LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute
LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute
LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute
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3. Materials and Methods.<br />
3.1 Generation of lentiviral vectors for the expression of shRNA’s.<br />
The pLKO.1 puro lentiviral vector (343) was modified to remove the Cla1 site in the<br />
stuffer downstream of the U6 promoter (1.9Kb). A 1 kb fragment containing a Multiple<br />
Cloning Site (MCS) was excised from pDesRedN1 with BshTI and EcoRI and cloned<br />
into pLKO.1 digested with the same enzymes to generate the pS18 lentiviral vector. The<br />
pLKO.1 vector was also modified to remove the stuffer and introduce a MCS for cloning<br />
shRNA casettes into pLKO.1 to generate pLKO.1 MCS.<br />
3.2 Generation of lentiviral vectors expressing shRNA and EGFP-f.<br />
The EGFP-f (Farnesylated EGFP) expression cassette was assembled in pBSK(-)<br />
(Stratagene). The sequences of all the oligonucleotides used for gene amplification are<br />
shown in Table 3.1. The EF1α promoter was amplified from pEF6MycHisA (Invitrogen)<br />
and cloned in pBSK digested with HindIII and EcoRI (NEB). Subsequently, the EGFP-f<br />
gene was amplified and cloned downstream of the EF1α promoter as an EcoRI and<br />
BamHI fragment. The resulting vector was digested with BamHI and NotI and the SV-40<br />
poly A signal, amplified from pEF6MycHisA, was inserted into the above vector using<br />
these restriction sites. A restriction site for BsuI5I (Fermentas) was introduced upstream<br />
of the NotI restriction site. The resultant vector is called pBSK –F2. The expression<br />
cassette generated contained BshTI and EcoRI sites that were required for cloning the<br />
shRNA oligonucleotides into pLKO.1 puro. To overcome this hurdle the BshTI and<br />
EcoRI sites were blunted using Klenow (NEB) and the fragment self-ligated resulting in<br />
the loss of both the BshTI and EcoRIrestriction sites . The resultant EGFP-f expression<br />
cassette was excised from pBSK (-) vector with BsuI5I and cloned in pLKO.1 MCS to<br />
generate pLKO.1 EGFP-f Puro.<br />
3.3 Generation of lentiviral vectors for cDNA expression.<br />
The EGFP-f expression cassette was assembled in pBSK(-) (Stratagene) to generate<br />
pBSK – F2 as described earlier . To generate the vector for gene expression studies the<br />
EGFP-f gene in pBSK-F2 was replaced with a MCS containing eleven unique sites to<br />
generate pBSK-FM3 (Table 3.2). The resultant expression cassette containing a MCS<br />
downstream of the EF1α promoter was excised from pBSK – FM3 vector with BsuI5I<br />
and cloned in pLKO.1 puro to generate the pEF.55 vector.<br />
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