Non-function of a Moloney murine leukaemia virus regulatory sequence in F9 embryonal carcinoma cells

Moloney murine leukaemia virus (M-MuLV) infection of embryonal carcinoma (EC) cells results in the integration of proviral DNA into the host cell genome, but not in virus production. One suggested explanation for the lack of viral gene expression in EC cells has been methylation of the integrated viral DNA. However, subsequent reports indicated that integration of the M-MuLV DNA occurs soon after infection, but that viral DNA methylation occurs considerably later. Nevertheless, viral gene expression is not observed even at early times. One possible explanation is that certain M-MuLV regulatory sequences do not function in EC cells. We now present evidence which supports this hypothesis.     

Virus-specific effects of interferon in embryonal carcinoma cells

Embryonal carcinoma (EC) cells are susceptible to infection by a variety of viruses, but do not become resistant to infection by Semliki Forest virus or vesicular stomatitis virus (VSV) on treatment with interferon. These observations have led to the conclusion that interferon does not induce an antiviral state in EC cells. We report here, however, that EC cells treated with interferon become resistant to infection by two picornaviruses and two ts mutants of VSV, whereas they remain sensitive to wild-type VSV, Sindbis and influenza virus infectin. These results suggest that a partial antiviral state is induced in EC cells by interferon and that the induced antiviral protein(s) interferes with the replication of specific viruses. A significant common feature of these viruses is their replication through structures containing double-stranded RNA (dsRNA).     

绵羊胚胎干细胞的分离与增殖培养

研究了囊胚(胚龄、处理方法)、传代方法(机械法和酶法)及培养液对绵羊类胚胎干细胞生长的影响,建立了绵羊类胚胎干细胞的分离培养体系.结果表明,实验获得的体外受精胚用于类胚胎干细胞的分离,具有较好的增殖及传代能力;囊胚的质量和胚龄影响绵羊类胚胎干细胞的贴壁及增殖;机械切割(半胚法)处理囊胚,可获得跟免疫法相近的内细胞团贴壁率(半胚法46%,免疫法53.6%);添加了一定浓度的胎牛血清、细胞生长因子、胰岛素及维生素C的DMEM高糖培养液可一定程度地促进类胚胎干细胞的增殖,适于绵羊类胚胎干细胞的培养.     

Interferon enhances 2-5A synthetase in embryonal carcinoma cells

Mouse teratocarcinomas provide a useful model of mammalian differentiation, because the malignant embryonal carcinoma (EC) stem cells of such tumours may produce various differential cell types in vivo or in vitro. Many EC cell lines have now been established and classified on the basis of their ability to differentiate in vivo into cell types characteristically derived from any of the three germ layers. There is convincing evidence that EC cells can neither produce interferon, nor respond to it by becoming resistant to virus, whereas differentiated cells derived from EC lines behave normally in both respects. We investigated the lack of responsiveness of EC cells towards interferon by measuring the levels of two double-stranded RNA-dependent enzyme activities recently shown to be enhanced by interferon. We report here that on treatment with interferon, EC cells show increased 2-5A synthetase levels comparable to those found in differentiated cells, while there is little or no effect on kinase activity in EC cells, in contrast to their differentiated counterparts.     

成纤维细胞和ES细胞在小鼠和兔去核卵母细胞内发育

以昆明白小鼠成纤维细胞和胚胎干（ES）细胞作为供核细胞,以昆明白小鼠和日本大耳白兔的MⅡ期去核卵母细胞作为受体,采用核移植方法,构楚了克隆胚胎．在同种克隆中,以ES细胞为供核细胞的克隆胚胎卵裂率明显低于以成纤维细胞为供核细胞的克隆胚胎卵裂率（24．4％相对于56．9％,P〈0．05）,1．8％的ES细胞克隆胚胎发育到囊胚阶段,而成纤维细胞克隆胚胎没能发育到囊胚阶段;在异种克隆中,以ES细胞为供核细胞的克隆胚胎卵裂率（89．6％）和囊胚发育率（18．8％）明显高于以成纤维细胞为供核细胞的克隆胚胎卵裂率（54．2％）和囊胚发育率（4．2％）．     

Role of ERas in promoting tumour-like properties in mouse embryonic stem cells

Embryonic stem (ES) cells are pluripotent cells derived from early mammalian embryos. Their immortality and rapid growth make them attractive sources for stem cell therapies; however, they produce tumours (teratomas) when transplanted, which could preclude their therapeutic usage. Why ES cells, which lack chromosomal abnormalities, possess tumour-like properties is largely unknown. Here we show that mouse ES cells specifically express a Ras-like gene, which we have named ERas. We show that human HRasp, which is a recognized pseudogene, does not contain reported base substitutions and instead encodes the human orthologue of ERas. This protein contains amino-acid residues identical to those present in active mutants of Ras and causes oncogenic transformation in NIH 3T3 cells. ERas interacts with phosphatidylinositol-3-OH kinase but not with Raf. ERas-null ES cells maintain pluripotency but show significantly reduced growth and tumorigenicity, which are rescued by expression of ERas complementary DNA or by activated phosphatidylinositol-3-OH kinase. We conclude that the transforming oncogene ERas is important in the tumour-like growth properties of ES cells.     

Participation of p53 cellular tumour antigen in transformation of normal embryonic cells

The cellular tumour antigen p53 is found at elevated levels in a wide variety of transformed cells (for reviews see refs 1, 2). Very little is yet known about the precise relationship of p53 to malignant transformation. Although the increase in p53 levels could be a secondary by-product of the transformed state, it is equally possible that p53 is actively involved in altering cellular growth properties, especially as it has been implicated in the regulation of normal cell proliferation. We sought to test whether p53 could behave in a manner similar to known genes in a biological test system, and we demonstrate here that p53 can cooperate with the activated Ha-ras oncogene to transform normal embryonic cells. The resultant foci contain cells of a markedly altered morphology which produce high levels of p53. Cell lines established from such foci elicit tumours in syngeneic animals.     

Targetted correction of a mutant HPRT gene in mouse embryonic stem cells

Two recent developments suggest a route to predetermined alterations in mammalian germlines. These are, first, the characterization of mouse embryonic stem (ES) cells that can still enter the germline after genetic manipulation in culture and second, the demonstration that homologous recombination between a native target chromosomal gene and exogenous DAN can be used in culture to modify specifically the target locus. We here use gene targetting functionally to correct the mutant hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in the ES cell line which has previously been isolated and used to produce an HPRT-deficient mouse. This modification of a chosen gene in pluripotent ES cells demonstrates the feasibility of this route to manipulating mammalian genomes in predetermined ways.     

Long-term proliferation of mouse primordial germ cells in culture.

Primordial germ cells (PGCs) are first identifiable as a population of about eight alkaline phosphatase-positive cells in the 7.0 days postcoitum mouse embryo. During the next 6 days of development they proliferate to give rise to the 25,000 cells that will establish the meiotic population. Steel factor is required for PGC survival both in vivo and in vitro and together with leukaemia inhibitory factor stimulates PGC proliferation in vitro. In feeder-dependent culture, PGCs will proliferate for up to 7 days, but their numbers eventually decline and their proliferative capacity is only a fraction of that seen in vivo. Here we report a further factor that stimulates PGC proliferation in vitro, basic fibroblast growth factor (bFGF). Furthermore, bFGF, in the presence of steel factor and leukaemia inhibitory factor, stimulates long-term proliferation of PGCs, leading to the derivation of large colonies of cells. These embryonic germ cells resemble embryonic stem cells, pluripotent cells derived from preimplantation embryos, or feeder-dependent embryonal carcinoma cells, pluripotent stem cells of PGC-derived tumours (teratomas and teratocarcinomas). To our knowledge, these results provide the first system for long-term culture of PGCs.     

Sequential activation of HOX2 homeobox genes by retinoic acid in human embryonal carcinoma cells

RETINOIC acid had been implicated as a natural morphogen in chicken and frog embryogenesis, and is presumed to act through the gene regulatory activity of a family of nuclear receptors. Homeobox genes, which specify positional information in Drosophila and possibly in vertebrate embryogenesis, are among the candidate responsive genes. We previously reported that retinoic acid specifically induces human homeobox gene (HOX) expression in the embryonal carcinoma cell line NT2/D1. We now show that the nine genes of the HOX2 cluster are differentially activated in NT2/D1 cells exposed to retinoic acid concentrations ranging from 10(-8) to 10(-5) M. Genes located in the 3' half of the cluster are induced at peak levels by 10(-8) M retinoic acid, whereas a concentration of 10(-6) to 10(-5) M is required to fully activate 5' genes. At both high and low retinoic acid concentrations, HOX2 genes are sequentially activated in embryonal carcinoma cells in the 3' to 5' direction.