LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

4.4 Generation of 14-3-3ε knockdown mice.
4.4.1 Testing the efficiency of cDNA and shRNA constructs.
A cDNA for the mouse 14-3-3ε gene product was cloned in pCDNA3.0 (Invitrogen)
downstream of the HA epitope as described in Materials and Methods. The ability of the
construct to express mouse 14-3-3ε was determined by transfection of different amounts
of the HA-tagged mouse 14-3-3ε construct into HCT116 cells. Western blotting with
antibodies to the HA epitope demonstrated that maximal expression of HA-14-3-3ε was
obtained when 1µg of the construct was transfected into HCT116 cells (Figure 4.4.1 A).
Cells transfected with an HA-tagged human 14-3-3γ and with pCDNA 3.0 served as
positive and negative controls respectively.
Three different shRNA constructs against 14-3-3ε were designed as described in
Materials and Methods. The ability of these constructs to inhibit the expression of
exogenously expressed 14-3-3ε was determined by co-transfection of the pLKO.1 based
shRNA constructs and HA-tagged mouse 14-3-3ε in HCT116 cells. Protein levels of HAmouse
14-3-3ε were determined by Western blotting with antibodies to the HA epitope.
The construct pLKO.1 E1 could inhibit the expression of HA-14-3-3ε, but the constructs
pLKO.1 E2 and E3 could not inhibit expression of HA-14-3-3ε (Figure 4.4.1 B). No
inhibition of HA-14-3-3ε expression was observed in cells transfected with the vector
control (Figure 4.4.1 B). Western blots for actin were performed to serve as loading
controls.
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