Cloning cells

At present, cell culture work is done mostly by hand by horticulturists in large greenhouses (Plate 3). Chemical engineers could greatly increase the usefulness of this method of plant propagation by developing efficient automated processes for producing plants from cloned cells. 

Gorin, M. B., Yancey, S. B., Cline, J., Revel, J. P. and Horwitz, J. (1984). The major intrinsic protein (MIP) of the bovine lens fiber membrane characterization and structure based on cDNA cloning, Cell, 39, 49-59. 

Holden, P. R. and Yeoman, M. M. 1994. Variation in the growth and biosynthetic activity of cloned cell-cultures of Capsicum frutescens and their response to an exogenously supplied elicitor. Plant Cell Tissue and Organ Culture, 38(1) 31-37. 

DiPaolo, J.A., Takano, K. and Popescu, N.C. (1972). Quantitation of chemically induced neoplastic transformation of Balb/c 3T3 cloned cell Unes, Cancer Res. 32,2686. 

Novick D, Cohen B, Rubinstein M. The human interferon oc/(3 receptor characterization and molecular cloning. Cell 1994 77 391-400. 

SCNT is a cloning strategy, originally reported by Campbell, et al. [33], in which nuclei are isolated from a donor s somatic cells, such as fibroblasts, and are transferred into enucleated oocytes from female donors [33]. By mechanisms yet to be uncovered, the cytoplasm of the oocytes reprograms the chromosomes of the somatic cell nucleus and the cloned cells develop into blastocysts, from which ESCs can be derived [34]. One of the landmarks of SCNT is the potential to generate isogeneic ESCs, carrying a set of chromosomes identical to that of an individual, and therefore unlikely to be rejected after transplantation into that individual [35]. 

The rate of elimination of a drug from the body. Process that involves removing the nucleus from an adult cell, transferring it to an unfertilized oocyte, destroying the genome of the oocyte and allowing the resulting cloned cell to develop. 

A major hurdle that must be overcome before enzymatic treatment can become a reality is the price of the enzyme [24]. Recent research has focused on improving the economics of enzymatic systems through the direct use of plants or plant materials that contain enzymes, the use of crude enzyme extracts, the development of cloned cells that can be stimulated to produce enzymes efficiently in reactor systems, and transgenic manipulation of plants to stimulate enzyme production. Specific examples of some of these efforts are described below. 

The factors which affect cells may be produced by the same cell as responds to them (an autocrine response shown by some tumour cells), or may be produced by a neighbouring cell type (a paracrine response mediated by the interleukins). It is sometimes only possible to distinguish autocrine and paracrine responses by cloning cells from a particular tissue, as a factor produced by one cell type may be processed by a second cell type before reacting with receptors on the first cell type. 

Hepes has been shown to be non-toxic to cells and can be used instead of bicarbonate in which case cells need not be maintained in an atmosphere of 5% C02 in air. However, as bicarbonate is essential for cloning cells (see 7.1) mixtures of Hepes and bicarbonate are used and the cells grown in an atmosphere of 2% C02 in air. Hepes is 4-(2-hydroxyethyl)-l-piperazineethane sulphonic acid. 

Thus McCoy s medium 5A (McCoy et al., 1959) has been used as a standard medium for cloning cells. It is based on BME amino acids and the vitamins from medium 199 (Appendix 1 Table 18). This was modified further to form RPMI (Rosswell Park Memorial Institute)-1629 (Appendix 1 Table 17) for long-term culture of leukaemic myoblasts (Armstrong, 1966). 

Ham s medium F12 has been designed specifically for cloning cells (Ham, 1965) and should be supplemented with 10-30% foetal bovine serum. Great care must be taken to prevent the medium becoming too alkaline by loss of C02. 

Release cells with 0.25% trypsin in PBS or BSS (the divalent ions improve plating efficiency) and disperse by gently pipetting up and down the minimum number of times. Trypsinisation at 4°C for 2-10 min has been recommended especially for cloning cells in low serum. 

Add 0.3 ml complete medium to each well and continue the incubation until the clone covers the surface of the well. The cloned cells may now be harvested by trypsinisation ( 4.2) and transferred to appropriate vessels. 

J. G. Rheinwald and H. Green, Formation of a keratinizing epithelium in culture by a cloned cell line derived from a teratoma, Cell 6, 331 -343 (1975). 

Zuscik MJ, Piascik MT, Perez DM. Cloning, cell-type specificity, and regulatory function of the mouse alb-adrenergic receptor promoter. Mol Pharmacol 1999 56 1288-1297. 

To test and evaluate novel therapeutic compounds inhibiting steps along the efferent portion of the HIV-1 life cycle, several chronically infected, cloned cell models have been developed (1). Because of several unique features, one cell model, OM-10.1, has proven particularly convenient and informative in the evaluation of efferent HIV-1 inhibitors. OM-10.1 cells were cloned following an acute HIV-1LAI infection of HL-60 promyelocytes (2). In a clonal fashion, these cells harbor a single HIV-1 provirus, constitutively express little... 

Kawakami Y, Klein TW, Newton C, Djeu JY, Dennert G, Specter S, Friedman H (1988a) Suppression by cannabinoids of a cloned cell line with natural killer cell activity. Proc Soc Exp Biol Med 187 355-359... 

Pyrococcus woesei[10°1 100 6.0 90 Purified/cloned/cell associated... 

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