THE EFFECT OF MATRIX ATTACHED REGIONS (MAR) AND SPECIALIZED CHROMATINSTRUCTURE (SCS) ON THE EXPRESSION OF GENE CONSTRUCTS IN CULTURED-CELLS AND IN TRANSGENIC MICE

The flanking sequences of several genes have been shown to direct a po sition independent expression of transgenes. Attempts to completely id entify the insulating sequences have failed so far. Some of these sequ ences contain a matrix attached region (MAR) located in the flanking p art of the genes. This article will show that the MARs in cultured cel ls located in the 3' OH region of the human apolipoprotein B100 (Ape B 100) acid within the SV40 genome were unable to stimulate and insulate transgene expression directed by the promoters from a rabbit whey aci dic protein (WAP) gene or from human cytomegalovirus (hCMV) early gene s. In transgenic mice, the MAR from the Apo B100 and SV40 genes did no t enhance the expression of a transgene containing the rabbit whey aci d protein (WAP) promotor, the late gene SV40 intron (VPI intron), the bovine growth hormone (bGH) cDNA and the SV40 late gene terminator. Th is construct was even toxic for embryos. Similarly, the specialized ch romatin structure (SCS) from the Drosophila 87A7 HSP70 gene reduced ch loramphenicol acetyl transferase (CAT) activity when added between a c ytomegalovirus (CMV) enhancer and a Herpes simplex thymidine kinase (T K) gene promoter. This inhibitory action was almost complete when a se cond SCS sequence was added before the CMV enhancer. Sequences from th e firefly luciferase and from the human gene cathepsin D cDNA used as control unexpectedly showed a similar inhibitory effect when added to the CMVTKCAT construct instead of SCS. When added before the CMV enhan cer and after the transcription terminator in the CMVTKCAT construct, the SCS sequence was unable to insulate the integrated gene as seen by the fact that the level of CAT in cell extracts were by no means corr elated with the number of copies in individual clones. From these data , it is concluded that i) a MAR containing the canonical AT rich seque nces does not amplify the expression of all gene constructs ii) AT ric h MAR sequences do not have per se an insulating effect iii) Drosophil a SCS from the 87A7 HSP70 gene has no insulating effect in all gene co nstructs (at least in mammalian cells) iv) and the addition of a DNA f ragment between an enhancer and a promoter in a gene construct cannot be used as a reliable test to evaluate its insulating property.