Creation And Maintenance Of Cell Lines Stably And Heterologously Expressing nAChR Of Defined Subunit Composition

Cell lines naturally expressing some important known nAChR subtypes have not yet been identified. In the case of nAChR subtypes found in the brain, this may be because mature, post-mitotic neurons have lost their susceptibility to neoplastic transformation, which may only occur in proliferating cells. This circumstance may change with advances in neuronal stem cell science and in creation of neuronal cell lines from transgenic animals in which oncogene expression is driven by neuron-specific promoter elements. Nevertheless, development of cell lines stably and het-erologously expressing specific nAChR subtypes has evolved to address this limitation in experimental resources. (Transient expression will not be discussed here.) Generation of heterologous expression systems also offers advantages in that nAChR subtypes can be characterized pharmacologically in isolation, in contrast to studies using many tissues or brain regions or even some clonal cell lines that naturally express more than one nAChR subtype. Creation of cell lines stably and heterolo-gously expressing nAChR has been fraught with failures, not in ability to express transgenes as message, but in ability of messages to be translated into nAChR protein subunits capable of assembling into ligand binding and/or surface-expressed, functional nAChR. Nevertheless, there have been some victories, and some of the details of strategy and technique in stable, heterologous expression of nAChR in mammalian cells are outlined here.

1.3.1 Vector Selection, Transfection, and Subcloning

Initial success in stably and heterologously expressing a7-nAChR18 can be attributed to selection of host cell (see next section) and selection of vector. The pCEP4 (Invitrogen) vector was chosen because it has the cytomegalovirus (CMV) promoter, which gives high constitutive expression of the downstream transgene of interest in human host cell lines. pCEP4 also contains genes coding for the Epstein-Barr virus nuclear antigen (EBNA) and origin of replication (ORI) allowing episomal replication of the vector in human cells. That is, a vector was chosen that would eliminate a requirement for insertion of the transgene into the host genome, where it might become silenced or subject to variable regulation of expression. This helped to ensure high copy number for the transgene and its stable expression under constant selective pressure via coordinate expression of the hygromycin resistance gene, all from the same plasmid.

Subsequently, there were succesful efforts in using integrating or episomal vectors for the stable expression of several different nAChR subunits as ligand binding and/or functional nAChR. (pcDNA3.1 (Invitrogen) is used as an integrating vector in our studies.)19-30 Particularly when the objective is to express nAChR composed as binary (or higher order) complexes of subunits (e.g., binary a402-nAChR, ternary a3a5p4-nAChR), availability of vectors containing different selection markers is useful. (pcDNA3.1 comes in zeocin, hygromycin, or G418 resistance forms.) In one of the two initial reports of stable expression of neuronal nAChR as ligand binding and functional sites,3132 a vector harboring an inducible promoter was used.31 We also have had reasons for expressing nAChR subunits from inducible promoters and have successfully used the Tet-on and Tet-off systems (Clontech) for this purpose.2728 These approaches require stable integration of both the pTet-off or pTet-on plasmids (containing tetracycline-sensitive regulatory elements and also conferring G418 resistance) and the pTRE plasmid (containing the gene of interest downstream from a tetracycline response element).

To introduce nAChR subunit cDNA(s) into host cells, no consensus exists in the literature as to whether electroporation, any of a number of homemade or commercially available lipofection aids, or calcium-phosphate precipitation is superior. The latter two techniques give higher initial survival of cells and are likely to be preferred for transient transfection studies (involving study of cells and nAChR that they make within a week of transfection). Electroporation is a more harsh approach (lower initial viability) that has to be custom-designed to optimize transfection efficiency balanced against cytotoxicity for every type of host cell, but requires an equipment investment rather than recurring costs for transfection reagents. In the laboratory, good and comparable success has been experienced using each of these approaches, but studies have not been done systematically. Typically, a 24- to 48-hour period of recovery from transfection is allowed, thus permitting transgenes of interest and genes conferring antibiotic resistance to be expressed before adding the appropriate antibiotic for positive selection of stable transfectants. Efficiency of transfection is then assessed based on numbers of cells and/or numbers of colonies of cells at different times after selection. Higher success in expression of multiple subunits occurs by isolating a stable mono- or poly-clone expressing one nAChR subunit and then using it as host for introduction of the second type of cDNA. Antibiotic kill curves (plots of untransfected cell survival as a function of time in the presence of selection antibiotic) are acquired for each prospective cell host before transfections are done; kill curves are also required to assess any synergistic effects on cell survival of treatment with more than one antibiotic.

Whether using episomal or integrating vectors, clones surviving selection are isolated while surviving cell densities are still low, either by using "ring cloning," "filter disc cloning," or a "stab-and-grab" technique. For ring cloning, a small amount of vaseline is applied to the bottom edge of 5-mm diameter cloning cylinders, which are placed to isolate targeted cell colonies. Medium within the cloning cylinders is removed, and 0.25% trypsin solution is applied until cells have lifted from the dish surface. Fresh medium is added, and the suspended cells are transferred to single wells in a 24-well tray. For filter disc cloning, medium is aspirated from the plate, and 5-mm diameter filter discs previously soaked in trypsin solution but wiped free of excess solvent are laid over visually identified colonies using flame-sterilized forceps. After 3 to 10 minutes, each filter disc is gently rubbed against the dish surface over the colony. Each disc with attached cells is then transferred to a well in a 24-well tray filled with fresh medium, shaken to dislodge cells, and removed if desired. The stab-and-grab technique simply involves aspiration to moistness of medium from a dish followed by positioning and manipulation of a pipette tip so that it wipes up some cells from a colony. The pipette tip is then dipped into fresh medium in a well in a 24-well tray and agitated to displace attached cells. Irrespective of the method for their physical isolation, cell clones are expanded for further subcloning as needed and screening for function and/or radioligand binding.

1.3.2 Host Cell Types Used and nAChR Subtypes Expressed

Systematic studies are still needed to determine roles that host cell types play in the success of nAChR subunit transfection and heterologous expression. Indeed, such studies might progress to identify critical molecular and cellular bases for regulation of receptor expression, thereby providing insight into phenomena such as chaperone-assisted protein folding, assembly, and trafficking. These studies also may provide a fundamental understanding of how and why some cells differ in their ability to cope with expression of nAChR subtypes differing in kinetics of channel opening and/or ion permeability.

Cognizance of the unpublished difficulties of nAChR expression in common host cell lines such as CHO and COS cells resulted in initial reasoning that transfection and heterologous expression of nAChR should be perfected first using cells that naturally make at least some nAChR subtype. Hence, it was decided to overexpress rat a7 subunits in the SH-SY5Y cell line known to naturally express human a7- and a3p4*-nAChR.1833 Bolstered by success with that approach and in expression of wild-type or mutant, human or chick a7 subunits in the same host cell line,19 attention was then turned to the SH-EP1 human epithelial cell line. This cell line was initially isolated from the same tumor that yielded the SH-SY5Y clone, but early control studies indicated that it was native nAChR-null,33 consistent with observations that the SH-EP1 and SH-SY5Y cell lines evolved divergent morphologies and chemical phenotypes as they were cloned.34 Nevertheless, neuronal and epithelial cells have a shared embryological lineage, and both types of cells exhibit polar morphologies (dendrite-soma/axon compared to apical/basolateral or tissu-lar/lumenal dispositions). Consequently, reasoning was that SH-EP1 epithelial cells might share abilities with neuronal cells to process and properly express complex transmembrane proteins. SH-EP1 cells have now been used successfully to stably and heterologously express functional nAChR composed of a7, a4 plus 02, or a4 plus 04 subunits;19, 27-30 preliminary data indicate successful expression of other homomeric, binary, and even ternary complexes in these epithelial cells.

Success in heterologous expression of nAChR has also been achieved using at least some subclones of HEK-293 human embryonic kidney and other fibroblast cells.20-22, 24-26 31 Data are available showing expression of chick a7-nAChR (mutant form) from the pCEP4 vector in HEK-293 cells as well as in IMR-32 human neuroblastoma cells, PC12 rat pheochromocytoma cells, and CATH.a mouse neuronal cells. Stability of nAChR expression in each of these cells has not been systematically evaluated. Nevertheless, these studies clearly indicate that many cell lines have the capacity to express nAChR heterologously. How they differ from cell lines that lack such a capacity is yet to be determined.

The issue of "stability" of transfection and uniformity in transgene expression across and within passages requires clarification and warrants discussion. Primary cell cultures, which presumably have not become immortalized, are commonly defined as those passaged ten times or less from initial seeding. Continuous cell lines are commonly defined as those cultures surviving over 24 passages (leaving a gray area in nomenclature for cells carried through passages 11 to 23). Clonal cell lines are those derived from a single cell. Flow cytometric studies show that chromosomal makeup and numbers can vary wildly as a tumor-derived cell line becomes established. Continuous cell lines usually arise when a particular chromosomal makeup stabilizes growth and cell phenotype. However, recombinations still can occur to give cells a competitive growth advantage but could result in loss of genes or gene expression of interest. The policy is not to carry any cell line for more than 6 months or about 30 passages (realizing that cells in stocks are no less than 5 passages and may be as far as 20 passages removed from the true first passage). Even established cell lines, such as PC12 or SH-SY5Y, sporadically show loss of nAChR when carried for more than 20 passages from the stocks. Engineered cell lines are considered to be stably transfected if they have been carried for at least 24 passages without evidence of loss of transfected gene expression. However, this does not ensure that all passages from the same frozen stock, or cells carried from the same stock in different laboratories, will express the same quantities of nAChR or will do so through 20 to 30 passages every time. Even if a cell line expresses the same amounts of nAChR subunit message, expression of nAChR as functional or ligand binding sites may still differ because of differences in expression of some other gene critical to synthesis, assembly, and/or maturation of nAChR. Continuous quality assurance is required to monitor transgene expression. However, it is not necessarily catastrophic if a cell line loses expression while being maintained, so long as frozen stocks of low passage cells are maintained in abundance to begin a fresh passage of cells.

While not systematically evaluated, experience indicates that there may be variations in levels of nAChR expression even across cells within the same passage and on the same dish. Heterogeneity in levels of nAChR expression returns quickly, even after cells identified for their high level of expression have been subcloned. It is not clear whether variability in nAChR expression reflects dependence on position in the cell cycle, cell-cell contacts, or other cellular features.

Colleagues practicing electrophysiology have remarked about physical differences in membranes of cells expressing different nAChR subtypes from transfected genes but derived from the same host cell stocks. It should not be surprising, given choice of a single clone from dozens arising from a transfection of 105 to 106 cells, that two clones isolated from a master stock might evolve differently during trans-fection, selection, subcloning, and passage to exhibit different properties while retaining capacity to express nAChR.

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