engleski

Immortalisation of primary tumor cells by transformation with hTERT telomerase gene

We used primary cultures of lung cancer as a model for transfection with telomerase gene. Human telomerase consists of two parts, an RNA part and a protein part. RNA part is encoded by hTR gene, and is expressed in all cells at all times. The protein part encoded by hTERT forms a catalytic subunit of human telomerase, which is not expressed in most cells. hTERT mRNA is 3644 bp long, with coding region of 3399 bp long, and gives a protein of 1132 amino acids. Therefore, to gain telomerase function and to immortalize cells, it is enough to insert only tha catalytic subunit gene, hTERT. Plasmid pBabe-puro-hTERT, where hTERT is located between two LTRs and puromycine resistance gene, was inserted into a special packaging Phoenix cell line where it was transcribed and the transcript was packed into retroviral particles. These particles are replication deficient, so they can be used for transfection of cell lines, but are unable to reproduce in them. The presence of hTERT transcript was proved directly on retroviral filtrate by performing reverse transcription reaction, and hTERT cDNA fragment 241 bp long was amplified by PCR. We proved that retroviral particles carrying hTERT are ready for transformation application.

primary cultures; telomerase; immortalisation

nije evidentirano