Immortalized neurons derived from tumors in transgenic mice

research news issue-specific enhancer el- T ements have been mapped for Immortalized neurons derived from tumors in many genes, but remain largely a mystery for genes that are ex- pressed specifically in neurons. Why should this be the case? There is a practical reason: for cells other than neurons, tissue- specific gene expression has been generally studied in transient ex- pression assays, which depend on the transfection of DNA into large numbers of recipient cells (usually established cell lines). These as- says quantify the activity of a reporter gene, often encoding an enzyme such as bacterial chloramphenicol acetyltransferase (CAT) 1, whose expression is di- rected by flanking DNA sequences of the gene being studied. In the past, neurons have been virtually impossible to study in this way: it is difficult to obtain pure populations of neurons in primary culture, especially in sufficient quantity to make transient expression assays worth performing, and neurons are also relatively intractable to DNA transfection. As there are almost no cell lines that represent specific types of neurons, in vitro studies of gene expression that is specific to the nervous system have been limited to genes that are expressed in tumors such as gliomas, neuro- blastomas and pheochroma- cytomas, which occur with reason- able frequency and from which cell lines can be established (for example, see Refs 2, 3). The alternative to using cell lines is to study tissue-specific gene expression in transgenic mice 4. This approach has advan- tages and disadvantages: the organ- ism itself does all the necessary tissue controls, but the approach is costly and, more importantly, it is slow. The time from initial egg transfer to tail blot is at least six weeks (this can be reduced to three weeks if polymerase chain reaction techniques are used to detect the presence of the trans- gene), and it then takes another seven to eight weeks before the next generation can be tested to determine if germ-line transform- ation has been achieved 5. In con- trast, transfection assays of tran- sient gene expression take only a few days. TINS, Vol. 14, No. 1, 1991 transgenic mice Despite the fact that generating a transgenic mouse is a slow pro- cess, it might nonetheless offer the most direct route from the identifi- cation of a gene that is expressed specifically in a particular type of neuron, to in vitro studies of gene regulation in that cell type. This is because transgenic mice can now be used to generate cell lines from particular types of neurons. In two recent studies 6,7, different groups have used relatively long stretches (approximately 2 kilobases) of 5' flanking DNA sequences from two genes expressed in the nervous system to direct oncogene ex- pression in transgenic mice. In both cases, transgenic mice devel- oped tumors in the appropriate regions of the nervous system and in both cases the investigators have been successful in establish- ing cultured cell lines with the properties of neurons from these tumors (Fig. 1). The use of transgenic mice to target tumorigenesis to a specific cell type was first demonstrated by Hanahan 8. Mice carrying a trans- gene that consisted of the 5' flank- ing sequences of the rat insulin II gene fused to the SV40 early region encoding large and small tumor antigens (Tag) specifically expressed Tag in the beta cells of the endocrine pancreas. Tumors developed with a high frequency (1-2% of the islets progressed to tumors) and retained at least one important property of the beta cells: they expressed insulin, so much so that transgenic mice bear- ing tumors died prematurely of severe hypoglycemia. Baetge and colleagues used the same strategy to target catechol- aminergic cells of the adrenal gland and retina, using 2 kilobases of the 5' flanking region of the hu- man phenylethanolamine N-methyl- transferase (PNMT) gene to direct Tag expression in transgenic mice 9. Targeted Tag expression led to the formation of adrenal and eye tumors. In a more recent study 6, they describe the estab- lishment of a neuronal cell line from an eye tumor of one of these Anne L. Calof transgenic mice. Cultured cells Neuroscience from this tumor (RT-1 cells) have a Program, 5C-5, Tufts neuronal morphology (phase-bright Universi~ Schoo/ of cell bodies and extensive pro- Medicine, 136 A venue, cesses), and express neuronal pro- Harrison Boston, MA 02111, teins such as GAP-43 and all three USA neurofilament subunits. The cells are also immunoreactive with two monoclonal antibodies that stain amacrine cells of the rat retina, VCI.1 and HPC-1 (Refs 10, 11). However, RT-1 cells do not ex- press the enzymes that are charac- teristic of catecholaminergic n e u r - ons: tyrosine hydroxylase (TH), the rate-limiting enzyme in cat- echolamine biosynthesis, and PNMT, the terminal enzyme in this pathway. The failure of RT-1 cells to express TH is perhaps not surprising; there is a population of PNMT-immunoreactive amacrine cells in the retina that lack detect- able TH 12, and these might very well be the cells that were immor- talized by the strategy used by Baetge and colleagues. Lack of PNMT expression is more disturbing, however, since it is presumably the PNMT pro- motet/enhancer that directs the continuous expression of Tag in these cells. The eye tumors them- selves are reported to lack cat- echolaminergic properties, so the failure of RT-1 cells to express PNMT is probably not a result of culture conditions. One possibility is that the cells immortalized in the eye tumors are not the PNMT- expressing amacrine cells of the retina at all. Ectopic expression of chimeric genes in transgenic mice has been observed, and might be the result of novel expression specificities dictated by the juxta- position of cis-acfing regulatory sequences from unrelated genes (see Ref. 13). Alternatively, as the authors suggest, Tag expression might somehow suppress expres- sion of the endogenous PNMT gene. If this is the case, however, suppression must be specific to retinal neurons, since the authors note that adrenal cell lines de- rived from tumors in the same © 1991, Elsevier Science Publishers Ltd, (UK) 0166- 2236/91/$02.00