Saturday, 10 July 2010

The episomal expression of the protein Lin28 in mammalian cells HEK 293 6E

Dec. 2009


The protein Lin28 and miRNA Let-7 form a cellular control system that is important regulator of gene expression that control both physiological and pathological processes such as development and cancer. Firstly, the miRNA Let-7 has been reported to play a tumour suppressor role by repression of some oncogenes (Hmga2, Ras and Myc). Secondly, Lin28 is a protein that blocks maturation of Let-7 and it is reported among the factors that reprogram human somatic cells to pluripotent stem cells (iPS). By adding, that Lin28 is highly expressed in undifferentiated cells and certain cancers, it could be an interesting protein for the development of the drugs. In this context, the purpose was to verify the episomal expression of the protein Lin28 in human embryonic kidney (HEK293-6E) cells despite the interactions between Let-7 and Lin28 that determine several embryonic cell transformations. To verify the production of recombinant protein (r-protein) Lin28, we used a system of r-protein production form by HEK293-6E cells (suspension culture), recombinant vector (pTT5SH8Q1-Lin28) with cytomegalovirus (CMV) promoter, linear polyethylenimine (PEI) as transfection agent and the culture medium serum-free (P). After the construction of recombinant vectors (pTT5SH8Q1-Lin28) we verified the production of Lin28 using three ratios DNA:PEI and two harvests of HEK293-6E cells (24h and 48h). The verification of the expression of Lin28 has been carried out by SDS-PAGE electrophoresis. The protein Lin28 was synthesised in all experimental conditions, but production was greater after 48h after transfection and a ratio DNA:PEI of 1:2. In conclusion, the protein Lin28 has been produced by episomal expression in HEK293-6E cells (suspension culture) using a recombinant vector (pTT5SH8Q1-Lin28).