Male and female mice derived from the same embryonic stem cell clone by tetraploid embryo complementation

We have devised a general strategy for producing female mice from 39,X0 embryonic stem (ES) cells derived from male cell lines carrying a targeted mutation of interest. We show that the Y chromosome is lost in 2% of subclones from 40,XY ES cell lines, making the identification of targeted 39,X0 subclones a routine procedure. After gene targeting, male and female mice carrying the mutation can be generated by tetraploid embryo complementation from the 40,XY ES cell line and its 39,X0 derivatives. A single intercross then produces homozygous mutant offspring. Because this strategy avoids outcrossing and therefore segregation of mutant alleles introduced into the ES cells, the time and expense required for production of experimental mutant animals from a targeted ES cell clone are substantially reduced. Our data also indicate that ES cells have inherently unstable karyotypes, but this instability does not interfere with production of adult ES cell tetraploid mice.     

Targeted mutation of the DNA methyltransferase gene results in embryonic lethality.

Gene targeting in embryonic stem (ES) cells has been used to mutate the murine DNA methyltransferase gene. ES cell lines homozygous for the mutation were generated by consecutive targeting of both wild-type alleles; the mutant cells were viable and showed no obvious abnormalities with respect to growth rate or morphology, and had only trace levels of DNA methyltransferase activity. A quantitative end-labeling assay showed that the level of m5C in the DNA of homozygous mutant cells was about one-third that of wild-type cells, and Southern blot analysis after cleavage of the DNA with a methylation-sensitive restriction endonuclease revealed substantial demethylation of endogenous retroviral DNA. The mutation was introduced into the germline of mice and found to cause a recessive lethal phenotype. Homozygous embryos were stunted, delayed in development, and did not survive past mid-gestation. The DNA of homozygous embryos showed a reduction of the level of m5C similar to that of homozygous ES cells. These results indicate that while a 3-fold reduction in levels of genomic m5C has no detectable effect on the viability or proliferation of ES cells in culture, a similar reduction of DNA methylation in embryos causes abnormal development and embryonic lethality.     

胚胎干细胞向造血细胞分化研究

胚胎干（embryonic stem,ES）细胞是来源于囊胚的内细胞团（inner cell mass,ICM）,具有发育的全能性或多能性,能嵌合到早期胚胎,在体内可以参与各种组织发育甚至包括生殖细胞;在体外分化培养条件下,可以顺序分化出各种组织细胞,与体内完整胚胎发育过程相符合,而且可以通过调节ES细胞某些基因的表达而调节其分化。因此,ES细胞是研究哺乳动物早期胚胎发育、细胞分化及其关键基因鉴定的理想模型。另外,胚胎生殖脊（embryonic germ,EG）细胞系也具有同样的生物学特性,它是由早期胚胎的原始生殖脊（primordial germ,PG）细胞建株而来。最近研究显示：ES细胞在体外不但可以分化为所有造血细胞系,而且还可以分化为具有长期增殖能力的造血干细胞。作者就胚胎干细胞向造血细胞和造血干细胞分化及其诱导因子和调控基因的表达作一综述。     

Disruption of the gene encoding the ubiquitin-conjugating enzyme UbcM4 has no effect on proliferation and in vitro differentiation of mouse embryonic stem cells

The ubiquitin-conjugating enzyme UbcM4, which is identical to the human enzyme UbcH7, was previously shown to be essential for normal mouse development. In order to study the possible role of UbcM4 for cell proliferation and in vitro differentiation, we here describe the establishment and characterization of fibroblast and embryonic stem cell lines with partial or complete inactivation of the UbcM4 gene. ES cell lines in which both alleles of the gene were inactivated by targeted mutagenesis showed no differences in growth rates, cell cycle progression and in vitro differentiation when compared to wild-type ES cells. Fibroblast cell lines with a partially inactivated UbcM4 gene were derived from embryos of the previously described A6 mouse mutant, where retrovirus integration has resulted in a recessive lethal mutation. As in the mutant embryos, steady levels of RNA and protein in the cell lines were reduced by about 70%. The mutant cell lines showed no differences in immortalization kinetics, growth rates and cell cycle progression when compared to wild-type fibroblasts. Taken together, our results strongly suggest that UbcM4-mediated ubiquitination and degradation are not necessary for proteins involved in the maintenance and growth of cells.     

Generation of normal lymphocyte populations by Rb-deficient embryonic stem cells

BACKGROUND: Mice homozygous for a loss-of-function mutation of the recombination-activating gene-2 (RAG 2), which is required for the rearrangement of antigen receptor genes, do not produce mature B and T lymphocytes. But chimeric mice that result from injection of normal embryonic stem (ES) cells into blastocysts from RAG2-deficient mice develop normal mature lymphocyte populations, all of which are derived from the injected ES cells; we have called this process RAG2-deficient blastocyst complementation. Using ES cells with homozygous mutations, RAG-2-deficient blastocyst complementation could provide a physiological assay with which to determine the potential role of almost any gene in the development and/or function of lymphocytes. To test the general utility of this system, we have used it to test the differentiation-potential of ES cells that harbor homozygous loss-of function mutations of their retinoblastoma susceptibility (Rb) gene loci. We chose Rb for this analysis because of its widespread function in the control of the cell cycle and cell differentiation, the adverse effect of homozygous germline mutations of Rb on hematopoiesis in fetal liver, and the embryonic lethality that results when the homozygous Rb mutation is introduced into the germline. RESULTS: Homozygous Rb mutant ES cells can develop into phenotypically normal, mature B and T lymphocytes in the RAG-2-deficient background. Strikingly, Rb-deficient B and T cells do not have major defects in either activation or function. CONCLUSION: We have demonstrated the efficacy of the RAG-2-deficient blastocyst complementation system for evaluating the role of critical genes in lymphocyte development. Our results indicate that Rb expression is not intrinsically required for B-cell or T-cell function, despite the normally high levels of Rb expressed in lymphoid cells.     

Establishment and therapeutic use of human embryonic stem cell lines

Embryonic stem (ES) cell lines, which are derived from the inner cell mass of blastocysts, proliferate indefinitely in vitro, retaining their potency to differentiate into various cell types derived from all of the three embryonic germ layers: the ectoderm, mesoderm and endoderm. Establishment of human ES cell lines in 1998 has indicated the great potential of ES cells for applications in medical research and other purposes such as cell transplantation therapy. Careful assessment of safety and effectiveness using proper animal models is required before such therapies can be attempted on human patients. Monkey ES cell lines provide valuable models for such research.     

Heterogeneity in imprinted methylation patterns of pluripotent embryonic germ cells derived from pre-migratory mouse germ cells

Pluripotent stem cells, termed embryonic germ (EG) cells, have been generated from both human and mouse primordial germ cells (PGCs). Like embryonic stem (ES) cells, EG cells have the potential to differentiate into all germ layer derivatives and may also be important for any future clinical applications. The development of PGCs in vivo is accompanied by major epigenetic changes including DNA demethylation and imprint erasure. We have investigated the DNA methylation pattern of several imprinted genes and repetitive elements in mouse EG cell lines before and after differentiation. Analysed cell lines were derived soon after PGC specification, “early”, in comparison with EG cells derived after PGC colonisation of the genital ridge, “late” and embryonic stem (ES) cell lines, derived from the inner cell mass (ICM). Early EG cell lines showed strikingly heterogeneous DNA methylation patterns, in contrast to the uniformity of methylation pattern seen in somatic cells (control), late EG cell and ES cell lines. We also observed that all analysed XX cell lines exhibited less methylation than XY. We suggest that this heterogeneity may reflect the changes in DNA methylation taking place in the germ cell lineage soon after specification.     

Using embryonic stem cells to introduce mutations into the mouse germ line

It is now possible, through the use of a number of experimental technologies, to transfer genetic information into mouse embryos to stably alter the genetic constitution of mice. This experimental approach, namely the generation of so-termed "transgenic" animals, is affording new insights into a wide variety of biological problems. This review focuses on one system for the generation of transgenic mice, which utilizes tissue culture cell lines of embryonic stem cells, termed ES cells. The remarkable property of ES cells is that they retain the potential to reform an embryo; when they are replaced inside a carrier embryo, they resume normal development and contribute to all the tissues of the live-born chimeric animal. Recent experiments, using a repertoire of gene transfer techniques, have shown that ES cells are amenable to a variety of experimental manipulations in tissue culture. Moreover, it has been demonstrated that these genetically altered cells can be transferred into the germ line of chimeric mice, thus allowing the production of unique strains of animals for study. The applications of the ES cell system are reviewed, with particular emphasis on their use for the generation of random insertional mutations using a retrovirally mediated mutagenesis approach. Finally, the use of ES cells in conjunction with the recently described technique of homologous recombination, or "gene targeting," is discussed. This technology allows the generation of animals carrying extremely precise genetic modifications of endogenous genes.     

BPOZ基因纯合缺失ES细胞系的建立及其生长和分化研究

研究BPOZ基因缺失对细胞生长和分化的影响.以高浓度的G418筛选BPOZ基因杂合缺失型ES细胞,PCR鉴定抗高浓度G418细胞克隆基因型;半定量RTPCR分析3种基因型ES细胞BPOZ基因的表达情况,分析3种基因型ES细胞Oct34基因的表达以明确ES细胞分化状态.利用3种基因型ES细胞进行细胞生长曲线和3H胸嘧啶核苷参入实验比较其生长速度和增殖能力.以裸鼠荷瘤实验和类胚体形成实验比较BPOZ基因纯合缺失型ES细胞与野生型ES细胞生长分化能力.结果表明,筛选获得两个BPOZ基因剔除的纯合ES细胞克隆;筛选得到的纯合ES细胞中BPOZ基因表达完全缺失,细胞处未分化状态.与野生型ES细胞相比,BPOZ基因纯合缺失型ES细胞生长受抑,增殖能力减弱.BPOZ基因纯合缺失型ES细胞可分化形成类胚体和具备来自3个不同胚层的细胞和组织的畸胎瘤.BPOZ基因剔除使ES细胞生长受抑,对ES细胞分化发育没有明显影响.     

Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells

Embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of the developing blastocyst that can proliferate extensively in vitro and are capable of adopting all the cell fates in a developing embryo. Clinical interest in the use of ES cells has been stimulated by studies showing that isolated human cells with ES properties from the inner cell mass or developing germ cells can provide a source of somatic precursors. Previous studies have defined in vitro conditions for promoting the development of specific somatic fates, specifically, hematopoietic, mesodermal, and neurectodermal. In this study, we present a method for obtaining dopaminergic (DA) and serotonergic neurons in high yield from mouse ES cells in vitro. Furthermore, we demonstrate that the ES cells can be obtained in unlimited numbers and that these neuron types are generated efficiently. We generated CNS progenitor populations from ES cells, expanded these cells and promoted their differentiation into dopaminergic and serotonergic neurons in the presence of mitogen and specific signaling molecules. The differentiation and maturation of neuronal cells was completed after mitogen withdrawal from the growth medium. This experimental system provides a powerful tool for analyzing the molecular mechanisms controlling the functions of these neurons in vitro and in vivo, and potentially for understanding and treating neurodegenerative and psychiatric diseases.     

Establishment of a human embryonic germ cell line and comparison with mouse and human embryonic stem cells

Human embryonic stem (ES) cells and embryonic germ (EG) cells are pluripotent and are invaluable material for in vitro studies of human embryogenesis and cell therapy. So far, only two groups have reported the establishment of human EG cell lines, whereas at least five human ES cell lines have been established. To see if human EG cell lines can be reproducibly established, we isolated primordial germ cells (PGCs) from gonadal ridges and mesenteries (9 weeks post-fertilization) and cultured them on mouse STO cells. As with mouse ES colonies, the PGC-derived cells have given rise to multilayered colonies without any differentiation over a year of continuous culture. They are karyotypically normal and express high levels of alkaline phosphatase, Oct-4, and several cell-surface markers. Histological and immunocytochemical analysis of embryoid bodies (EBs) formed from floating cultures of the PGC-derived cell colonies revealed ectodermal, endodermal, and mesodermal tissues. When the EBs were cultured in the presence of insulin, transferrin, sodium selenite, and fibronectin for 1 week, markers of primitive neuroectoderm were expressed in cells within the EBs as well as in cells growing out from the EBs. These observations indicate that our PGC-derived cells satisfy the criteria for pluripotent stem cells and hence may be EG cells.     

Progress towards cell-mediated gene transfer in zebrafish.

Although the zebrafish possesses several favourable characteristics that make it an ideal model for genetic studies of vertebrate development, one disadvantage of this model system is the absence of methods for the production of gene knockouts. The authors' laboratory, and others, are working to develop zebrafish pluripotent embryonic stem (ES) and primordial germ cell (PGC) cultures that can be used for cell-mediated gene transfer and the production of knockout mutant lines of fish. Progress has been made in developing short-term cell cultures that possess the ability to contribute to multiple tissues, including the germ line of a host embryo, and transgenic lines of zebrafish have been established using the embryo cell cultures. Work is in progress to extend the length of time that the embryo cells can be maintained in culture without losing their ability to generate germ-line chimeras.     

Production of mouse ES cells homozygous for Cdk5-phosphorylated site mutation in c-Src alleles

c-Src-null mutants have not provided a full understanding of the cellular functions of c-Src, reflecting the functional redundancy among Src family members. c-Src is phosphorylated by cyclin-dependent kinase 1 (Cdk1) and Cdk5 at Ser75 in the unique amino terminal c-Src-specific domain. The specific roles of c-Src may be assessed by establishing mouse embryonic stem (ES) cells homozygous for a point mutation at Ser75. Mammalian homozygous cultured cells with a point mutation, however, have not yet been produced by gene targeting. Here we show an efficient procedure for producing ES cell clones bearing a homozygous Ser75 to Asp mutation in the c-src gene. This procedure was developed by combining two previously reported strategies: our procedure for introducing a point mutation into one allele with no exogenous sequence, and the high-geneticin (G418) selection procedure for introducing a mutation into both alleles. The mutant clones expressed the same levels of c-Src protein and autophosphorylation activity as wild-type cells, but the mutant c-Src was not phosphorylated on Ser75 during mitosis. This procedure is feasible for generating cells homozygous for a subtle mutation in most genes, and is expected to be applicable to other somatic cell lines.     

High-grade transgenic somatic chimeras from chicken embryonic stem cells

Male and female embryonic stem (ES) cell lines were derived from the area pellucidae of Stage X (EG&K) chicken embryos. These ES cell lines were grown in culture for extended periods of time and the majority of the cells retained a diploid karyotype. When reintroduced into Stage VI-X (EG&K) recipient embryos, the cES cells were able to contribute to all somatic tissues. By combining irradiation of the recipient embryo with exposure of the cES cells to the embryonic environment in diapause, a high frequency and extent of chimerism was obtained. High-grade chimeras, indistinguishable from the donor phenotype by feather pigmentation, were produced. A transgene encoding GFP was incorporated into the genome of cES cells under control of the ubiquitous promoter CX and GFP was widely expressed in somatic tissues. Although cES cells made extensive contributions to the somatic tissues, contribution to the germline was not observed.     

从胚胎干细胞到视网膜

视网膜退行性病变影响着全世界数百万人。然而,视网膜是人体再生能力很差的一类组织,成年机体无法自我更新那些病变中丢失的视网膜细胞,导致视网膜退行性病变的不可逆性。因此,恢复患者视觉将依赖于引入外源细胞替代丢失的视网膜神经元。胚胎干细胞（ES细胞）具有无限的自我更新能力和形成机体所有类型细胞的巨大分化潜力。这两个特性使得ES细胞成为细胞替代疗法的理想供体细胞。近年来,人们在探索将ES和诱导多能干细胞（iPS细胞）体外定向诱导分化为视网膜神经元,甚至整个视网膜方面已取得多项进展,并且体外形成的视网膜细胞可以与宿主视网膜整合。在此篇综述中,首先简要概括哺乳动物视网膜的组织结构、发育过程和调控机制,然后,重点阐述近年来科研工作者探索ES/iPS细胞体外诱导分化为视网膜细胞和组织的研究进展。     

Mouse embryonic stem (ES) cell lines established from neuronal cell-derived cloned blastocysts

We have established mouse embryonic stem (ES) cell lines from blastocysts derived by transfer of nuclei of fetal neuronal cells. These neuronal cell-derived embryonic cell lines had properties that characterize them as ES cells, including typical cell markers and alkaline phosphatase activity. Moreover, the cells had a normal karyotype and were pluripotent, as they were capable of differentiating into all three germ layers. Although they were derived from neuronal donor nuclei, the cells no longer expressed neuronal markers; however, they were capable of differentiating into cells with neuronal characteristics. These results suggest that the clone-derived cells have fully acquired an ES cell character. Thus, ES cells can be derived from embryos resulting from nuclear transfer, which results in reprogramming of the genetic information and acquisition of pluripotency. ES cells established from somatic cell-derived blastocysts could be useful not only as research tools for studying reprogramming but also as models for cell-based transplantation therapy.     

New approach for the establishment of mouse early embryonic stem cells and induction of their differentiation

Eleven early embryonic stem (EES) cell lines were established using a new novel method. Two cell stage embryos from the ddY mouse strain were cultured in alpha-MEM supplemented with 10% fetal calf serum (FCS) and embryotrophic factors (ETFs) and allowed to develop to the trilaminal germ disc embryonic stage. Only small round cells (EES cells) were isolated by the colony isolating technique and subsequently cultured in the same medium containing the ETFs and leukemia inhibitory factors (LIF-10 ng/ml). The newly established embryonic stem (ES) cells isolated from inner cell mass of blastocysts differentiated from two cell stage embryo in culture. The EES and ES cell lines were maintained in an undifferentiated state using Ham's F12 medium supplemented with 10% FCS and 1 ng/ml of LIF. The EES cells maintained their normal genetic and morphological features as well as their potential to differentiate into a broad spectrum of cell types as well as their ability to contribute to all cell lineages in chimeric mice. Moreover, these cell lines changed and differentiated into various kinds of cells by removing LIF and by the addition of ETFs to the vitro culture system. All 11 EES cell lines and 3 ES cell lines formed embryoid bodies; however, cell line EES-4 formed tube-like structures which extended, anastomosed with each other, and finally formed networks when the LIF were absent. Primitive germ organ-like structures composed of 3 germ layers were recognized in the cultures following the administration of ETFs. In conclusion, the new method devised by us is a novel, easy and reliable technique for establishing EES cell lines.     

Exclusive transmission of the embryonic stem cell‐derived genome through the mouse germline

Gene targeting in embryonic stem (ES) cells remains best practice for introducing complex mutations into the mouse germline. One aspect in this multistep process that has not been streamlined with regard to the logistics and ethics of mouse breeding is the efficiency of germline transmission: the transmission of the ES cell‐derived genome through the germline of chimeras to their offspring. A method whereby male chimeras transmit exclusively the genome of the injected ES cells to their offspring has been developed. The new technology, referred to as goGermline, entails injecting ES cells into blastocysts produced by superovulated homozygous Tsc22d3 floxed females mated with homozygous ROSA26‐Cre males. This cross produces males that are sterile due to a complete cell‐autonomous defect in spermatogenesis. The resulting male chimeras can be sterile but when fertile, they transmit the ES cell‐derived genome to 100% of their offspring. The method was validated extensively and in two laboratories for gene‐targeted ES clones that were derived from the commonly used parental ES cell lines Bruce4, E14, and JM8A3. The complete elimination of the collateral birth of undesired, non‐ES cell‐derived offspring in goGermline technology fulfills the reduction imperative of the 3R principle of humane experimental technique with animals. genesis 54:326–333, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.     

Self-renewal vs. differentiation of mouse embryonic stem cells

Embryonic stem (ES) cells are typically derived from the inner cell mass of the preimplantation blastocyst and can both self-renew and differentiate into all the cells and tissues of the embryo. Because they are pluripotent, ES cells have been used extensively to analyze gene function in development via gene targeting. The embryonic stem cell is also an unsurpassed starting material to begin to understand a critical, largely inaccessible period of development. If their differentiation could be controlled, they would also be an important source of cells for transplantation to replace cells lost through disease or injury or to replace missing hormones or genes. Traditionally, ES cells have been differentiated in suspension culture as embryoid bodies, named because of their similarity to the early postimplantation-staged embryo. Unlike the pristine organization of the early embryo, differentiation in embryoid bodies appears to be largely unpatterned, although multiple cell types form. It has recently been possible to separate the desired cell types from differentiating ES cells in embryoid bodies by using cell-type-restricted promoters driving expression of either antibiotic resistance genes or fluorophores such as EGFP. In combination with growth factor exposure, highly differentiated cell types have successfully been derived from ES cells. Recent technological advances such as RNA interference to knock down gene expression in ES cells are also producing enriched populations of cells and elucidating gene function in early development.