Fate of embryonal carcinoma cells injected into postimplantation mouse embryos

Embryonal carcinoma (EC) cells, stem cells of teratocarcinoma, represent an excellent model to study the developmental mechanisms that, inappropriately reactivated, can drive tumorigenesis. EC cells are very aggressive, and grow rapidly when injected into adult syngeneic mice. However, when injected into blastocysts, they revert to normality, giving rise to chimeric animals. In order to study the ability of postimplantation embryonic environment to "normalize" tumorigenic cells, and to study their homing, we transplanted F9, Nulli-SCC1, and P19 EC cells into 8 to 15-day allogenic CD1 mouse embryos, into allogenic CD1 newborns, and into syngeneic adult mice, and evaluated tumor formation, spreading, and homing. We found that, although at all embryonic stages successful transplantation occurred, the chances of developing tumors after birth increased with the time of injection of EC cells into the embryo. In addition, using enhanced green fluorescent protein-expressing F9 cells, we demonstrated that the cells not giving rise to tumors remained latent and could be tracked down in tissues during adulthood. Our data indicate that the embryonic environment retains a certain ability to "normalize" tumor cells also during post-implantation development. This could occur through yet unknown epigenetic signals triggering EC cells' differentiation.     

Injection of murine embryonal carcinoma cells and embryo-derived pluripotential cells into mouse blastocysts

Two pluripotential mouse cell lines, the OTT 6050-derived cell line TCE and the embryo-derived stem cell line BLC-1, were injected into blastocysts to analyze their developmental potential. The contribution of TCE cells to the embryo was found to be limited and sporadic. There was no indication of a preferential colonization of extraembryonal membranes or developmentally related tissues in adult chimeras. BLC-1 cells failed to colonize the embryo. This indicates that a normal karyotype, pluripotency, and cell surface markers which are shared by cells of early embryos are not necessarily sufficient markers for their ability to participate in embryogenesis.     

Fate of DNA injected into early Drosophila embryos

Plasmid DNA injected into early Drosophila embryos becomes enclosed in nuclei or nucleus-like structures where it remains at least throughout embryonic development. The fraction remaining in the cytoplasm is gradually degraded. The nuclear fraction is converted to a high-molecular-weight form consisting largely but not exclusively of tandem oligomers. Some of it, however, can occasionally become integrated in the genome. Extensive DNA replication takes place but few, if any, molecules are able to initiate a second round of replication.     

Fate of tetraploid cells in 4n<-->2n chimeric mouse blastocysts

Previous studies have shown that tetraploid (4n) cells rarely contribute to the derivatives of the epiblast lineage of mid-gestation 4n<-->2n mouse chimeras. The aim of the present study was to determine when and how 4n cells were excluded from the epiblast lineage of such chimeras. The contributions of GFP-positive cells to different tissues of 4n<-->2n chimeric blastocysts labelled with tauGFP were analysed at E3.5 and E4.5 using confocal microscopy. More advanced E5.5 and E7.5 chimeric blastocysts were analysed after a period of diapause to allow further growth without implantation. Tetraploid cells were not initially excluded from the epiblast in 4n<-->2n chimeric blastocysts and they contributed to all four blastocyst tissues at all of the blastocyst stages examined. Four steps affected the allocation and fate of 4n cells in chimeras, resulting in their exclusion from the epiblast lineage by mid-gestation. (1) Fewer 4n cells were allocated to the inner cell mass than trophectoderm. (2) The blastocyst cavity tended to form among the 4n cells, causing more 4n cells to be allocated to the hypoblast and mural trophectoderm than the epiblast and polar trophectoderm, respectively. (3) 4n cells were depleted from the hypoblast and mural trophectoderm, where initially they were relatively enriched. (4) After implantation 4n cells must be lost preferentially from the epiblast lineage. Relevance of these results to the aetiology of human confined placental mosaicism and possible implications for the interpretation of mouse tetraploid complementation studies of the site of gene action are discussed.     

Fate and structure of DNA microinjected into mouse TK L cells

Co-microinjection of single linearized molecules of plasmids containing the human β-globin gene (pRK1) and the herpes simplex virus (HSV) type I thymidine kinase gene (pX1) into the mouse TK^? L cell nucleus results in covalent linkage between these (or derived) molecules within the nucleus as revealed by Southern blotting, plasmid rescue, and recovery of plasmid-derived DNA from a Charon 4A phage library of cellular DNA. The microinjected DNA is predominantly found as high molecular weight DNA as determined by Hirt fractionation. Southern blotting data and recombinants from the Charon 4A library suggest that the plasmid DNA is in the form of a head-to-tail linear concatamer of up to 80 copies. Passage of these microinjected cells in selective medium (HAT) results in coordinate amplification of both plasmids, which are maintained in an approx. 3:1 molar ratio of pRK1 to pX1-derived molecules. Hybridization in situ shows the DNA to be integrated on a translocation chromosome, t(4;4). Integration does not appear to be site-specific, since plasmid DNA from another microinjected cell line, C2B, appears on a different translocation chromosome, t(8?;14). Plasmid rescue experiments confirm a previous finding that passage of pBR322 DNA through eukaryotic cells may result in deletions of normally stable plasmid DNA upon subsequent transformation of E. coli. These deletions appear to occur in the bacteria, and originate in a 128 bp region between the Sal I and Hae II sites of pBR322.     

Fate of exogenous recombinant plasmids introduced into mouse and human cells.

We have constructed a number of plasmids selectable in both E. coli and mouse or human cells. Human DNA sequences were inserted and the recombinant plasmids were used to transfect either mouse or human cells by the Ca-phosphate precipitation technique. We have observed that: (i) competent cells uptake large amounts of plasmid DNA; (ii) input plasmids persist in transformed mammalian cells as free unreplicating circular molecules for up to 20 generations; such persistence does not depend on the presence of selective markers; (iii) plasmids incorporated into mouse L-cells undergo widespread rearrangements (in the absence of replication) entailing mostly deletions of both human and bacterial sequences which yield smaller products; the latter appear to be more stable in a subsequent transformation cycle. Surprisingly such rearrangements are almost totally absent in transformed human KB-cells. This property of human KB-cells may prove useful for the development of a vector apt at cloning and expressing human DNA sequences. Unlike what has been observed in yeast, no "autonomously replicating sequence" can be detected in mammalian cells by randomly cloning human DNA sequences into a selectable plasmid and screening for an increased transformation efficiency.     

On the fate of primordial germ cells injected into early mouse embryos

Primordial germ cells (PGCs) are the founder cells of the germline. Via gametogenesis and fertilisation this lineage generates a new embryo in the next generation. PGCs are also the cell of origin of multilineage teratocarcinomas. In vitro, mouse PGCs can give rise to embryonic germ (EG) cells – pluripotent stem cells that can contribute to primary chimaeras when introduced into pre-implantation embryos. Thus, PGCs can give rise to pluripotent cells in the course of the developmental cycle, during teratocarcinogenesis and by in vitro culture. However, there is no evidence that PGCs can differentiate directly into somatic cell types. Furthermore, it is generally assumed that PGCs do not contribute to chimaeras following injection into the early mouse embryo. However, these data have never been formally published. Here, we present the primary data from the original PGC-injection experiments performed 40 years ago, alongside results from more recent studies in three separate laboratories. These results have informed and influenced current models of the relationship between pluripotency and the germline cycle. Current technologies allow further experiments to confirm and expand upon these findings and allow definitive conclusions as to the developmental potency of PGCs.     

The fate of embryonal-carcinoma cells in mouse blastocysts

Double-labeled embryonal-carcinoma (ECa) cells were injected into blastocysts or incorporated into blastocysts by aggregation, and their fate after various periods of time in culture was investigated. ECa-247 cells labeled with fluorescent microscopheres were easily identified in whole blastocysts. These blastocysts were embedded in plastic, serially sectioned, and prepared for autoradiography. The 3H-thymidine label on the embryonal-carcinoma cells allowed precise localization of the cancer-derived cells. ECa-247 cells preferentially localized in the mural trophectoderm, with a few being seen in primitive endoderm and, even more rarely, in the inner cell mass. Selected autoradiograms were re-embedded and thin sectioned for transmission electron microscopy. The cancer-derived cells were found to have differentiated in accordance with their localization.     

Generation of rat-mouse chimeras by introducing single cells of rat inner cell masses into mouse blastocysts

正In the field of developmental biology and regenerative medicine,mammalian interspecific chimeras have been proved very useful for investigating early embryonic development and the immune system establishment,and extended to a promising potential for human organ generation(Rossant et al.,1982).In the early     

Differential effects of p-nitrophenyl-D-xylosides on mouse blastocysts and uterine epithelial cells

A number of studies have implicated glycoconjugates in cell recognition events associated with implantation of mammalian blastocysts into the uterus. We have found that p-nitrophenyl-D-xylosides inhibit mouse embryo attachment and outgrowth on monolayers of uterine epithelial cells when cocultured in vitro. Inhibition of attachment and trophoblast formation by alpha- and beta-xylosides was observed in embryos cultured on tissue culture plastic in serum containing medium or on monolayers of epithelial cells. The biochemical basis for this inhibition has been investigated. Consistent with their accepted mode of action, beta- but not alpha-D-xylosides greatly stimulated glycosaminoglycan chain production by uterine epithelial cells and likewise reduced proteoglycan assembly. In contrast, both alpha- and beta-anomers selectively inhibited embryo attachment and outgrowth without stimulating glycosaminoglycan chain production by embryos. The inhibitory effect of the xylosides on embryos was reversible and did not require concentrations that reduced the rate of protein synthesis. Both alpha- and beta-D-xylosides inhibited the synthesis of proteoglycans including heparan sulfate as well as certain other glycoconjugates by embryos. Collectively, these data indicate that proper assembly of glycoconjugates, including proteoglycans, is required for implantation-related processes, although the inhibition of embryo outgrowth by xylosides may be by an as yet uncharacterized mechanism.     

Somatic cell hybrids between Friend erythroleukemia cells and mouse hepatoma cells.

Somatic cell hybrids between hepatoma and Friend erythroleukemia parental cells were studied for the expression of liver-specific and erythroid properties. Several independent clones were isolated using HAT selection and were shown to be true hybrids by isozyme and chromosome analysis. All displayed a complete extinction of hemoglobin and globin mRNA production, but a retention of albumin and transferrin secretion. The data suggest that erythroid differntiation is being actively inhibited by the hepatoma genome. Possible mechanisms that might explain these results are discussed in the light of current hypotheses regarding the mechanism of cell differentiation.     

A technique for quantifying the amount of macromolecule injected into cells of the early mouse embryo

A simple technique for the quantification of the volume injected into an individual mouse egg or blastomere is described. The method does not seem to have deleterious effects on development, allows the progeny of the injected cell to be identified and permits the measurement of the injected volume to be made up to 48 h after the time of injection.