Would it be feasible to treat multiple independent domains as a concatamer? E.g.,
Forward 5' = Sense1 -- Sense2 -- hairpin -- AntiSense2 -- AntiSense1 -- term
Reverse 5' = term -- S1 -- S2 -- hairpin -- AS2 -- AS1
Alternatively, I'm considering altering our plasmid to have multiple expression cassettes. I've never done this before so ideally I would like to talk this through with someone to make sure I'm thinking about this correctly. Let's say I'm using pLKO.1 plasmid with an expression cassette of U6 and insert between AgeI and EcoRI.
To modify this plasmid to contain two expression cassettes I would have to:
1) amplify downstream of U6
2) cut (1) with AgeI
3) independently ligate shRNAs with fragment from (2)
4) pool and ligate fragments from (3)
5) ligate (4) into linearized pLKO.1 vector
Or am I thinking about this the wrong way?
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