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. 1999 Feb 16;96(4):1333–1338. doi: 10.1073/pnas.96.4.1333

Figure 4.

Figure 4

Functional complementation of the molybdate-dependent murine cell line L929. L929 cells were transfected with constructs expressing the full-length gephyrin cDNA clone P1 (+geph), the C-terminal truncated gephyrin (+gephΔC) and Cnx1 (+cnx1), and pcDNA3 plasmid without insert (−) as a control. (A Upper) PCR amplification of defined parts of the gephyrin and cnx1 cDNA sequences in different transgenic cell lines. Amplifications were performed by using genomic DNA of two independent pools of transformants (P1, P2), of untransfected L929 cells in the negative control (n.c.), and plasmid DNA of the different expression constructs as positive control (p.c.). (Lower) Analysis of transgene expression by RNase protection assays in cells stably transfected. In the case of the L929 cells stably transfected with the gephyrin expression constructs (geph and gephΔC), an increase of the gephyrin mRNA level in comparison to the untransfected L929 cells can be seen, demonstrating the expression of the transgene. (B) Moco content of transfected L929 cells. The columns show Moco activity obtained from multiple nit-1 complementation assays of NR6P cells (black bar), L929 cells (stippled bars) grown in the presence (+Mo) or absence (−Mo) of 1 mM sodium molybdate and pools of at least 100 stable transformants (gray bars). 100% of wild-type Moco content represent a NADPH–nitrate reductase activity of 51 ± 4.5 nmol/mg protein⋅h in the nit-1 complementation assay. (C) Antisense expression of gephyrin cDNA in murine cells. For gephyrin depletion, cells of the murine wildtype cell line NR6P were transfected with the pcDNA3 gephyrin antisense expression construct and as a negative control with pcDNA3 without insert (indicated by −), and stable transformants were selected. The colums show Moco contents of L929 cells, untransfected NR6P cells, NR6P cells transfected with control vector (NR6P−), and cell clones derived from the transfection with the antisense construct (NR6Pgeph antisense clone A and clone B) grown on normal medium (−Mo, white and gray bars) and on medium supplemented with 1 mM sodium molybdate for 12 h (+Mo, stippled and black bars). Values are expressed in % of the Moco content of the untransformed NR6P wild-type cells (51 ± 4.5 nmol/mg protein⋅h).