Team:MIPT MSU/Proof Of Concept
This section was the last to fill due to protracted experiments and work with iGEM Wiki-pages. As a result, the data obtained is not included in the Results and Engineering Success sections.
We tested five types of constructs and demonstrated the efficiency of expression in MCF7 cells.
The following constructions were used:
- 5’ - psi(short) - IRES - GFP - lacZ -3’
- 5’ - psi(short) - GFP -lacZ - 3’
- 5’ - psi(long) - IRES -GFP - lacZ - 3’
- 5’ - psi(long) - GFP - lacZ -3’
- 5’ - IRES - GFP - lacZ - 3’
Control plasmid constructs containing GFP luminescence brightly (Figure I, control). GFP from constructs 1, 2, 5 was expressed, but the luminescence of the cells was less intense. Interestingly, constructs with a long 5'UTR practically do not express GFP (Figure I, constructs 3 and 4). This may be correlated to the presence of a long 5'UTR in RNA transcripts. What is the reason for the difference in the level of expression remains to be clarified in our further experiments.
2 days after nucleofection, we collected the supernatant of five MCF7 culture cells, which contained our vesicles. We immediately added this supernatant with vesicles to the HT29 cells containing the syncytin receptor. After 1 day, we observed GFP luminescence in target HT-29 cells, to which 1st vesicle constructions (5'-psi (short) -IRES-GFP-lacZ-3 ') were added. While no GFP luminescence was observed in HT-29 cells, to which vesicles containing other types of constructs were added. In the case of constructions №3 (5'-psi (long) -IRES-GFP-lacZ) and 4 (5'-psi (long) -GFP-lacZ-3 '), no luminescence was observed (due to the low production rate of vesicles in the MCF-7 cells). In construct №5, absence of luminescence was an expected result, as construction lacked the packaging psi-signal (therefore no GFP mRNA got into vesicles), and served as a negative control. In the case of construct №2 (5'-psi (short) -GFP-lacZ-3 '), no luminescence was observed, probably due to the absence of ribosome landing site(IRES), subsequently decreasing the translation of GFP in such constructs. Thus, we preliminary confirmed the operability of our system, and in the future, we can continue to work not only on the GFP mRNA, but also on other types of mRNA and miRNA.
HT-29 cells treated with vesicles with 5’-psi(short)-IRES-GFP-lacZ-3’ construction. Expression of GFP was observed after 1 day after treatment.
