LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

developing testes to infect spermatogonial stem cells as described in Material and
Methods. The 14-3-3ε fore-founder mouse (indicated by the hatched box and arrow) was
mated with normal female mice of the same strain (Figure 4.4.18). The pups obtained
were then screened for the presence of RFP by performing polymerase chain reactions
with RFP specific primers on genomic DNA. A PCR reaction for the mouse patch (Ptc)
gene was performed as a loading control. Five out of seven animals showed the presence
of the RFP transgene (Figure 4.4.18). Stable lines will be generated and the ability of
doxycyclin to inhibit 14-3-3ε expression will be tested at a later date.
Figure 4.4.17: HA-14-3-3ε-R is resistant to degradation by E1 shRNA construct. 50µg
protein extracts from HCT116 cells co-transfected with shRNA constructs targeting 14-3-
3ε along with wild type or mutant 14-3-3ε were resolved on a 10% SDS-PAGE gel and
Western blots were performed using antibodies to GFP and actin. Note that 14-3-3ε-R
mutant is resistant to degradation by E1 shRNA construct as compared to WT-14-3-3ε.
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