Tesi etd-07252012-230620 |
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Tipo di tesi
Tesi di laurea magistrale
Autore
ELIA, ILARIA
URN
etd-07252012-230620
Titolo
Expression of Magic-F1 to induce myogenesis in C2C12 myogenic cells and embryonic stem cells
Dipartimento
BIOLOGIA
Corso di studi
BIOLOGIA MOLECOLARE E CELLULARE
Relatori
correlatore Prof.ssa Dente, Luciana
relatore Prof.ssa Batistoni, Renata
correlatore Prof.ssa Marracci, Silvia
relatore Prof.ssa Batistoni, Renata
correlatore Prof.ssa Marracci, Silvia
Parole chiave
- Magic-F1
- cloning
- cell culture
- immunofluorescence
- Real Time PCR
Data inizio appello
13/09/2012
Consultabilità
Non consultabile
Data di rilascio
13/09/2052
Riassunto
Magic-F1 is a recombinant protein derived from the human hepatocyte growth factor (HGF). Differently from HGF, Magic-F1 enhances the myogenic differentiation process.
To evaluate the effect of Magic-F1 in C2C12 cells I infected these cells with lentiviral vectors carrying Magic-F1 cDNA or turbo Red Fluorescence Protein (tRFP) as control.
C2C12 cells were differentiated into myotubes in serum starvation condition for 7 days. In these cells, Magic-F1 accelerated the myotube formation, as shown by immunofluorescence analysis of myosin heavy chain (MHC) protein.
I generated transgenic ES cells expressing Magic-F1 or GFP as control. Forced aggregation by centrifugation of ES cells resulted into embryoid bodies (EB) formation. Since the expression of the transgene is under the control of a tetracycline inducible promoter, doxycycline was added to induce Magic-F1 or GFP expression. The presence of Magic-F1 induced an higher expression of Pax3 and Pax7 genes at day 5, 7 and 9 of EB differentiation and stimulated myotube formation, enhancing the myogenic commitment of transgenic ES cells.
In conclusion, my results demonstrate that Magic-F1 is an engineered factor that provides pro-differentative clues towards myogenic commitments.
To evaluate the effect of Magic-F1 in C2C12 cells I infected these cells with lentiviral vectors carrying Magic-F1 cDNA or turbo Red Fluorescence Protein (tRFP) as control.
C2C12 cells were differentiated into myotubes in serum starvation condition for 7 days. In these cells, Magic-F1 accelerated the myotube formation, as shown by immunofluorescence analysis of myosin heavy chain (MHC) protein.
I generated transgenic ES cells expressing Magic-F1 or GFP as control. Forced aggregation by centrifugation of ES cells resulted into embryoid bodies (EB) formation. Since the expression of the transgene is under the control of a tetracycline inducible promoter, doxycycline was added to induce Magic-F1 or GFP expression. The presence of Magic-F1 induced an higher expression of Pax3 and Pax7 genes at day 5, 7 and 9 of EB differentiation and stimulated myotube formation, enhancing the myogenic commitment of transgenic ES cells.
In conclusion, my results demonstrate that Magic-F1 is an engineered factor that provides pro-differentative clues towards myogenic commitments.
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