2.1.1. Targeted Cell Lines
One way to make certain that all cell lines produced in an experiment contain the same site of integration of the immortalizing gene is to create cell lines from transgenic animals. Since all cells derived from such an animal will share the same insertion site, at least this source of variability in the biology of cell lines will be brought under more stringent control. The possibility of making cell lines from such transgenic animals was recognized from the earliest studies in which immortalizing genes were inserted into the animal genome. For example, the first studies indicating that expression of oncogenes would disrupt normal development (24) showed that mice expressing SV40 TAg under the control of the metallothionein promotor developed tumors of the choroid plexus and showed that cell lines could be isolated from the transformed tissue. Studies in which the 5' regulatory sequences of the insulin gene were used to control the in vivo expression of SV40 TAg showed that it was possible to use tissue-specific promoters to cause an immortalizing gene to be expressed precisely in the cell type of interest (25). Further studies demonstrated that targeted oncogene expression allowed isolation of cell lines from a variety of tissues in which the oncogene caused neoplastic development in vivo (e.g., 26,27). It has also been found that it was not necessary for full transformation to occur in order to allow generation of cell lines from affected tissue. For example, nonmalignant hepatocyte cell lines (which become increasingly transformed with further growth in culture) can also be isolated from mice in which SV40 TAg expression was driven by the mouse metallothionein promoter sequences (28,29). The use of targeted expression of immortalizing genes to create cell lines of particular interest to neurobiologists has been recently demonstrated by studies of Hammang et al. (30) and Mellon et al. (31).
Despite the success of targeted transgene expression in the generation of neural cell lines, there are two major drawbacks to the use of this technique.
First, in all cases reported thus far, cell lines have been isolated from tumor tissue. Since tumor formation is associated with the acquisition of multiple genetic aberrations (e.g., 26,32-36), it is likely that the resultant cell lines differ from their normal counterparts in a variety of important ways. Indeed, given that the acquisition of cooperating mutations is necessary to allow transformed growth to occur (37-39), it is almost inconceiveable that the resultant cell lines do not have multiple mutations. Furthermore, since expression of a single activated immortalizing gene seems to be sufficient to generate the phenotype of a benign tumor in vivo (35), it is likely that these cells will have undergone a protracted period of abnormal growth in vivo prior to the point of having generated a tumor. The extent to which such cells can be expected to mimic their untransformed ancestors is unknown.
The second drawback associated with the approach of specifically targeting oncogene expression to individual cell types is the necessity of identifying cell-type-specific promoter elements for every cell type of interest. At present, there are relatively few cell types for which appropriate 5'-regulatory elements have been identified. In addition, since many of the cell-type-specific proteins identified thus far are expressed at relatively late stages of differentiation, it may be difficult to target oncogene expression to precursor populations by utilizing the promoter regions for such proteins. In specific regard to the generation of precursor cell lines, it may be more useful to use promoter regions associated with such developmentally regulated genes as the homeobox proteins.
An approach to cell line production that would overcome many of the difficulties described thus far would be to create a strain of transgenic animals in which expression of a conditionally active immortalizing gene was regulated in such a way that the gene was not functionally active in vivo (and thus did not perturb normal development), but could be turned on in cells isolated from any tissue of the body by simple manipulation of tissue-culture conditions. Ideally, the oncogene utilized should be capable of immortalizing as wide a range of cell types as possible, and the regulatory regions used to control expression of this gene should also be capable of functioning in the widest possible range of cell types. Theoretically, transgenic animals of this generic class would allow the direct derivation of cell lines from a wide variety of tissues simply by dissection and growth of cells in an appropriate in vitro milieu. In addition, each cell line generated would possess an identical integration site of the immortalizing gene. Such animals would also have two further distinct advantages. First, the presence of the immortalizing gene in the genome of the transgenic animal would not cause neoplastic development in situ, because functionally active protein would not be expressed in vivo at levels sufficient to perturb normal development. Second, expression of the functionally active immortalizing gene could be turned off again after desired cell lines were obtained in tissue culture, thus allowing study of normal processes of differentiation either in vitro or following cell transplantation in vivo.
The first transgenic animals expressing the ideal characteristics described above are the H-2KbtsA58 transgenic mice (40). These mice harbor the tsA58 TAg gene under the control of the H-2Kb Class I antigen promoter (41-43). The combination of existing studies on the effects of TAg in tissue culture and in transgenic animals indicates clearly that this gene expresses its immortalizing function in a very wide variety of cell types (e.g. 9,27,44-48). In addition, expression from the H-2Kb promoter can be induced or enhanced in almost all cell types by exposure of cells to interferons (48-50). Thus, in cells or tissues (such as brain) that normally express little or no Class I antigens, exposure to interferons activates transcription from this promoter. Moreover, cells that con-stitutively express high levels of Class I antigens can still be induced to express even higher levels of expression by exposure to interferons.
In initial experiments on the H-2KbtsA58 transgenic mice, 34 different transgenic founder animals were created (40). Skin fibroblasts from normal and transgenic animals were placed in culture at 33°C, the permissive temperature for tsA58 TAg, in the presence of y-interferon (IFN-y), which is known to increase expression from the H-2Kb promoter. Skin fibroblasts derived from the transgenic mice all readily yielded proliferating cultures that could be continuously passaged when grown in permissive conditions. In contrast, fibroblasts stopped dividing within a limited number of passages, with a significant decline in even this limited passage number seen in cultures established from older animals.
The conditionality of growth observed in the fibroblasts derived from transgenic animals was correlated with the levels of tsA58 TAg. In all cultures, the level of tsA58 TAg was reduced by increasing temperature and/or by removal of IFN-y. It is interesting that when the most conditional cultures were grown at 33°C in the absence of IFN-y, a condition where these cells did not grow, low levels of TAg could still be detected. This suggests that the level of TAg produced in these conditions was below the threshold needed to support continued rapid cell growth or to allow single-cell cloning.
In general, H-2KbtsA58 transgenic mice appear to undergo normal development. However, these animals do routinely exhibit hyperplastic development of the thymus. This enlargement appears to be owing to hyperplasia rather than to malignant transformation, since thymic histology, T-cell repertoire, and T-cell clonality were all normal in the transgenic animals, and cells derived from enlarged thymuses did not generate tumors in syngeneic recipients. Despite their enlargement in vivo, thymuses of transgenic mice yielded conditionally immortal cultures containing cells of both epithelial and fibroblastic morphologies. Both morphological cell types could be readily cloned, exhibited optimal growth in fully permissive conditions, and did not grow in nonpermissive conditions. Clones that exhibited epithelial-like morphologies expressed cytokeratin and had the ability to rosette T-lymphocytes. Thus, in our initial experiments, we were able to derive conditionally immortal lines of epithelial cells, as well as of fibroblasts, readily from these mice (40).
One founder animal survived to the age of 6 mo and fathered multiple offspring, which harbor a functional transgene. Sibling matings of transgenic offspring generated homozygous animals that have been bred successfully through many generations. As discussed below, this transgenic mouse strain has been a ready source of novel cell lines.
2.1.3. Astrocyte Clones Derived from H-2KbtsA58 Transgenic Mice Express Properties of Glial Scar Tissue
As part of our investigation of the use of H-2KbteA58 transgenic for neuroscience research, we generated clonal astrocyte cell lines that exhibit the properties of glial scar tissue. Such cell lines are of potential importance, since it is believed that glial scarring within the CNS may play an important role in inhibiting regrowth of axons (51-62) and perhaps also in inhibiting repair of extensive breakdown of myelin in the CNS (as occurs in multiple sclerosis; 63).
To generate clonal lines of conditionally immortal astrocytes, cortical astro-cytes were purified from neonatal H-2KbteA58 transgenic mice by simple and well-established methods (64), with the only modificiation being that cultures were grown at 33 °C in the presence of 25 U/mL of IFN-y. To generate clonal cell lines, cultures of purified astrocytes were infected with the BAG retrovirus, which expresses bacterial P-galactosidase and the neomycin resistance gene (65). Subsequent selection in medium containing G418 allowed the ready generation of clonal colonies. Four of these clones were chosen for detailed analysis
All four astrocyte lines chosen for further analysis expressed glial fibrillary acidic protein (GFAP, an astrocyte-specific cytoskeletal protein), although at lower levels than that seen in primary cultures of mouse cortical astrocytes. Characterization of expression of membrane and extracellular matrix molecules indicated that all of these clones expressed a phenotype much like that associated with glial scar tissue (66). All four lines expressed tenascin, laminin, and chondroitin sulfate proteoglycans, all of which are present in some glial populations during development and are particularly expressed in CNS lesions (67-70).
The extent of neurite outgrowth promoted by our astrocyte cell lines was consistent with the possibility that these cells expressed a phenotype functionally similar to glial scar tissue, in that monolayers of the four transgenic astro-
cyte lines were less effective than monolayers of primary cortical astrocytes at promoting outgrowth of cerebellar neurons. The mean total neurite length on all four astrocyte lines was less than 50% of that seen on primary astrocyte monolayers. Despite this dramatic failure to promote growth, the astrocyte cell lines did not cause the cerebellar neurons to clump together or fasciculate, as has been reported for neurons of the CNS growing on fibroblasts or meningeal cells derived from the CNS (64-71). Thus, although the extent of neurite outgrowth was markedly reduced on monolayers of our H-2KbtóA58 astrocyte cell lines, the organization of neuronal cell bodies and processes on the surfaces of these astrocytes suggested that they still expressed glial, rather than nonglial, surface properties.
The four transgenic astrocyte cell lines were also less effective than primary astrocytes at supporting outgrowth of neurites from dorsal root ganglion neurons derived from 7-d postnatal rats. However, there was no inhibition of the growth of dorsal root ganglion neurons derived from ganglia of 18-d-old embryos. In this respect also, the astrocyte cell lines appeared to behave like scar tissue, which is thought to be markedly less inhibitory for the growth of immature neurons as compared with mature neurons (58).
As previously observed for purified cortical astrocytes (72,73), all of the clonal astrocyte cell lines derived from H-2KbtóA58 transgenic mice produced platelet-derived growth factor (PDGF) and promoted the division of O-2A (74) progenitors in vitro. However, the astrocyte cell lines (66) differed markedly from primary astrocytes in their support of O-2A progenitor migration. O-2A progenitors formed small, tight colonies on monolayers of all four astrocyte lines after 7 d of growth, whereas on primary cortical astrocytes, the O-2A progenitor cells were distributed much more evenly over the entire monolayer. The possibility that this failure to migrate represented a functional inhibition of migration was tested by preparing confrontation experiments. O-2A progenitors were either plated onto primary astrocytes and allowed to migrate onto the astrocyte cell lines, or were plated onto the astrocyte cell lines and allowed to migrate onto primary astrocytes. The interface between primary and transgenic astrocyte monolayers could be clearly seen owing to the lower level of GFAP expression in the transgenic astrocytes.
In experiments where O-2A progenitor cells growing on primary astrocytes were challenged with a monolayer of transgenic astrocyte lines, very few progenitors crossed the interface between the primary and transgenic astrocytes. Instead, the progenitor cells appeared to migrate to the astrocyte interface, but no further, frequently aligning their processes along the interface. The apparent failure of O-2A progenitors to cross the interface from primary to trans-
genic astrocytes was not the result of a failure to cross an astrocyte interface per se. In the majority of cases where O-2A progenitors were plated onto transgenic astrocyte monolayers and challenged with monolayers of primary astro-cytes, the progenitors were able to cross the astrocyte interface and migrate considerable distances over the primary astrocyte monolayer.
The availability of clonal cell lines that exhibit properties much like that of glial scar tissue offers us a simple in vitro system for analyzing the biochemical cues that hinder neurite outgrowth from mature neurons and for identifying factors that might inhibit migration of O-2A progenitors in demyelinated lesions.
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