hNT BlueNeurons are ideal for use in transplantation studies
Michael McGrogan * Guadalupe Gonzalez * Waldemar Lernhardt
Layton BioScience, Inc. Gilroy, California
Stratagene's hNT BlueNeurons express beta-galactosidase at high levels and are derived from a clone of the NT2 teratocarcinoma stably transfected with a lacZ gene expression vector. Because they are easily distinguished from surrounding tissue by in situ staining for beta-galactosidase, hNT BlueNeurons are ideal for use in transplantation studies.
The NT2 cell line, derived from a human teratocarcinoma, exhibits properties indicative of a committed neuronal precursor stage. NT2 cells can be induced by retinoic acid (RA) to differentiate in vitro into postmitotic central nervous system (CNS) neurons, hNT neurons* (figure 1). In the course of RAinduced differentiation, NT2 cells lose neuroepithelial markers and telomerase activity, acquire many neuronal markers, begin to express glutamate receptor channels, change the metabolism of amyloid precursor proteins and become permissive for infection by the human immunodeficiency virus (HIV).
An increasing number of reports describe transplantation of hNT neurons into rodent brains.15 hNT neurons successfully integrate into surrounding tissue and establish synapses. Although hNT neurons can be identified in transplanted tissue by staining with antibodies specific for human markers, an additional method of identification would allow immunostaining for multiple markers. The lacZ gene was chosen as a marker to be introduced into hNT neurons because it is very stable and does not appear to interfere with cell metabolism. In addition, staining protocols for beta-galactosidase are easy to perform.
Previous research has shown that postmitotic hNT neurons can be successfully transfected 6,7 and expression of the transgene can be followed for several weeks. Nonetheless, because the transgene is most likely not integrated, such a transfection can not be considered stable. In order to achieve stable transfection, it was necessary to transfect NT2 precursor cells, select stably transfected clones and differentiate the clones into hNT neurons.
The expression vector used for transfecting NT2 precursor cells with the lacZ gene had to fulfill several crucial criteria: integration, not interfering with the differentiation process during the 6 week RA treatment and expressing the transgene in postmitotic neurons at a high level. The best results were achieved in transfections using the vector pSNRSV (figure 2), which contains a neomycinresistance gene controlled by the SV40 early promoter and a lacZ gene controlled by the Rous sarcoma virus (RSV) promoter.
NT2 cells were transfected with the pSNRSV expression vector as prescribed by the manual for Stratagene's Transfection MBS Mammalian Transfection Kit. Forty-eight hours posttransfection, cells were split 1:40 and after an additional 24 hours, G418 was added to the medium. Following 3 weeks of selection in neomycincontaining medium, clones were picked, grown in 6-well plates and analyzed for beta-galactosidase expression using Stratagene's Beta -Galactosidase Assay Kit. Figure 3 shows beta-galactosidase activity in four representative clones, and their potential for differentiation into neurons was evaluated. Clones were selected according to their ability to proliferate at a sufficient rate and their ability to stably express beta-galactosidase.
Clones B6, C4 and C6 were chosen to be induced with RA. Cells were cultured in medium containing 10 mg/ml RA for 6 weeks (see Figure 3), and the resulting neurons were harvested and assayed for beta-galactosidase activity as described. As seen in figure 4, clone B6 appeared to have the least activity (about 2 fold above background) but yielded the largest number of neurons among the three clones that were chosen for differentiation.
Figure 5A & 5B
Figure 5A shows in situ staining of control replate 2 cells (after 6 weeks of RA induction, figure 1) that had not been transfected. Neurons were stained with Xgal using Stratagene's In Situ beta-galactosidase Staining Kit. Upon staining, the neurons appear as clusters of small cells that sit on top of accessory cells; a slight blue tinge of background beta-galactosidase activity is apparent. Figure 5B shows transfected B6 clone replate 2 cells. These neurons appear as dark blue small cells in clusters that are easily distinguished from the accessory cell layer. Although both cell types arise from the same transfected precursor cells, the staining of the neurons results in a darker blue color than the staining of the accessory cells. Furthermore, the staining pattern of neurons is uniform and homogeneous.
Figure 6A &6B
Figure 6A shows transfected clone B6 replate 3 neurons after 6 weeks of RA induction, treatment with mitotic inhibitors and purification (figure 1). This photomicrograph shows two clusters of neurons that are stained intensely blue. Again, the blue staining appears uniform and homogeneous. This is also apparent in Figure 6B, a larger magnification of the cluster on the upper left of Figure 6A. These results indicate that the beta-galactosidase gene is expressed at a sufficient level to yield intense blue staining of the transfected neurons.
hNT BlueNeurons have high levels of beta-galactosidase activity and stain dark blue in standard assays. This feature is especially useful for transplantation studies of CNS tissues, where the ability to stain for beta-galactosidase activity allows hNT BlueNeurons to be easily distinguished from surrounding cells or tissues.
Trojanowski, J.Q., et al. (1993) Exp. Neurol. 122: 283294.
Mantione, J.R., et al. (1995) Brain Res. 671: 333337.
Kleppner, S.R., et al. (1995) J. Comp. Neurol. 357: 618632.
Miyazano, M., Lee, V.M.Y., and Trojanowski, J.Q. (1995) Lab. Investigat. 73: 111.
Santoshi, K., et al. (1995) Lab. Investigat. 73: 636648.
Lee, V.M., McGrogan, M.P., Lernhardt, W., and Huvar, A. (1994) Strategies 7: 2831.
McGrogan, M.P., Simonson, C., and Lernhardt, W. (1995) Strategies 8: 5657.