A number of general principles emerge from our experiments to date. One point of central importance is that we think it most appropriate to utilize heterozygotes for the generation of cell lines in most (and possibly all) circumstances. The successful derivation of cell lines from heterozygotes offers a number of practical advantages, not least of which is the fact that it is not necessary to maintain a homozygous breeding colony if one's intention is to use only animals from a limited series of dissections. Instead, male stud homozygotes can be mated with normal females, with cells of interest being derived from the heterozygous F1 animals. It is easy to obtain a steady stream of heterozygous animals with a few homozygous stud males and a harem of normal females.
It does seem reasonable to consider whether cell types that have proven difficult to grow from heterozygous animals may be more readily obtained from homozygotes, owing to the higher level of TAg expression achieved per unit of interferon applied. Thus far, however, our tendency has been to expend more effort on determining appropriate growth conditions for each cell type of interest. Part of the reason for choosing this approach is that we are primarily interested in using cells derived from H-2KbtsA58 transgenic animals as a tool for exploring the cellular biology of normal cells, and we prefer to express the minimal amount of TAg needed to achieve immortalization.
A second useful observation we have made is that one can often (although not invariably) turn off tsTAg function by shifting temperature only up to 37°C. This reduces the need for extra incubators from 2 (33°C and 39.5°C) to 1 (33°C), with regular 37°C incubators being used to turn off TAg activity. For a large number of laboratories, this second advantage is particularly useful. More importantly, the lack of an absolute requirement for shift to 39.5°C means that transplantation in vivo has a higher chance of turning off functional expression of tsTAg, thus allowing cells to differentiate normally in situ.
A further point to note is that we have not seen any great differences in effectiveness of IFN-y from different manufacturers. Moreover, it should be possible to substitute any other inducer of Class I antigen expression (e.g., other interferons) to promote expression from the H-2Kb promoter, in the event that IFN-y is specifically detrimental to the growth of the cells of interest (a problem we have not encountered).
Although it appears likely that the H-2KbtsA58 transgenic mice and their generic relatives will greatly facilitate cell line production, it is important to note that the probability of successfully generating a cell line of interest is still enhanced by knowledge about the biological properties of the cell of interest. Such a result is expected from previous studies on cell lines produced by retroviral infection of cultured cells. For example, even though expression of TAg in Schwann cells simplifies the growth factor requirements of these cells, promotion of cell division in the immortalized Schwann cells still requires the presence of two out of three of the mitogenic stimuli used in the growth of primary Schwann cells (4). To date, our studies with cells derived from the H-2KbtsA58 transgenic mice have indicated that generation of novel cell lines is indeed a straightforward matter for many tissues. However, when attempting to grow cells for which little is known about the control of division in the cell type of interest, it has also become clear that experimentation is required to determine suitable conditions for optimizing cell line production.
In addition to the cells discussed thus far, it also has been possible to isolate cell lines from an increasing variety of tissue. Primitive kidney cells have been isolated (84) and characterized for their chloride conductance properties (85). Hippocampal progenitor cells have also been described with the ability to generate neurons (86,87). Isolation of colonic and intestinal epithelial cells (88) has been followed by the derivation of cell lines from a cross-breed of H-2KbtsA58 transgenics with Min mice, yielding colonic epithelial cells with mutations in the APC gene (89). Vascular smooth muscle lines (90) and biliary epithelial cell lines (91) also have been recently described. In addition, it is possible to target expression of tsA58 to specific lineages, thus yielding very specific cells lines (e.g., 92). Thus, the H-2KbtsA58 transgenic mice appear to be useful in the manner we originally envisaged. Moreover, the success of our transplantation studies with myoblast lines derived from H-2KbtsA58 mice suggests that these animals might provide a generally useful source of cell lines suitable for application in studies in which transplantation and incorporation into normal tissue are a desired goal.
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