study we examined the interplay between serum, leukemia inhibitory factor (LIF), retinoic acid, and dibutyrl cyclic adenosine monophosphate (dbcAMP) in affecting IOUD2 embryonic stem cell self-renewal and differentiation as assessed by Oct4 expression, and cell proliferation as measured by total cell protein. Removal of LIF, reduced levels of fetal calf serum (FCS), and addition of retinoic acid all induced embryonic stem cell differentiation as measured by reduced Oct4 expression. Lower levels of retinoic acid (0.1–10 nM) promoted the formation of epithelial-like cells, whereas higher levels (100–10,000 nM) favored differentiation into fibroblastic-like cells. The effects of dbcAMP varied with the presence or absence of FCS and LIF and the concentration of dbcAMP. In FCS-containing media, a low level of dbcAMP (100 μM) increased self-renewal in the absence of LIF, but it had no effect in its presence. In contrast, at higher concentrations (1000 μM dbcAMP), regardless of LIF, differentiation was promoted. A similar effect of dbcAMP was seen in the presence of retinoic acid. In media without FCS but with serum replacement supplements, there was no effect of dbcAMP. This study shows that the Oct4 expression system of IOUD2 cells provides a novel, simple method for quantifying cellular differentiation.