A NOVEL GFPNEO VECTOR DESIGNED FOR THE ISOLATION AND ANALYSIS OF ENHANCER ELEMENTS IN TRANSFECTED MAMMALIAN-CELLS

We have designed a new approach to the direct cloning and rapid analys is of mammalian enhancer elements by fusing green fluorescent protein and neomycinphosphotransferase under the control of a thymidine kinase minimal promoter. DNA fragments containing known or potential enhance r elements can be inserted into a polylinker upstream of GFPneo and re -isolated from stably transfected cell lines by a direct transgene-spe cific polymerase chain reaction (PCR), for further analysis. C2C12 mus cle cells were transfected with four vectors containing the GFPneo fus ion gene regulated by the cytomegalovirus promoter, the myoD distal co re enhancer and myoblast- and myotube-specific enhancers from the desm in gene. GFPneo shows robust epifluorescence by microscopy and flow cy tometry and retains sufficient neo activity to permit selection of G41 8-resistant clones. The fluorescence signal pattern of GFPneo expresse d under the control of the desmin enhancers mirrors their transcriptio nal profile during myogenic differentiation. This finding demonstrates the value of GFPneo as a tool to analyse differentiation stage-specif ic regulatory DNA elements in stably transfected mammalian cell lines. We were able to re-isolate the myoD enhancer mediating GFPneo express ion from a stably transfected C2C12 clone by a transgene-specific PCR reaction, demonstrating the feasibility of using this new vector syste m for the isolation of regulatory sequences. (C) 1998 Elsevier Science B.V. All rights reserved.