( stable and does not appear to interfer...)hNT BlueNeurons Express b-Galactosidase at High ,Levels

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.

figure 2

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.

Generating Stably Transfected NT2 Clones

Figure 3

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
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