LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

3. Materials and Methods.
3.1 Generation of lentiviral vectors for the expression of shRNA’s.
The pLKO.1 puro lentiviral vector (343) was modified to remove the Cla1 site in the
stuffer downstream of the U6 promoter (1.9Kb). A 1 kb fragment containing a Multiple
Cloning Site (MCS) was excised from pDesRedN1 with BshTI and EcoRI and cloned
into pLKO.1 digested with the same enzymes to generate the pS18 lentiviral vector. The
pLKO.1 vector was also modified to remove the stuffer and introduce a MCS for cloning
shRNA casettes into pLKO.1 to generate pLKO.1 MCS.
3.2 Generation of lentiviral vectors expressing shRNA and EGFP-f.
The EGFP-f (Farnesylated EGFP) expression cassette was assembled in pBSK(-)
(Stratagene). The sequences of all the oligonucleotides used for gene amplification are
shown in Table 3.1. The EF1α promoter was amplified from pEF6MycHisA (Invitrogen)
and cloned in pBSK digested with HindIII and EcoRI (NEB). Subsequently, the EGFP-f
gene was amplified and cloned downstream of the EF1α promoter as an EcoRI and
BamHI fragment. The resulting vector was digested with BamHI and NotI and the SV-40
poly A signal, amplified from pEF6MycHisA, was inserted into the above vector using
these restriction sites. A restriction site for BsuI5I (Fermentas) was introduced upstream
of the NotI restriction site. The resultant vector is called pBSK –F2. The expression
cassette generated contained BshTI and EcoRI sites that were required for cloning the
shRNA oligonucleotides into pLKO.1 puro. To overcome this hurdle the BshTI and
EcoRI sites were blunted using Klenow (NEB) and the fragment self-ligated resulting in
the loss of both the BshTI and EcoRIrestriction sites . The resultant EGFP-f expression
cassette was excised from pBSK (-) vector with BsuI5I and cloned in pLKO.1 MCS to
generate pLKO.1 EGFP-f Puro.
3.3 Generation of lentiviral vectors for cDNA expression.
The EGFP-f expression cassette was assembled in pBSK(-) (Stratagene) to generate
pBSK – F2 as described earlier . To generate the vector for gene expression studies the
EGFP-f gene in pBSK-F2 was replaced with a MCS containing eleven unique sites to
generate pBSK-FM3 (Table 3.2). The resultant expression cassette containing a MCS
downstream of the EF1α promoter was excised from pBSK – FM3 vector with BsuI5I
and cloned in pLKO.1 puro to generate the pEF.55 vector.
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