Estudo de elementos moduladores da expressão gênica em diferentes linhagens de células de mamífero em cultura

AUTOR(ES)
DATA DE PUBLICAÇÃO

2008

RESUMO

It had been constructed plasmids with the promoter of CMV and the luciferase reporter gene comprising the following features: without the CMV intron A (IA) (pGLCMV I-), with the full IA (pGLCMV) or the deleted versions of it (pGLCMV200, pGLCMV400 and pGLCMV600), and Z-DNA inducer sequences upstream the promoter. These constructions had been transiently transfected in CHOK1, COS-7, HepG2 and HEK-293 cells. The whole intron A resulted in a 100 to 1000- fold increase in the activity of luciferase compared to the intronless version, depending on the cell line tested. The pGLCMV200 construction had also enhanced in 3 to 6-fold the luciferase activity compared to the wild-type IA. Following the results obtained with the construction above, the IA deleted in 600 pb had improved the luciferase activity no more than 4 times, depending on the cell line used. The pGLCMV400 construction, instead, resulted in a reduction of gene expression to levels similar to the intronless construction. Taken together, these data suggest that there are a binding region for an inhibitory transcription factor comprising the 200 pb fragment. The data of the activity of luciferase had been corroborated with experiments of RT-qPCR, which had correlated the increases of luciferase activity for the different constructions above with the increase of the expression of the gene of luciferase. To verify the effect of splicing in the constructions with the deleted IA, a RT-PCR experiment was carried through showing that the removal of the donor splicing site might have a relation with the incorrect processing of exons 1 and 2 of the CMV IE gene. Amongst the Z-DNAinducer sequences cloned upstream the CMV promoter, the Z3 sequence resulted in a 2- fold increase of the transient luciferase activity when compared to the control-sequence Z5. As a result, it had been developed stable clones of CHO-K1 cells with this sequence in order to verify if the sequence in question exerts its activity by means of the formation of Z-DNA in the adjacent region to the promoter. In these stable clones, it had been transiently transfected the pMACIA scFvZ22NLS plasmid, that codes for an antibody fragment anti-Z-DNA. The values of the luciferase activity had increased 3 times when this element in trans it was added to the expression system, indicating that this sequence really forms Z-DNA and that the effect observed occurs in transcriptional levels. Therefore, in this work it was shown that the intron A and its innovative deletions, as well as Z-DNA-inductive sequences, can improve the gene expression, being important tools for the optimization of clinical relevant protein expression in mammalian cells.

ASSUNTO(S)

expressão heteróloga biologia molecular intron z-dna

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