Expression of the neurofilament protein NF-H in L cells

We have inserted a Not1-Sal1 fragment of the mouse gene coding for the neurofilament protein NF-H behind the dexamethasone-inducible transcription promoter of MMTV in a vector derived from pMAMneo (Clonetech Labs). This construct, which includes all four exons of the NF-H gene, was amplified and incorporated into liposomes for transfection of L cells. Transfectants were selected in G418-containing medium and cloned. Clones were grown in serum-containing medium and screened for expression of the NF-H mRNA by extraction of total RNA, generation of cDNAs by reverse transcription, and amplification of a 900-base portion of the NF-H cDNA by PCR. Positive clones were detected by the presence of a band of the correct size on agarose gels. This was confirmed by Southern blotting of the gels probed with a 185-base segment of the amplified region. Immunofluorescent analysis of two positive clones, C33 and C34, showed that C33 cells grown in serum-containing medium or in serum-free medium in the presence of dexamethasone have a network of SMI32 (Sternberger/Meyer Inc.--monoclonal antibody against a nonphosphorylated epitope on NF-H)-positive filaments with the same distribution as filaments stained with antibodies to vimentin, while C34 cells do not react with antibodies against neurofilament proteins. Neither clone reacted with antibodies against highly phosphorylated NF-H (SMI31).