Embryo cells protocol

Landolph JR (1985) Chemical transformation of C3H/10T1/2 Cl 8 mouse embryo cells historical background, assessment of the transformation assay and evaluation and optimization of the transformation assay protocol. In IARC Scientific Publication No 67, pp 185-198... 

Endo and co-workers at Ehime University, Matsuyama, Japan, have led the development of the most promising eukaryotic cell-free system to date, based on wheat embryos. A significant advance made by this group was the development of pEU expression vectors that have overcome many of the difficulties associated with mRNA synthesis for translation in a eukaryotic system [8]. In addition to extensive optimization of reaction conditions that have seen improvements in protein synthesis rates, Endo and colleagues have improved wheat extract embryo preparation protocols to enhance the stability of these systems to a remarkable extent [9]. When coupled with the dialysis mode of reaction, Endo et al. were able to maintain translational activity in a coupled transcription/ translation wheat embryo reaction for 150 hours, producing 5 mg of enzymatically active protein per mb reaction mixture [10]. This again represents a serious alternative to in vivo methods of large-scale protein production. 

Custer L, Gibson DP, Aardema MJ, Le Boeuf RA. A refined protocol for conducting the low pH 6.7 Syrian hamster embryo (SHE) cell transformation assay. Mutat Res 2000 455 129-39. 

The implementation of animal test protocols in the 1980s has been accompanied by the development of a host of alternative methods to study adverse effects of chemicals on reproductive and developmental parameters. For example, rat whole embryo culture stems from the seventies (16), as does the rat limb bud organ culture (17) and rat limb bud and brain micromass was developed in the eighties (18). An elegant nonvertebrate alternative model used regeneration of polyps of Hydra atUnuata from dissociated cells (19). Animal-free in vitro alternatives include those employing the proliferation of a human embryonic palatal mesenchymal cell line (20), the attachment of a mouse ovarian tumor cell line (21), and the differentiation of a neuroblastoma cell line (22) and a embryonal carcinoma cell line (23). Various overviews of methods have been published over the years (24). The predictability of... 

This chapter contains protocols for high-throughput protein production based on the cell-free system prepared from eukaryote wheat embryos. 

A different approach has been investigated within the EU FP7 ESNATS project (www.esnats.eu FP7 2009-2013) that is the development of alternative tests based only on one single cell model, the embryonic stem cells (ES cells), taking advantage of their ability to mimic early embryo development and to differentiate in all three germ layers under specific differentiation protocols. The aim of the ESNATS project is to develop a novel all-in-one toxicity test platform, preferably based on human ES cells, to overcome interspecies variations. Several differentiation protocols have been optimized and adapted to the purpose of defining robust standard operation... 

Figure 4. Demonstration of the development of a transcriptionally repressive state by nuclear transplantation. In the first experimental protocol, the injected nucleus was transplanted to an S-phase-arrested, one-cell embryo that was then analyzed at a time that corresponded to the mid-late two-cell stage. In the second protocol, the injected nucleus was transplanted to an S-phase-arrested, two-cell embryo that was analyzed at a time that corresponded to the four-cell stage, whereas in the third protocol the injected nucleus was transplanted to a two-cell blastomere that divided. In the last protocol, the injected two-cell blastomere in G2 was transplanted to an S-phase-arrested, one-cell embryo that was then at a time that corresponded to the mid-late two-cell stage. The data are expressed relative to the amount of activity observed for the tk promoter-containing reporter gene in the S-phase-arrested one-cell embryos, and were taken from Henery et al., 1995.

Fig. 1. Examples of fluorescence preparations of Drosophila whole mounts using the protocols is described in this chapter. All confocal images were obtained with a LeicaTCS4D confocal microscope, (a) Confocal optical section of a D. melanogaster embryo whole mount at blastoderm stage double stained with phalloidin—rhodamine (red) and DAPI (blue) to allow simultaneous visualization of nuclei and cortical actin around cell membranes. Anterior is to the left.

One approach to overcome the transplant rejection of human embryonic stem (ES) cells is to derive them by nuclear transfer of the patients own cells. In the absence of an efficient protocol for human somatic cell nuclear transfer (SCNT), several critical steps must be optimized, namely reprogramming time, activation method, and in vitro culture conditions. Reprogramming time was defined as the time between cell fusion and oocyte activation to permit proper embryonic development. A 2 h reprogramming time led to 25% of the recon-stracted embryos developing to blastocysts. In SCNT, in the absence of sperm-mediated activation, an artificial stimulus is needed to initiate embryo development. Addition of 10 pM ionophore for 5 min, and incubation with 2.0 mM 6-dimethyl aminopurine for 4 h, was the most efficient chemical activation protocol for human SCNT embryos. Encouragingly, inefficiencies in embryo culture have been overcome by supplementing... 

The best-characterized cell-free systems derived from crude extracts are the E. coli (prokaryotic) and wheat embryo (eukaryotic) systems. The extract preparation protocols used are central to the productivity of any cell-free system. 

Liquid crystal configuration within the embryo, animal disease models, and diseased human tissues are all cytoplasmic with Maltese-Crosses situated in cells of various tissues, especially in the luminal portion of kidney during diseases and embiyonic blood. Further investigation into liquid crystal involvement in disease through its embryonic mechanisms is expected to generate new diagnostic protocols for liquid crystal related diseases, such as ARMD, Steatohepatitis, Atherosclerotic lesions, and Fabry-Anderson. 

The distribution of graft-derived cells may be analyzed in whole mount or on serial sections of the embryo if the cells are marked by lacZ transgene. For protocols used to visualize the transgenic cells, see Chapter 10. 

Serbedzija GN, Bronner-Fraser M, Fraser SE (1989) A vital dye analysis of timing and pathways of avian neural crest cell migration. Development 106 809-816. Kinder SJ, Tan S-S, Tam PPL (2000). Cell grafting and fate mapping of the early-somite-stage mouse embryo. In Methods in molecular biology, vol.l, pp. 425 37, Developmental Biology Protocols, vol. 135. 

Rat cryopreservation is less well researched. This protocol describes a method for freezing one-cell rat embryos (15). 

Protocols and descriptions detailing the introduction of cells into mice by aggregation of ES cells with preimplantation embryos are provided elsewhere (40). Our own lab protocols can be obtained through the World Wide Web at http //www.mshri.on.ca/develop/nagy/nagy.htm. 

This protocol allows for the staining of E. coli P-galactosidase antibody and NBT-BCIP histochemical detection of alkaline phosphatase. It is designed to detect alkaline phosphatase in primordial germ cells in /acZ-expressing embryos. All staining should be done at room temperature unless otherwise indicated and on 6-8 0,m sections. 

The injected embryos can be examined for the presence and distribution of /acZ-positive cells at a later embryonic age or at some perinatal or postnatal time point. Since it is beyond the scope of this chapter to describe individual perfusion protocols for animals of every developmental age, a protocol that can be followed for adult animals will be given in the greatest detail. 

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